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CN116940673A - 可编程转座酶及其用途 - Google Patents

可编程转座酶及其用途 Download PDF

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CN116940673A
CN116940673A CN202180093884.8A CN202180093884A CN116940673A CN 116940673 A CN116940673 A CN 116940673A CN 202180093884 A CN202180093884 A CN 202180093884A CN 116940673 A CN116940673 A CN 116940673A
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M·盖尔卡戈尔
A·桑切斯-梅希亚斯加西亚
M·帕拉斯马西米亚
D·伊万西奇杰尔马诺维奇
A·拉赫迈
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Universitat Pompeu Fabra UPF
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Abstract

本公开提供了基于组合物的有效且准确的可编程基因递送技术，所述组合物包含(i)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；和(ii)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由其组成，其中所述转座酶是修饰的高活性PiggyBac。

Description

可编程转座酶及其用途
技术领域
本发明涉及基因编辑和基因治疗领域。
背景技术
许多疾病，例如癌症、发育障碍和一些感染，都具有共同的遗传和表观遗传畸变。基因治疗旨在将遗传物质引入细胞中，直接靶向和编辑基因组，以纠正基因功能失调的细胞，从而治愈相关疾病。
基因编辑工具箱在过去几年中得到了显著扩展，它是除基因治疗之外的一种具有前景的工具，可以修复缺陷基因，从而治疗有此需要的受试者的疾病。
传统上，基因编辑基于人工核酸内切酶的设计，该酶可在基因组中的目标序列中诱导双链断裂(DSB)1。细胞通过两种主要途径之一修复DSB：非同源末端连接(NHEJ)或同源引导修复(HDR)2。最近，开发了不依赖于DSB的编辑。基于用脱氨酶即碱基编辑器(BE)3直接编辑DNA碱基和借助逆转录酶(RT)即先导编辑器(PE)4原位替换DNA碱基的方法已成为可能。
然而，病理性基因缺陷的范围可以从几个碱基到大的缺失。碱基编辑器或先导编辑器仅靶向少量碱基，并且基于HDR的编辑对于尺寸的放大能力弱5。已经开发了基于NHEJ的方法，例如同源性非依赖性靶向整合(HITI)6。这种方法已被证明可用于数千个碱基的插入，但对于非常大的编辑仍然无效5。尽管HITI可能能够递送外显子，但它的效力可能不足以稳健递送诸如肌营养不良蛋白(～14kb)或层粘连蛋白-ɑ2(～9kb)的基因的cDNA。
在细菌中已描述了高准确度的CRISPR可编程转座子7,8，但它们不适用于哺乳动物细胞。之前尝试将锌指或酿脓链球菌(Streptococcus pyogenes)Cas9(SpCas9)与哺乳动物兼容的PiggyBac(PB)或睡美人转座酶递送系统融合，但准确度水平相对较低9-11。由于效率随尺寸的放大能力好，PB系统是用于基因治疗的一种有吸引力的工具，它是一种不依赖于突变的技术，并且由于对DNA修复机制的依赖性低而适用于任何组织。
因此，仍然需要开发在体外、离体或离体用于哺乳动物细胞的靶向基因递送的新系统。
发明内容
某些可编程转座酶及其在靶向基因编辑中的用途已经公开于WO2020250181中，其内容通过引用并入本文。
本公开现提供进一步的基于组合物的有效且准确的可编程基因递送技术，所述组合物包含(i)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合并切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；和(ii)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由其组成，其中所述转座酶是修饰的高活性PiggyBac(hyperactive PiggyBac)。这种技术能够递送小核酸片段，也能够递送大核酸片段。发明人已经在哺乳动物细胞和体内小鼠肝脏中测试了这项技术，并且令人惊讶地在所有测试中均实现了高效率(5-10％)的定点整合(site directed integration)。
在一个实施方案中，组合物包含(i)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合并切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；和(ii)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由其组成，其中所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变。
在一个实施方案中，第一蛋白和第二蛋白融合在一起以形成融合蛋白，任选地通过接头。在一个实施方案中，第一蛋白融合至第二蛋白的C末端，任选地通过接头。
在一个实施方案中，所述转座酶是修饰的高活性PiggyBac，其与未修饰的高活性PiggyBac相比包含增加切除活性(excision activity)的一个或多个氨基酸突变，和/或与未修饰的高活性PiggyBac相比，包含降低DNA结合活性的一个或多个氨基酸突变。
在一个实施方案中，所述一个或多个氨基酸突变不由R372A、K375A和D450N组成。在一个实施方案中，所述一个或多个氨基酸突变选自M194、D450、T560、S564、S573、S592或F594位置处的增加切除活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代M194V和/或D450N。在一个实施方案中，所述一个或多个氨基酸突变选自M194或D450位置处的增加切除活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代M194V和/或D450N。在一个实施方案中，所述一个或多个氨基酸突变选自在位置R275、R277、R347、R372、K375、R376、E377和/或E380处的降低DNA结合活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代R275A、R277、R347S、R372A、K375A、R376A、E377A和/或E380A。在一个实施方案中，所述一个或多个氨基酸突变选自在位置R372、K375、R376、E377和/或E380处的降低DNA结合活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代R372A、K375A、R376A、E377A和/或E380A。
在一个实施方案中，修饰的高活性PiggyBac包含双突变N347S和D450N，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBoc的氨基酸编号。在一个实施方案中，修饰的高活性PiggyBac突变包括以下氨基酸取代之一或氨基酸取代的组合：R372A/K375A/R376A/D450N、K375A/R376A/E377A/E380A/D450N、R372A/K375A/R376A/E377A/E380A/D450N、M194V、R376A、E377A、E380A、M194V/R372A/K375A、S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、T43I/R372A/K375A/A411T/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G；位置编号对应于SEQ ID NO:9的高活性PiggyBac的氨基酸编号，通常所述修饰的转座酶具有选自SEQID NO:2-8、10-18和135-149中任一个的氨基酸序列。
在一个实施方案中，组合物进一步包含第三蛋白或编码所述第三蛋白的核酸构建体，所述第三蛋白包含第二转座酶或由第二转座酶组成，其中所述第二转座酶是SEQ IDNO:9的高活性PiggyBac，或者是与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变的修饰的高活性PiggyBac。在一个实施方案中，第一、第二和第三蛋白融合在一起以形成三重融合蛋白，任选地通过接头。
在一个实施方案中，第一蛋白包含RNA引导的核酸酶或切口酶或锌指核酸酶，或由其组成。在一个实施方案中，所述第一蛋白是核酸酶蛋白，其包含活性DNA切割结构域和引导RNA结合结构域，并且与SEQ ID NO:31的酿脓链球菌(Streptococcus pyogenes)Cas9(SpCas9)、SEQ ID NO:72的金黄色葡萄球菌(Staphylococcus aureus)Cas9(SaCas9)、SEQID NO:74的Cpf1、SEQ ID NO:29的空肠弯曲杆菌(Campylobacter jejuni)Cas9(CjCas9)、SEQ ID NO:70的酿脓链球菌Cas9切口酶(nCas9)、SEQ ID NO:75的CasX或SEQ ID NO:76的金黄色葡萄球菌Cas9切口酶具有至少80％、90％、95％、99％或至少100％的同一性；优选地，其中所述第一蛋白是选自SEQ ID NO:72的金黄色葡萄球菌Cas9(SaCas9)和SEQ ID NO:31的酿脓链球菌Cas9(SpCas9)的Cas9蛋白。
在一个实施方案中，组合物进一步包含引导RNA和用于插入基因组中的外源核酸。
在一个实施方案中，转座酶与RNA结合蛋白融合，所述RNA结合蛋白能够与引导RNA中包含的至少一种特异性RNA序列结合；任选地，其中所述RNA结合蛋白是MS2噬菌体外壳蛋白(MCP)并且其中引导RNA包含MS2 RNA四环结合序列，优选与SEQ ID NO:153具有至少75％的同一性。
在一个实施方案中，外源核酸是大DNA片段，通常具有5kb至25kb的大小，更优选8kb至20kb的大小。
在一个实施方案中，组合物包含在纳米颗粒中。
本发明还涉及编码本文公开的任何一种融合蛋白的核酸，通常以信使RNA(mRNA)的形式。
本发明还涉及用于将外源核酸序列位点特异性整合至细胞基因组中的体外方法，该方法包括将本发明的组合物、引导RNA和外源核酸递送至细胞。
本发明还涉及本发明的组合物、引导RNA和外源核酸，用于通过将外源核酸序列位点特异性整合至细胞基因组中来治疗疾病。
附图说明
图1：可编程转座酶技术：cas9(红色)与工程化的PB转座酶结构域(粉色)相结合。实验中使用的突变及其在PB核心模型中的相应位置(位置563位于Ct上，其不包含在模型中)的表。
图2：A：依赖于cas9变体的可编程转座酶。与死cas9(dcas9)或切口酶cas9(ncas9)融合物相比，核酸酶cas9和PB融合物在靶向和整体插入方面显示出更好的结果。蓝色表示靶向插入，黄色表示脱靶插入(off-target insertion)。B：可编程转座酶对PB变体的依赖性。DNA结合减少的切除增强突变体呈现了最佳的中靶：脱靶比率(橙色)。在AAV位点(绿色)和TRAC位点(蓝色)处进行中靶插入(On-target insertion)。C：不同接头的测试。接头长度和拓扑学不会显著影响Spcas9和PB融合物的中靶活性(on-target activity)。
图3：Hershey报告细胞系：HEK293T细胞系经工程化以含有GFP的C末端片段，其前面有一个剪接接受体(splicing acceptor)和gRNA靶位点。组合CAG启动子、GFP的N末端片段和后接的剪接供体来生成PB转座子。灰色三角形：PB ITR；SA：剪接接受体；SD：剪接供体；靶标：靶向插入位点；*插入过程破坏ITR。
图4：A，可编程转座酶对PB变体的依赖性。切除增强突变体450在减少DNA结合的不同突变的背景下，呈现处最佳的中靶。R372和R376同时突变为A的耐受性不佳。尽管E377不参与DNA结合，但突变为A可能有利于避免K375和R376突变为A时该区域中负电荷的积聚。B：由于与靶DNA的结合减少，R372A/K375A降低了PB的整合活性(也如D450N所观察到的)。脱靶整合的测试在进行中。
图5：双链断裂和可编程DNA结合结构域在靶向插入中的作用。插入位点中双链断裂和PB的共定位是有效的中靶插入所必需的。
图6：多种插入的桑格测序验证(参见图2a中通过NGS测量的更全面的分布)。ITRTTAA在靶向插入过程中丢失。NGG Pam以红色突出显示。
图7：没有cas9时的插入活性PB K375A_R376A_E377A_E380A_D450N。为了进一步研究靶向插入机制，在没有cas9的情况下克隆hyPB K375A_R376A_E377A_E380A_D450N，并在hek293T细胞中使用RFP转座子测试了其插入效率与hyPB WT的比较。结果显示该突变体在没有融合cas9的情况下没有插入活性。
图8：A：使用Guide-seq表征靶向插入位点。可编程转座酶通过多个插入缺失使ITR位点失活，从而产生不可逆插入。B：使用Guide-seq表征整个插入位点。在TCR基因座上仅检测中靶插入(上图)。显示了4个克隆的桑格测序(下图)。
图9：Guide-seq的插入分析的可编程转座酶表征表明，hyPB突变体与Cas9组合进行准确的转座子插入。
图10：Cas9-hyPB R372A-K375A-D450N与其他靶向插入平台(例如Cas9诱导的HDR)(使用300bp同源臂)的基准测试(Benchmarking)。
图11：Cas9-hyPB R372A-K375A-D450N在小鼠肝脏中的体内分布。报告了通过qPCR测量的相对拷贝数。
图12：使用不同的Cas变体如CasX、CjCas9 Cpf1或SaCas9对可编程转座酶进行工程化，其中一些在靶位点的可编程插入方面取得了与SpCas9类似的结果。测试的每种Cas变体均使用3种独立的gRNA靶向分裂GFP报告细胞系(split GFP reporter cell line)的特定靶标区域。
图13：通过Cas9和单gRNA(gRNA-TCR1或AAVS1-3)或通过切口酶Cas9和靶向临近位置的两种gRNA(gRNA-TCR1和AAVS1-3)的双链DNA断裂，以及与修饰的hyPB(突变体R372A-K375A-D405N)融合的可编程DNA结合结构域(ZnF)，导致了靶向插入。插入位点中双链断裂和PB的共定位是有效的中靶插入所必需的。这可以通过核酸酶Cas9或切口酶Cas9双切割来实现。
图14：可编程转座酶可以被工程化为两个hyPB结构域和Cas9核酸酶的二聚体多肽，与Cas9-hyPB相比产生更好的可编程插入。分裂GFP报告细胞系用于将分裂GFP转座子可编程插入至靶位点。hyPB R372A-K375A-D450N的突变体已用于与Cas9的单体或二聚体融合。条件：1：仅使用hyPB作为插入机制的阴性对照；2：在pcDNA表达载体中的Cas9-hyPBR372A-K375A-D450N为阳性对照；3：在慢病毒表达载体中的Cas9-hyPB R372A-K375A-D450N为阳性对照；4：Cas9核酸酶在C末端与两个单元的hyPB R372A-K375A-D450N融合；图5：Cas9核酸酶与两个单元的hyPB R372A-K375A-D450N融合，一个在C末端，另一个在N末端。
图15：细胞的多轮选择，其中发生可编程转座从而允许从文库中选择最佳突变组合。我们鉴定了几种突变，其与Cas9融合时比Cas9-hyPB R372A-K375A-D450N具有更好的富集能力和可编程插入能力。
图16：中靶效率随着选择轮次的增加而增加。将从每个循环中选择的大量变体与靶向AAVS1的gRNA和1/2GFP转座子共转染至报告细胞系中。通过PB拷贝数校正质粒数量，以将克隆效率标准化。
图17：(A)所选择的前几个候选者的中靶效率。根据上一轮中选择的96个随机克隆中最高的中靶活性，选择了6个单独的候选者。将单独的中靶活性与Cas9-hyPB R372A-K375A-D450N进行比较。(B)标识显示在所选择的前几个中靶活性变体中的主要PB残基。
图18：Cas9-hyPB R372A-K375A-D450N(FiCAT)与同源独立靶向插入(HITI)的基准测试。
图19：使用四种不同核酸酶蛋白的FiCAT R372A-K375A-D450N的可编程插入活性。SpCas9用作仅使用gRNA-TRAC-1进行可编程插入的对照(左)。每种核酸酶与三种独立的gRNA(1-3)一起使用，用于在1/2GFP报告细胞系中进行靶向插入。
图20：微环荧光素酶转座子的肝脏整合。通过流体动力学注射递送微环荧光素酶转座子、靶向Rosa26基因座的sgRNA和FiCAT(Cas9-hyPB R372A-K375A-D450N)mRNA，并监测荧光素酶信号。
图21：(a)CasX(左)和Cpf1(中)的编辑活性。(b)SaCas9(左)、CjCas9(中)的编辑活性。显示了两次技术重复的具有插入缺失读段的平均值％+/-SD，N＝3次生物学重复的代表性图像。靶向TRAC-1位点的SpCas9用作参考(右)。
图22：中靶效率随选择轮次提高。(A)将从每轮中选择的大量变体与靶向AAVS1的gRNA和1/2GFP转座子共转染至报告细胞系中。通过PB拷贝数校正质粒数量，以将克隆效率标准化。(B)产生表达每轮的大量变体的慢病毒，并用于感染报告细胞系。
图23：用gRNA tcr1和1/2GFP MC转座子共转染，在4和5轮cas9_PB文库富集后从大量变体中分离出的单个突变体相对于FiCAT(hyPB R372A-K375A-D450N)的特异性靶标整合。
图24：与SpCas9或SaCas9融合的二聚体hyPB R372A-K375A-D450N的可编程插入活性，用于在1/2GFP报告细胞系中进行靶向插入。
图25：1/2GFP报告细胞系中用于靶向插入的可编程插入活性的相对比较。(A)与SpCas9蛋白融合的hyPB R372A-K375A-D450N(左)和与MCP蛋白融合的hyPB R372A-K375A-D450N且单独添加SpCas9(右)之间的比较。(B)与MCP蛋白融合的3种hyPB突变体(R372A-K375A-D450N，R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)且单独添加SpCas9时之间的比较。
图26：1/2GFP报告细胞系中用于靶向插入的可编程插入活性的比较。(A)hyPBR372A-K375A-D450N和SpCas9蛋白的共表达(左)和包含hyPB R372A-K375A-D450N和SpCas9蛋白的融合蛋白(右)之间的比较。(B)与SpCas9共表达的3种hyPB突变体(R372A-K375A-D450N，R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)之间的相对比较。
图27：在具有包含SpCas和hyPB R372A-K375A-D450N的第一融合蛋白与包含MCP蛋白和hyPB突变体(R372A-K375A-D450N，R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)的第二融合蛋白共表达的1/2GFP报告细胞系中，用于靶向插入的可编程插入活性的相对比较。
图28：在具有包含SpCas和hyPB R372A-K375A-D450N的融合蛋白以及3种hyPB突变体R372A-K375A-D450N、R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L的共表达的1/2GFP报告细胞系中，用于靶向插入的可编程插入活性的相对比较。
图29：与hyPB R272A-K275A-D450N的二聚体融合的SpCas9(左)和与第一hyPBR272A-K275A-D450N和第二hyPB突变体融合的SpCas9(右)在1/2GFP报告细胞系中的用于靶向插入的可编程插入活性的比较。
定义
如本文所用，单数形式“a”、“an”和“所述(the)”包括单数和复数指代，除非上下文另有明确指示。因此，例如，“药剂(an agent)”包括单个药剂和多个此类药剂。
术语“核酸序列”和“核苷酸序列”可以互换使用，是指由单体核苷酸组成或包含单体核苷酸的任何分子。核酸可以是寡核苷酸或多核苷酸。核苷酸序列可以是DNA、RNA或其混合物。核苷酸序列可以是化学修饰的或人工的。核苷酸序列包括肽核酸(PNA)、吗啉代核酸和锁核酸(LNA)以及二醇核酸(GNA)和苏糖核酸(TNA)。这些序列中的每一个均通过分子主链的变化而与天然存在的DNA或RNA区分。另外，可以使用硫代磷酸核苷酸。其他脱氧核苷酸类似物包括但不限于可用于本公开的核苷酸中的甲基膦酸酯、氨基磷酸酯、二硫代磷酸酯、N3'P5'-氨基磷酸酯和寡核糖核苷酸硫代磷酸酯以及它们的2'-O-烯丙基类似物和2'-O-甲基核糖核苷酸甲基膦酸酯。
术语“转基因”是指外源核酸序列，特别是编码基因产物的外源DNA或cDNA。基因产物可以是RNA、肽或蛋白。除了基因产物的编码区(CDS)之外，转基因可以包括或连接一种或多种操作序列以促进或增强表达，例如启动子、增强子、应答元件、报告元件、隔绝元件、聚腺苷酸化信号和/或其他功能元件。除非另有说明，否则本公开的实施方案可以利用任何已知的合适的启动子、增强子、应答元件、报告元件、隔绝元件(insulator element)、聚腺苷酸化信号和/或其他功能元件。合适的元件和序列对于本领域技术人员来说是公知的。
术语“多肽”、“肽”和“蛋白”可互换使用，是指氨基酸残基的聚合物。该术语也适用于其中的一个或多个氨基酸是相应天然存在氨基酸的化学类似物或修饰衍生物的氨基酸聚合物。
术语“结合蛋白”是指能够非共价结合另一分子的蛋白。结合蛋白可以结合例如DNA分子(DNA结合蛋白)、RNA分子(RNA结合蛋白)和/或蛋白分子(蛋白结合蛋白)。就蛋白结合蛋白而言，它可以与一个或多个分子的同一蛋白结合，形成同二聚体、同三聚体等；和/或它可以结合一种或多种不同蛋白的一个或多个分子。结合蛋白可以具有多于一种类型的结合活性。例如，锌指蛋白具有DNA结合、RNA结合和蛋白结合活性。
术语“Cas9”或“Cas9核酸酶”是指包含Cas9蛋白或其片段(例如，包含Cas9的活性或非活性DNA切割结构域和/或Cas9的gRNA结合结构域的蛋白)的RNA引导的核酸酶。Cas9核酸酶有时也称为casn1核酸酶或CRISPR(成簇规则间隔短回文重复序列)相关核酸酶。CRISPR是一种适应性免疫系统，提供对抗移动的遗传元件(病毒、转座元件和接合质粒)的保护作用。CRISPR簇包含间隔区，它们是与先前的移动元件互补的序列，并靶向入侵核酸。CRISPR簇被转录并加工成CRISPR RNA(crRNA)。在II型CRISPR系统中，pre-crRNA的正确加工需要反式编码的小RNA(tracrRNA)、内源核糖核酸酶3(rnc)和Cas9蛋白。tracrRNA可作为核糖核酸3辅助的pre-crRNA加工的向导。随后，Cas9/crRNA/tracrRNA对与间隔区互补的线性或环状dsDNA靶标进行核酸内切酶式切割(endonucleolytically)。与crRNA不互补的靶标链首先进行核酸内切酶式切割，然后进行3'-5'核酸外切酶式修剪。在自然界中，DNA结合和切割通常需要蛋白和两种RNA。然而，可以工程改造单引导RNA(“sgRNA”或简称“gRNA”)，从而将crRNA和tracrRNA两者的各个方面合并到单个RNA物质中。
Cas9识别CRISPR重复序列中的短基序(PAM或原型间隔区相邻基序)，以帮助区分自我与非我。Cas9核酸酶序列和结构是本领域技术人员公知的。Cas9直系同源物已在多种物种中得到描述，包括但不限于酿脓链球菌和嗜热链球菌。基于本公开内容，其他合适的Cas9核酸酶和序列对于本领域技术人员来说将是显而易见的，并且这样的Cas9核酸酶和序列包括来自Chylinski等人，2013(RNA Biol.10(5):726-37)中公开的生物体和基因座的Cas9序列，其全部内容通过引用并入本文。
在一些实施方案中，Cas9核酸酶具有无活性(例如，失活的)DNA切割结构域。核酸酶失活的Cas9蛋白可以互换地称为“白可以互换地蛋白(指核酸酶“死”的Cas9)。用于产生具有无活性DNA切割结构域的Cas9蛋白(或其片段)的方法是本领域已知的(参见，例如，Jinek等,2012.Science.337(6096):816-821；Qi等,2013.Cell.152(5):1173-83，其整体内容通过引用并入本文)。
术语“锌指蛋白”是指蛋白或较大蛋白内的结构域，其通过一个或多个锌指以序列特异性方式结合DNA，所述锌指是通过锌离子的配位而稳定的锌指蛋白的结合结构域内的氨基酸序列区域。术语“锌指蛋白”通常缩写为“ZFP”。
术语“锌指核酸酶”是指通过将锌指DNA结合结构域与DNA切割结构域融合而产生的人工限制酶。锌指结构域可以被工程化以靶向特定的目标DNA序列，这使得锌指核酸酶能够靶向复杂基因组内的独特序列。“锌指核酸酶”通常缩写为“ZFN”或“ZNP”。
本文所用的术语“氨基酸序列”或“多肽”或“蛋白”是指氨基酸残基的聚合物。除非另有说明，否则氨基酸残基的聚合物可以是任何长度。
本文使用的术语“外源”是指天然不存在于细胞中、但可以通过一种或多种遗传、生物化学或其他方法引入细胞中的分子。细胞中的天然存在也可以根据细胞的特定发育阶段和环境条件来确定。因此，例如，仅在肌肉的胚胎发育期间存在的分子对于成体肌肉细胞来说是外源分子。类似地，由热激诱导的分子对于非热激细胞来说是外源分子。外源分子可包含例如功能障碍的内源分子的功能版本或正常功能的内源分子的功能障碍版本。
相比之下，“内源”分子是通常在特定环境条件下在特定发育阶段存在于特定细胞中的分子。例如，内源核酸可包含染色体，线粒体、叶绿体或其他细胞器的基因组，或天然存在的游离核酸。其他的内源分子可以包括蛋白，例如转录因子和酶。
“靶序列”或“靶核酸序列”或“靶位点”是定义核酸(例如基因组中的核酸)的一部分的序列，只要存在足够的结合条件，结合分子将与其结合。例如，序列5'-GAATTC-3'是EcoRI限制性核酸内切酶的靶位点。
术语“融合物”是指其中有两个或更多个亚基分子连接的分子。在一些实施方案中，两者之间的连接是共价的；或者，两者之间的连接可以是非共价的，并且依赖于例如分子间相互作用。亚基分子可以是相同化学类型的分子，或者可以是不同化学类型的分子。
术语“融合蛋白”是指包含来自至少两种不同蛋白的蛋白结构域的杂合多肽。例如，一个蛋白结构域可以位于融合蛋白的氨基末端(N末端)部分或羧基末端(C末端)蛋白，从而分别形成“氨基末端融合蛋白”或“羧基末端融合蛋白”。在优选的实施方案中，融合蛋白是可以完全由核酸序列编码的单链多肽，并且包括通过肽连接而共价连接或任选地通过肽接头共价连接的至少两个蛋白结构域。
本文所用的术语“基因”或“基因组”包括编码基因产物的DNA区域，以及调节基因产物产生的所有DNA区域，无论此类调节序列是否与编码和/或转录序列相邻。因此，基因包括但不一定限于启动子序列、终止子、翻译调节序列例如核糖体结合位点和内部核糖体进入位点、增强子、沉默子、隔绝物、边界元件、复制起点、基质附着位点和基因座控制区。
术语“真核”细胞包括但不限于真菌细胞(例如酵母)、植物细胞、动物细胞、哺乳动物细胞和人类细胞(例如T细胞)。
本文所用的术语“连接的”是指两个或更多个组件(例如序列元件)的并置，其中这些组件被布置成使得两个组件正常发挥作用并且允许至少一个组件能够介导施加在至少一个其他组件上的功能的可能性。
蛋白、多肽或核酸的“功能片段”分别是其序列与全长蛋白、多肽或核酸不同，但保留与全长蛋白、多肽或核酸相同的功能的蛋白、多肽或核酸。功能片段可以具有与相应天然分子相比更多、更少或相同数量的残基，和/或可以含有一个或多个氨基酸或核苷酸取代。
本文所用的术语“转染”是指将核酸(DNA或RNA)引入真核或原核细胞或生物体中。
术语“切割”是指DNA分子的共价主链的断裂。切割可以通过多种方法引发，包括但不限于磷酸二酯键的酶促水解或化学水解。单链切割和双链切割都是可能的，并且双链-切割可以作为两个不同的单链切割事件的结果而发生。DNA切割可能导致产生平末端或交叠末端。在某些实施方案中，融合多肽用于靶向双链DNA切割。
术语“特异性”是指选择性结合与所选序列具有一定程度的序列同一性的序列的能力。
术语“插入”和“整合”是指将核酸序列添加到第二核酸序列中或添加到基因组或其部分中。与插入或整合有关的术语“特异性”、“位点特异性”、“靶向”和“中靶(on-targeted)”在本文中可互换使用，是指将核酸插入到第二核酸的特定位点中或插入到基因组或其部分的特定位点中。相比之下，术语“随机”、“非靶向”和“脱靶(off-targeted)”是指核酸非特异性且非目的性地插入到不希望的位点。术语“总”或“总体”是指插入的总数。
术语“突变”是指序列(例如核酸或氨基酸序列)内的残基被另一残基取代；和/或核酸或氨基酸序列内一个或多个残基的缺失或插入。本文通常通过指明原始残基、随后指明该残基在序列中的位置、然后指明新取代的残基的身份来描述突变。本文提供的氨基酸取代(突变)的各种方法是本领域公知的，并且由例如Green&Sambrook,2012(Molecularcloning:a laboratory manual(第4版).Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.)提供。在优选的实施方案中，术语蛋白中的突变是指氨基酸取代。
术语“转座酶”是指与转座子末端结合并通过剪切-和-粘贴机制或复制转座机制催化其移动至基因组的另一部分的酶。
术语“修饰的”是指与相应的未修饰的蛋白或核酸序列不同的蛋白或核酸序列。
术语“接头”是指连接两个相邻分子或部分的化学基团或分子。
本文使用的术语“载体”和“质粒”是指可以携带例如感兴趣的第二多核苷酸和例如可以将基因序列转移至靶细胞的任何多核苷酸。因此，该术语包括克隆和表达媒介物以及整合载体。具体地，本文所用的术语“表达载体”是指能够引导核酸表达的任何多核苷酸。在一些方面，术语“载体”和“质粒”与术语“核酸构建体”互换使用。
如本文所用，两个序列之间的百分比同一性是序列共有的相同位置的数量的函数(即，％同一性＝相同位置的数量/位置总数×100)，其中考虑需要引入的空位数量和每个空位的长度以实现两个序列的最佳比对。序列的比较和两个序列之间的百分比同一性的确定可以使用数学算法来完成，如下所述。
两个氨基酸序列之间的百分比同一性可以使用E.Meyers和W.Miller(Comput.Appl.Biosci.,4:11-17,1988)的算法来确定，该算法已被并入ALIGN程序(版本2.0)，使用PAM120权重残基表，空位长度罚分为12，空位罚分为4。或者，两个氨基酸序列之间的百分比同一性可以使用Needleman和Wunsch(J.Mol,Biol.48:444-453,1970)算法来确定，该算法已被并入GCG软件包(可在http://www.gcg.com获取)中的GAP程序，使用Blossom62矩阵或PAM250矩阵，空位权重为16、14、12、10、8、6或4，长度权重为1、2、3、4、5或6。
两个核苷酸氨基酸序列之间的百分比同一性也可以使用例如算法来确定，所述算法例如用于核酸序列的BLASTN程序，使用默认字长(W)为11，期望值(E)为10，M＝5，N＝4，并将两条链进行比较。
本文所用的术语“重组的”或“工程化的”是指人工产生的蛋白或核酸序列。
本文使用的术语“受试者”是指个体生物体，例如个体哺乳动物。在一些实施方案中，受试者是人。在一些实施方案中，受试者是非人哺乳动物。在一些实施方案中，受试者是非人灵长类动物。在一些实施方案中，受试者是啮齿动物。在一些实施方案中，受试者是绵羊、山羊、牛、猫或狗。在一些实施方案中，受试者是脊椎动物、两栖动物、爬行动物、鱼、昆虫、两翼昆虫(fly)或线虫。在一些实施方案中，受试者是研究动物。
术语“治疗”是指旨在逆转、减轻疾病或病症或其一种或多种症状，延迟其发作，或抑制其进展的临床干预，如本文所述。本文所用的术语“治疗”是指旨在逆转、减轻疾病或病症或其一种或多种症状，延迟其发作，或抑制其进展的临床干预，如本文所述。在一些实施方案中，治疗可以在已经发展出一种或多种症状之后和/或在已经诊断出疾病之后施用。在其他实施方案中，治疗可以在没有症状的情况下施用，例如以预防症状、降低发展出症状的可能性、或延迟症状的发作或抑制疾病的发作或进展。例如，可以在症状发作之前对易感个体施用治疗(例如，根据症状史和/或根据遗传因素或其他易感因素)。还可以在症状缓解后继续治疗，例如以预防或延迟其复发。
发明详述
本发明涉及一种组合物，其包含：
(i)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合并切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；
(ii)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成；并且
其中所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变。
目前的基因组工程化工具，包括工程化锌指蛋白(ZFP)、转录激活子样效应核酸酶(TALEN)以及最近的RNA引导的DNA核酸酶(例如Cas9)，可实现基因组中的序列特异性DNA切割。这种可编程切割可通过非同源末端连接(NHEJ)导致切割位点处DNA的突变，或通过同源引导修复(HDR)替换切割位点周围的DNA。
在一个实施方案中，位点特异性DNA结合蛋白选自包含以下或由以下组成的组：RNA引导的DNA核酸酶、锌指蛋白和转录激活子样效应物核酸酶。
在一个实施方案中，位点特异性DNA结合蛋白选自包含以下或由以下组成的组：RNA引导的DNA核酸酶和锌指蛋白。
在一个实施方案中，位点特异性DNA结合蛋白是RNA引导的核酸酶。
在一个实施方案中，位点特异性DNA结合蛋白是Cas9蛋白(例如但不限于酿脓链球菌Cas9(SpCas9)、金黄色葡萄球菌Cas9(SaCas9)或空肠弯曲杆菌Cas9(CjCas9)；下文将描述一些其他合适的实例)或其变体(例如，切口酶Cas9(nCas9)或死Cas9(dCas9))、Cas12a蛋白、Cas12b蛋白、Cpf1蛋白或CasX蛋白，包括其变体和功能片段。
在一个实施方案中，位点特异性DNA结合蛋白是Cas9蛋白，包括其变体和功能片段。
CRISPR-Cas9系统是一种通过序列特异性双链断裂(DSB)使基因失活或修饰基因的高效工具。这些DSB被细胞DNA损伤响应机制所识别，并且可以通过内源DSB修复途径进行修复。主要的修复途径是非同源末端连接(NHEJ)，它通常会导致小的插入和/或缺失，从而产生移码突变并破坏基因的功能。该途径可用于产生基因敲除突变。或者，在存在修复模板(例如，包含编码层粘连蛋白-α2蛋白、其功能变体或片段的转基因或由其组成的核酸构建体)的情况下，所述损害可以通过同源引导修复(HDR)无缝地修复。然而，尽管取得了显着的进展，但使用HDR介导的基因组编辑引入准确的基因修饰的效率远低于NHEJ介导的基因破坏。此外，HDR途径的大量多kb替换面临问题，需要选择和/或大量细胞分选。因此，HDR途径的主要应用目前仅限于基因内关键区域的局部替换，而不用于大的全长基因的替换。如上所述，本发明弥补了该缺陷。
在一个实施方案中，Cas9蛋白包含(i)活性DNA切割结构域和(ii)引导RNA结合结构域。
在已知的Cas9蛋白中，酿脓链球菌Cas9蛋白已被广泛用作基因组工程化的工具。该Cas9蛋白是一种大的多结构域蛋白，其包含两个不同的核酸酶结构域。
在一个实施方案中，Cas9蛋白选自包含以下的组或由以下组成的组：SEQ ID NO:19的来自溃疡棒状杆菌(Corynebacterium ulcerans)的Cas9蛋白(NCBI参考号：NC_015683.1,NC_017317.1)；SEQ ID NO:20的来自白喉棒状杆菌(Corynebacteriumdiphtheria)的Cas9蛋白(NCBI参考号：NC_016782.1,NC_016786.1)；SEQ ID NO:21的来自梅毒螺原体(Spiroplasma syrphidicola)的Cas9蛋白(NCBI参考号：NC_021284.1)；SEQ IDNO:22的来自中间普氏菌(Prevotella intermedia)的Cas9蛋白(NCBI参考号：NC_017861.1)；SEQ ID NO:23的来自台湾螺原体(Spiroplasma taiwanense)的Cas9蛋白(NCBI参考号：NC_021846.1)；SEQ ID NO:24的来自海豚链球菌(Streptococcus iniae)的Cas9蛋白(NCBI参考号：NC_021314.1)；SEQ ID NO:25的来自Belliella baltica的Cas9蛋白(NCBI参考号：NC_018010.1)；SEQ ID NO:26的来自热带冷弯菌(Psychroflexus torquisi)的Cas9蛋白(NCBI参考号：NC_018721.1)；SEQ ID NO:27的来自嗜热链球菌(Streptococcusthermophilus)的Cas9蛋白(NCBI参考号：YP_820832.1)；SEQ ID NO:28的来自英诺克李斯特菌(Listeria innocua)的Cas9蛋白(NCBI参考号：NP_472073.1)；SEQ ID NO:29的来自空肠弯曲杆菌的Cas9蛋白(CjCas9)(NCBI参考号：YP_002344900.1)(由SEQ ID NO:81编码)；SEQ ID NO:30的来自脑膜炎奈瑟菌(Neisseria meningitidis)的Cas9蛋白(NCBI参考号：YP_002342100.1)；SEQ ID NO:72的来自金黄色葡萄球菌Cas9蛋白(SaCas9)(由SEQ ID NO:77编码)；和SEQ ID NO:31的来自酿脓链球菌的Cas9蛋白(SpCas9)(NCBI参考号：NC_017053.1)。
在一个实施方案中，当本文提及野生型Cas9蛋白时，除非另有说明，否则所述野生型Cas9蛋白对应于来自SEQ ID NO:31的酿脓链球菌的Cas9(spCas9)。
在一个实施方案中，Cas9蛋白可以是“Cas9变体”。如本文所用，“Cas9变体”是与如本文所述的Cas9蛋白具有同源性的蛋白，并且包括其片段。
在一个实施方案中，Cas9变体可以与SEQ ID NO:31的野生型Cas9蛋白或与SEQ IDNO:19-30或72的任何其他Cas9蛋白具有至少约70％的同一性、至少约80％的同一性、至少约90％的同一性、至少约95％的同一性、至少约96％的同一性、至少约97％的同一性、至少约98％的同一性、至少约99％的同一性、至少约99.5％的同一性或至少约99.9％的同一性。
在一个实施方案中，Cas9变体包含具有一个或数个氨基酸取代的Cas9蛋白的氨基酸序列。例如，已知Cas9的DNA切割结构域包括两个亚结构域：HNH核酸酶亚结构域和RuvC1亚结构域。HNH亚结构域切割与gRNA互补的链，而RuvC1亚结构域切割非互补链。
这些亚结构域内的突变可以沉默Cas9的核酸酶活性。例如，已知取代D10A和H841A使SEQ ID NO:31的酿脓链球菌Cas9蛋白的核酸酶活性完全失活，产生死Cas9(dCas9)，其仍然保留以sgRNA编程的方式结合DNA的能力。原则上，当与另一个蛋白或结构域融合时，dCas9可以简单地通过与适当的sgRNA共表达，而将该蛋白靶向几乎任何DNA序列。在一个实施方案中，dCas9蛋白由与SEQ ID NO:66具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，dCas9蛋白包含与SEQ ID NO:71具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列，或由其组成。
关于Cas9切口酶(nCas9)，它是Cas9核酸酶的变体，不同之处在于RuvC核酸酶结构域中的点突变(D10A)，这使其能够将DNA切口而不是切割。在一个实施方案中，nCas9蛋白由与SEQ ID NO:65具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，nCas9蛋白包含与SEQ ID NO:70具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列，或由其组成。在一些实施方案中，SaCas9切口酶(SanCas9)由与SEQ ID NO:80具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一些实施方案中，SaCas9切口酶(SanCas9)包含与SEQID NO:76具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。
在一个实施方案中，Cas9变体包含Cas9的片段，使得该片段与SEQ ID NO:31的野生型Cas9蛋白的相应片段或SEQ ID NO:19-30或72的任何其他Cas9蛋白的相应片段至少约70％同一(identical)、至少约80％同一、至少约90％同一、至少约95％同一、至少约96％同一、至少约97％同一、至少约98％同一、至少约99％同一、至少约99.5％同一或至少约99.9％同一。
在一个实施方案中，Cas9变体仅包含DNA切割结构域或引导RNA结合结构域的其中之一。
在一个实施方案中，示例性Cas9变体是人源化Cas9(hCas9)或其变体或功能片段。如本文所用，术语“人源化Cas9”或“hCas9”是指针对人细胞的序列优化的Cas9蛋白。
在一个实施方案中，hCas9蛋白由与SEQ ID NO:64具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，hCas9蛋白包含与SEQ ID NO:69具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。
在一个实施方案中，位点特异性DNA结合蛋白是cpf1蛋白。在一个实施方案中，cpfl蛋白由与SEQ ID NO:78具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，cpfl蛋白包含与SEQ ID NO:74具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。
在一个实施方案中，位点特异性DNA结合蛋白是CasX蛋白。在一个实施方案中，CasX由与SEQ ID NO:79具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的核酸序列编码。在一个实施方案中，CasX包含与SEQ ID NO:75具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。
如下文将进一步详述地，本公开的某些方面还涉及包含编码位点特异性DNA结合蛋白的核酸构建体的载体或质粒(例如，表达载体、包装载体等)，所述位点特异性DNA结合蛋白特别是RNA引导的核酸酶，特别是本文描述的任何Cas9蛋白；所述载体或质粒优选适合于在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。
在一个实施方案中，位点特异性DNA结合蛋白是锌指蛋白(ZFP)。
锌指蛋白是能够以序列特异性方式与DNA结合的蛋白。ZFP在真核生物中分布不均匀。ZFP已被鉴定为参与DNA识别、RNA结合和蛋白结合。考虑到折叠结构域中蛋白主链的整体形状，锌指蛋白的某些分类基于“折叠基团”。最常见的锌指“折叠基团”是C2H2或Cys2His2样(“经典锌指”)、高音谱号锌指(treble clef)和带状锌指(zinc ribbon)。表征这些蛋白的代表性基序公开于Li&Liu,2020(Int J Mol Sci.21(4):1361)的表1中，该表通过引用并入本文。
ZFP可以是能够与基因组中的特定基因组DNA序列结合的任何ZFP、其变体或功能片段。ZFP的非限制性实例包括包含选自C2H2锌指、gag结节锌指(gag knuckle)、高音谱号锌指、带状锌指、Zn2/Cys6样锌指或TAZ2结构域样锌指或其任何组合的折叠基团或锌指基序的ZFP。在一个实施方案中，ZFP是C2H2锌指蛋白。
在一个实施方案中，ZFP是工程化的ZFP。工程化锌指阵列可以与DNA切割结构域(通常是FokI的切割结构域)融合以生成锌指核酸酶。这样的锌指-FokI融合物已成为操纵基因组的有用试剂。
ZFP可包含2、3、4、5、6、7、8、9、10、11、12或更多个锌指结构域。ZFP可包含2至12、2至10、2至8、3至8、4至8或5至8个锌指结构域。在一个实施方案中，ZFP包含6个锌指结构域。
常见的模块组装方法包括组合各自可识别3个碱基对的DNA序列的单独的锌指，以生成识别长度为9个碱基对至18个碱基对的靶位点的3指、4指、5指或6指阵列。另一种方法使用2指模块来生成具有多达六个单独锌指的锌指阵列。
在一个实施方案中，ZFP的结合结构域可被工程化以结合感兴趣的序列。与天然存在的ZFP相比，工程化的锌指结合结构域可以具有改进的结合特异性。
在一个实施方案中，编码ZFP的示例性核酸序列包含SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36或SEQ ID NO:38，或由其组成。在一个实施方案中，由这些序列编码的示例性氨基酸序列包含SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37或SEQ ID NO:39，或由其组成。
在一个实施方案中，ZFP包含与SEQ ID NO:33、35、37或39中任一个具有至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或约100％序列同一性的氨基酸序列。
在一个实施方案中，ZFP不具有Gal4 DNA结合结构域。Gal4结合CGG-N11-CCG，其中N可以是任何碱基。该蛋白是半乳糖诱导基因(例如GAL1、GAL2、GAL7、GAL10和MEL1)基因表达的正调节物，而这些半乳糖诱导基因编码用于将半乳糖转化为葡萄糖的酶。它识别这些基因的上游激活序列(UAS-G)中的17个碱基对的序列。因此，Gal4识别基因组中的短且非常频繁的序列，因此不具有位点特异性。在一个实施方案中，ZFP具有被工程化为具有位点特异性的Gal4 DNA结合结构域。
如下文将进一步详述地，本公开的某些方面涉及包含编码位点特异性DNA结合蛋白、由其是本文所述的ZFP的核酸构建体的载体或质粒(例如，表达载体、包装载体等)；所述载体或质粒优选适合于在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。
根据本发明，第二蛋白包含转座酶或由转座酶组成。
转座子是可以进行转座的染色体片段，例如，在宿主DNA中不存在互补序列时可以作为整体被转位的DNA。转座子可用于在人细胞中进行长距离DNA工程化。哺乳动物细胞中使用的常见转座子系统包括但不限于从失活的转座子重建的睡美人(SB)，以及从粉纹夜蛾(Trichoplusia)中分离的PiggyBac(PB)。PiggyBac与SB相比具有更高的转座活性，并且可以被无痕切除。
天然DNA转座子通常包含编码转座酶蛋白的单个基因，其侧翼是携带转座酶结合位点的反向末端重复序列(ITR)。在它们的转座过程中，转座酶蛋白识别这些ITR，以催化该元件的切除以及随后以随机方式在别处重新整合。此外，这些转座子中的一些可以改造用于基因治疗方案，将它们用作双组分系统，其中质粒包含表达盒，其中放置在转座子ITR之间的目标DNA序列可以经由共转染质粒引导而被引入宿主基因组中，所述共转染质粒含有编码转座酶的序列或其体外合成的mRNA。根据本公开，基于转座子的系统用于有效介导转基因作为治疗基因在细胞中的稳定整合和持续表达。
本公开的转座酶或修饰的转座酶可以是能够将外源核酸插入基因组的特定位点的任何转座酶。本公开的一些方面提供了使用本文描述的方法和策略设计的转座酶融合蛋白。本公开的一些实施方案提供了编码此类转座酶或修饰的转座酶和/或包含其的融合蛋白的核酸。本公开的一些实施方案提供了包含编码转座酶或修饰的转座酶和/或包含其的融合蛋白的此类核酸构建体的质粒或表达载体。
转座酶的非限制性实例包括Frog Prince、睡美人(Sleeping Beauty)、高活性睡美人、PiggyBac和高活性PiggyBac。
在一个实施方案中，转座酶是高活性PiggyBac转座酶。在一些实施方案中，转座酶是对应于SEQ ID NO:9或由SEQ ID NO:67编码的高活性PiggyBac转座酶(在本公开中也称为hyPB或简称为PB)。
在一个实施方案中，转座酶是修饰的高活性PiggyBac转座酶。
如本文所用，“修饰的高活性PiggyBac转座酶(modified hyperactive PiggyBactransposase)”是指与SEQ ID NO:9的野生型高活性PiggyBac转座酶相比包含一个或多个氨基酸取代，通常不超过1、2、3、4、5、6、7、8、9或10个氨基酸取代的转座酶。更具体地，修饰的高活性PiggyBac包含(i)与野生型高活性PiggyBac转座酶相比增加切除活性的一个或多个氨基酸取代，和/或(ii)与野生型高活性PiggyBac转座酶相比降低DNA结合活性的一个或多个氨基酸取代。在一个实施方案中，修饰的高活性PiggyBac转座酶包含与SEQ ID NO:9所示序列具有至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的氨基酸序列。
在一些实施方案中，高活性PiggyBac转座酶的所述一个或多个突变不由三重突变R372A/K375A/D450N组成，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号。
在一些实施方案中，修饰的高活性PiggyBac包含增加切除活性的一个或多个氨基酸突变。
在一些实施方案中，修饰的高活性Piggybac包含增加切除活性的一个或多个氨基酸突变，所述氨基酸突变选自氨基酸位置编号[194-200]、[214-222]、[434-442]或[446-456]定义的区域内的氨基酸突变，例如在位置D198、D201、R202、M212和/或S213处的氨基酸取代；所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。
在一些实施方案中，修饰的高活性Piggybac包含增加切除活性的一个或多个氨基酸突变，所述氨基酸突变选自位置450、560、564、573、589、592和/或594处的氨基酸突变；所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。
在一些实施方案中，修饰的高活性PiggyBac包含增加切除活性的一个或多个氨基酸突变，所述氨基酸突变选自M194和/或D450位置处的氨基酸突变，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号，优选地，氨基酸取代选自M194V和/或D450N。
在一些实施方案中，修饰的高活性PiggyBac包含降低DNA结合活性的一个或多个氨基酸突变。
在一些实施方案中，修饰的高活性PiggyBac包含降低DNA结合活性的一个或多个氨基酸突变，所述氨基酸突变选自位置254、275、277、347、372、375和/或465处的氨基酸突变；所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。
在一些实施方案中，修饰的高活性PiggyBac包含降低DNA结合活性的一个或多个氨基酸突变，所述氨基酸突变选自R275、N347、R372、K375、R376、E377和E380，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。
在一些实施方案中，修饰的高活性PiggyBac包含降低DNA结合活性的一个或多个氨基酸突变，所述氨基酸突变选自R372、K375、R376、E377和E380，所述位置编号对应于SEQID NO:9的未修饰的高活性Piggybac的氨基酸编号，优选选自氨基酸取代R372A、K375A、R376A、E377A和/或E380A。
在一些实施方案中，修饰的高活性PiggyBac包含降低DNA结合活性的一个或多个氨基酸突变，所述氨基酸突变选自N347、R372和K375，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号，优选选自氨基酸取代N347S、N347A、R372A、K375A，更优选选自氨基酸取代N347S、N347A。
在一些实施方案中，修饰的高活性Piggybac包含增加切除活性的一个或多个氨基酸突变，如上文所定义；以及降低DNA结合活性的一个或多个氨基酸突变，如上文所定义。
在一些实施方案中，修饰的高活性Piggybac包括位置D450处的增加切除活性的至少一个氨基酸取代以，和位置N347、R372和K375处的降低DNA结合活性的至少两个氨基酸取代以，优选地，所述修饰的高活性Piggybac转座酶包括双突变N347S和D450N或三重突变D450N、R372A和K375A，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。在更优选的实施方案中，修饰的高活性Piggybac转座酶包括双突变N347S和D450N，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。
在一些实施方案中，前述实施方案中公开的修饰的高活性Piggybac进一步包含在由氨基酸位置编号[158-169]定义的区域中的至少一个突变，例如A166S；和/或在位置Y527、R518、K525、N463处的至少一个突变。
通常，所述修饰的高活性Piggybac包含与SEQ ID NO:1的修饰的高活性Piggybac具有至少85％、至少90％、至少95％同一性或100％同一性的氨基酸序列。
在一些实施方案中，所述修饰的高活性Piggybac是SEQ ID NO:9的高活性Piggybac的变体，其与SEQ ID NO:9相比具有一个或多个氨基酸取代，通常具有不超过1、2、3、4、5、6、7、8、9或10个氨基酸取代。
在一些实施方案中，所述修饰的高活性Piggybac包含在以下位置的一个或多个氨基酸突变：34、43、117、202、230、245、268、275、277、287、290、315、325、341、346、347、350、351、356、357、388、409、411、412、432、447、460、461、465、517、560、564、571、573、576、586、587、589、592和/或594，所述位置编号对应于高活性PiggyBac序列(SEQ ID NO:9)的氨基酸编号。
在一些实施方案中，所述修饰的高活性PiggyBac包含以下突变或突变的组合：V34M、T43I、Y177H、R202K、S230N、R245A、D268N、K287A、K290A、K287A/K290A、R315A、G325A、R341A、D346N、N347A、N347S、T350A、S351E、S351P、S351A、K356E、N357A、R388A、K409A、A411T、K412A、K432A、D447A、D447N、D450N、R460A、K461A、W465A、S517A、T560A、S564P、S571N、S573A、K576A、H586A、I587A、M589V、S592G或F594L、D450N/R372A/K375A、R275A/R277A、K409A/K412A、R460A/K461A、R275A/R277A/N347S/K375A/T560A/S573A/M589V/S592G和R245A/R275A/R277A/R372A/W465A。
在一些实施方案中，所述修饰的高活性PiggyBac包含以下氨基酸取代或氨基酸取代的组合：R372A/K375A/D450N、R372A/K375A/R376A/D450N、K375A/R376A/E377A/E380A/D450N、R372A/K375A/R376A/E377A/E380A/D450N、M194V、M194V/R372A/K375A、S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、T43I/R372A/K375A/A411T/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G，所述位置编号对应于高活性PiggyBac序列(SEQ ID NO:9)的氨基酸编号。
本公开使用的非常优选的修饰的高活性PiggyBac转座酶包括修饰的高活性PiggyBac，其包含以下氨基酸取代的组合：R372A/K375A/D450N、S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、T43I/R372A/K375A/A411T/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G和R275A/325A/R372A/T560A，位置编号对应于高活性的PiggyBac序列的氨基酸编号(SEQ ID NO:9)。
在一些实施方案中，所述修饰的高活性PiggyBac包含以下氨基酸取代或氨基酸取代的组合：R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G，位置编号对应于高活性PiggyBac序列(SEQ IDNO:9)的氨基酸编号。
在更优选的实施方案中，所述修饰的高活性PiggyBac包含以下氨基酸取代的组合：N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G，位置编号对应于高活性PiggyBac序列(SEQ ID NO:9)的氨基酸编号。
在一些实施方案中，所述修饰的转座酶具有选自SEQ ID NO:1-8、10-18和135-149中任一项的氨基酸序列。
在一些实施方案中，所述修饰的转座酶具有选自SEQ ID NO:1-8和10-18中任一项的氨基酸序列。
在一些实施方案中，所述修饰的转座酶具有选自SEQ ID NO:90-99中任一项的氨基酸序列。
在一些实施方案中，所述修饰的转座酶具有选自SEQ ID NO:135-149中任一项的氨基酸序列。在一些实施方案中，所述修饰的转座酶具有选自SEQ ID NO:135-140中任一项的氨基酸序列。在一些实施方案中，所述修饰的转座酶具有选自SEQ ID NO:141-149中任一项的氨基酸序列。
在一些实施方案中，修饰的转座酶相对于hyPB的可包含参与保守催化三联体的一个或多个突变，例如在对应于SEQ ID NO:9或SEQ ID NO:11的氨基酸编号的氨基酸268和/或346处(例如，D268N和/或D346N)。
在一些实施方案中，修饰的转座酶相对于hyPB的可包含对切除至关重要的一个或多个突变，例如在对应于SEQ ID NO:9或SEQ ID NO:12的氨基酸编号的氨基酸287、287/290和/或460/461处(例如K287A、K287A/K290A和/或R460A/K461A)。
在一些实施方案中，修饰的转座酶相对于hyPB的可包含参与靶标连接的一个或多个突变，例如在对应于SEQ ID NO:9或SEQ ID NO:13的氨基酸编号的氨基酸351、356和/或379处(例如，S351E、S351P、S351A和/或K356E)。
在一些实施方案中，修饰的转座酶相对于hyPB可包含对于整合至关重要的一个或多个突变，例如在对应于SEQ ID NO:9或SEQ ID NO:14的氨基酸编号的氨基酸560、564、571、573、589、592和/或594处(例如，T560A、S564P、S571N、S573A、M589V、S592G和/或F594L)。
在一些实施方案中，修饰的转座酶相对于hyPB的可包含参与对齐(alignmeng)的一个或多个突变，例如在对应于SEQ ID NO:9或SEQ ID NO:15的氨基酸编号的氨基酸325、347、350、357和/或465处(例如，G325A、N347A、N347S、T350A和/或W465A)。
在一些实施方案中，修饰的转座酶相对于hyPB可包含高度保守的一个或多个突变，例如在对应于SEQ ID NO:9或SEQ ID NO:16的氨基酸编号的氨基酸576和/或587处(例如，K576A和/或I587A)。
在一些实施方案中，修饰的转座酶相对于hyPB可包含参与Zn2+结合的一个或多个突变，例如对应于SEQ ID NO:9或SEQ ID NO:17的氨基酸编号的586(例如，H586A)。
在一些实施方案中，可编程转座酶相对于hyPB的可包含参与整合的一个或多个突变，例如对应于SEQ ID NO:9或SEQ ID NO:18的氨基酸编号的315、341、372和/或375(例如，R315A、R341A、R372A和/或K375A)。
在一些实施方案中，修饰的高活性PiggyBac包含与SEQ ID NO:9中所示序列至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％同一的氨基酸序列。在一些实施方案中，与高活性PiggyBac相比，修饰的高活性PiggyBac因其对DNA整合到基因组中的高特异性而被选择。在一些实施方案中，修饰的高活性PiggyBac包含相对于SEQ IDNO:9、10、11、12、13、14、15、16、17或18具有本文公开的的一个或多个修饰的氨基酸序列，并且分别与SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17或18所示序列保留至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的同一性。
在一些实施方案中，高活性PiggyBac转座酶由与SEQ ID NO:67具有至少85％、90％、95％、96％、97％、98％、99％或100％序列同一性的核酸序列编码。在一些实施方案中，SB100转座酶由与SEQ ID NO:68具有至少85％、90％、95％、96％、97％、98％、99％或100％序列同一性的核酸序列编码。
在一些实施方案中，SB100转座酶包含与SEQ ID NO:73具有至少85％、90％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
在一些实施方案中，修饰的转座酶是包含一个或多个突变的修饰的睡美人转座酶。在一些实施方案中，高活性睡美人转座酶或SB100中的一个或多个突变对应于：SEQ IDNO:9或SEQ ID NO:73的L25F、R36A、I42K、G59D、I212K、N245S、K252A和Q271L。
在某些实施方案中，修饰的转座酶不是Himar1C9突变体。
本公开的某些方面涉及包含核酸构建体的载体或质粒(例如，表达载体或包装载体)，所述核酸构建体包含适合于在宿主细胞(例如，哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞)中表达的本公开的转座酶或修饰的转座酶。在一些实施方案中，修饰的转座酶表达为与Cas9的融合蛋白。在一些实施方案中，修饰的转座酶与来自单独载体的Cas9共表达，但递送至相同细胞。在一些实施方案中，修饰的转座酶或包含其的融合蛋白被包装在慢病毒颗粒中以递送至细胞。
如实施例所示，新开发的高活性PiggyBac转座酶突变文库已用于鉴定执行特异性靶向转座的修饰的高活性PiggyBac。使用这样的文库鉴定了具有阳性靶向转座的修饰的高活性PiggyBac。
在一些实施方案中，修饰的高活性PiggyBac转座酶可包含选自以下的一个或多个氨基酸的突变：245、275、277、325、347、351、372、375、388、450、465、560、564、573、589、592、594，对应于SEQ ID NO:9的氨基酸编号。
在一些实施方案中，修饰的高活性PiggyBac转座酶突变可包含选自以下的一个或多个氨基酸修饰：R245A、R275A、R277A、R275A/R277A、G325A、N347A、N347S、S351E、S351P、S351A、R372A、K375A、R388A、D450N、W465A、T560A、S564P、S573A、M589V、S592G或F594L，对应于SEQ ID NO:9的氨基酸编号。
在一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰D450N。
在一个实施方案中，修饰的高活性PiggyBac转座酶对应于SEQ ID NO:1并且包含氨基酸修饰R372A、K375A和D450。
在一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰R245A和D450。
在一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰R245A、G325A和S573P。
在一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰R245A、G325A、D450和S573P。
在一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰N347S或N347A。
在一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰N347S和D450N。
在另一个实施方案中，修饰的高活性PiggyBac转座酶包含对应于SEQ ID NO:9的氨基酸编号的氨基酸修饰N347A和D450N。在一个实施方案中，该修饰的高活性PiggyBac转座酶包含SEQ ID NO:137的氨基酸序列。
如前文所述，本文提供了修饰的高活性PiggyBac转座酶，其可以与本文公开的元件融合，但也可以单独使用或与不同元件组合使用。所述转座酶已由发明人产生。因此，提供了修饰的高活性PiggyBac转座酶，其包含氨基酸序列SEQ ID NO:9，其中：
-34位的氨基酸是V或M，
-43位的氨基酸是T或I，
-177位的氨基酸是Y或H，
-202位的氨基酸是R或K，
-230位的氨基酸是S或N，
-245位的氨基酸是A，
-268位的氨基酸是D或N，
-277位的氨基酸是R或A，
-275位的氨基酸是R或A，
-277位的氨基酸是R或A，
-325位的氨基酸是A或G，
-347位的氨基酸是S或A，
-351位的氨基酸是E、P或A，
-372位的氨基酸是R或A，
-375位的氨基酸是K或A，
-388位的氨基酸是R或A，
-409位的氨基酸是K或A，
-411位的氨基酸是A或T，
-412位的氨基酸是K或A，
-450位的氨基酸是D或N，
-460位的氨基酸是R或A，
-465位的氨基酸是W或A，
-517位的氨基酸是S或A，
-560位的氨基酸是T或A，
-564位的氨基酸是P或S，
-571位的氨基酸是S或N，
-573位的氨基酸是S或A，
-576位的氨基酸是K或A，
-586位的氨基酸是H或A，
-587位的氨基酸是I或A，
-589位的氨基酸是M或V，
-592位的氨基酸是G或S，和/或，
-594位的氨基酸是L或F。
本公开还涉及本文提供的修饰的高活性PiggyBac转座酶用作药物，特别是在基因治疗中，离体或体内。
在一个实施方案中，第一蛋白包含能够结合并切割靶核酸序列的位点特异性DNA结合蛋白或由其组成(如上所述)，并且第二蛋白包含转座酶或由转座酶组成(如上所述)，第一蛋白和第二蛋白直接或通过接头间接融合在一起以形成融合蛋白。
一方面涉及位点特异性DNA结合蛋白而另一方面涉及转座酶的任何实施方案，经必要修改后都适用于本文描述的融合蛋白的情况。
因此，在一个实施方案中，融合蛋白包含以下或由以下组成：
(i)第一蛋白，包含RNA引导的DNA核酸酶、锌指蛋白或转录激活子样效应核酸酶，或由其组成，如上所述，和
(ii)第二蛋白，包含转座酶或由转座酶组成，所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变，如上所述。
在一个实施方案中，融合蛋白包含以下或由以下组成：
(i)第一蛋白，包含RNA引导的DNA核酸酶或锌指蛋白，或由其组成，如上所述，和
(ii)第二蛋白，包含转座酶或由转座酶组成，所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变，如上所述。
在一个实施方案中，融合蛋白包含以下或由以下组成：
(i)第一蛋白，包含RNA引导的DNA核酸酶或由其组成，如上所述，和
(ii)第二蛋白，包含转座酶或由转座酶组成，所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变，如上所述。
在一个实施方案中，融合蛋白包含以下或由以下组成：
(i)第一蛋白，包含Cas9蛋白或其变体或由Cas9蛋白或其变体组成，如上所述，和
(ii)第二蛋白，包含转座酶或由转座酶组成，所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变，如上所述。
在一个实施方案中，第一蛋白和第二蛋白可以以任一顺序在融合蛋白中定向。
在一个实施方案中，融合蛋白包含直接或通过接头间接融合在第二蛋白的C末端的第一蛋白，或由其组成。换言之，融合蛋白从N末端到C末端包含以下或由以下组成：(i)第二蛋白(即，转座酶)；(ii)任选地，接头；(iii)第一蛋白(即，位点特异性DNA结合蛋白，优选RNA引导的DNA核酸酶；更优选Cas9蛋白或其变体)。
在一个实施方案中，融合蛋白包含直接或通过接头间接融合在第二蛋白的N末端的第一蛋白，或由其组成。换言之，融合蛋白从N末端到C末端包含以下或由以下组成：(i)第一蛋白(即，位点特异性DNA结合蛋白，优选RNA引导的DNA核酸酶；更优选Cas9蛋白或其变体)；(ii)任选地，接头；和(iii)第二蛋白(即，转座酶)。
在一个实施方案中，融合蛋白包含接头。
接头的合适实例包括第一蛋白和第二蛋白之间(以任何顺序)的肽接头。
在一个实施方案中，肽接头选自包含以下或由以下组成的组：(GGS)n、SEQ ID NO:133的(GGGGS)n、(G)n、SEQ ID NO:134的(EAAAK)n、XTEN接头和(XP)n基序，以及任何这些的组合，其中n独立地是1至50之间的整数。
在一个实施方案中，接头的长度为12至24个氨基酸，或者由长度为36至72个核苷酸的核酸序列编码。
在一个实施方案中，接头是XTEN接头或(GGS)n接头。
在一个实施方案中，接头选自表1所示的接头。
表1：接头
在一个实施方案中，接头包含选自包含以下的组或由以下组成的组的氨基酸序列：SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ IDNO:59、SEQ ID NO:61、SEQ ID NO:63或其任何组合；分别由SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62的示例性核酸序列编码。
在一个实施方案中，接头包含SEQ ID NO:49的氨基酸序列或由SEQ ID NO:49的氨基酸序列组成；其由SEQ ID NO:48的示例性核酸序列编码。
本文还提供了从本公开中提供的任何核酸构建体的表达获得的融合蛋白。
在一个实施方案中，融合蛋白是三重融合蛋白。
这样的三重融合蛋白可包含以下或由以下组成：
一个第一蛋白(即，一个位点特异性DNA结合蛋白)和两个第二蛋白(即，两个转座酶)；或者
两个第一蛋白(即，两个位点特异性DNA结合蛋白)和一个第二蛋白(即，一个转座酶)。
在一个实施方案中，三重融合物包含一个第一蛋白(即，一个位点特异性DNA结合蛋白)和两个第二蛋白(即，两个转座酶)或由其组成，并且三重融合物从N末端到C末端包含：
(i)位点特异性DNA结合蛋白，(ii)第一转座酶，(iii)第二转座酶；或者
(i)第一转座酶，(ii)位点特异性DNA结合蛋白，(iii)第二转座酶；或者
(i)第一转座酶，(ii)第二转座酶，(iii)位点特异性DNA结合蛋白。
在一个实施方案中，第一和第二转座酶是相同的。在一个实施方案中，第一和第二转座酶是不同的。例如，第一转座酶可以是高活性PiggyBac转座酶，第二转座酶可以是修饰的高活性PiggyBac转座酶，其选自本文所述的任何修饰的高活性PiggyBac转座酶。或者，第一和第二转座酶可以均为修饰的高活性PiggyBac转座酶，但各自携带不同的取代或不同的取代组合，如本文所述。
在一个实施方案中，第一和第二转座酶能够形成功能性二聚体。
在一个实施方案中，三重融合物包含两个第一蛋白(即，两个位点特异性DNA结合蛋白)和一个第二蛋白(即，一个转座酶)或由其组成，并且三重融合物从N末端到C末端包含：
(i)第一位点特异性DNA结合蛋白，(ii)第二位点特异性DNA结合蛋白，(iii)转座酶；或者
(i)第一位点特异性DNA结合蛋白，(ii)转座酶，(iii)第二位点特异性DNA结合蛋白；或者
(i)转座酶，(ii)第一位点特异性DNA结合蛋白，(iii)第二位点特异性DNA结合蛋白。
在一个实施方案中，第一和第二位点特异性DNA结合蛋白是相同的。在一个实施方案中，第一和第二位点特异性DNA结合蛋白是不同的。例如，第一位点特异性DNA结合蛋白可以是Cas9蛋白，第二位点特异性DNA结合蛋白可以是Cas9蛋白的变体，其选自本文所述的任何Cas9蛋白变体。或者，第一和第二位点特异性DNA结合蛋白可以均是Cas9蛋白变体，但各自是不同的变体。
在一个实施方案中，三重融合蛋白任选地包含其两个蛋白之间或三个蛋白之间的接头。
本文还公开了融合蛋白，包含：
(i)第二蛋白或编码所述第二蛋白的核酸构建体，其包含转座酶或由转座酶组成，如上所述，和
(ii)RNA结合蛋白或编码所述RNA结合蛋白的核酸构建体，其能够结合至少一个特定RNA序列。
在一个实施方案中，融合蛋白包含接头，如上所述。
在一个实施方案中，第二蛋白包含转座酶或由转座酶组成，所述转座酶是SEQ IDNO:9的高活性PiggyBac。在一个实施方案中，第二蛋白包含转座酶或由转座酶组成，所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变。特别地，修饰的高活性PiggyBac可以是本文公开的那些中的任一种。
在一个实施方案中，转座酶/RNA结合蛋白融合物可以进一步融合第一蛋白，所述第一蛋白包含位点特异性DNA结合蛋白或由位点特异性DNA结合蛋白组成，如上所述。
在一些实施方案中，RNA结合蛋白是MS2噬菌体外壳蛋白(MCP)或其片段。
在一些实施方案中，MCP与SEQ ID NO:151(例如由SEQ ID NO:150的核酸序列编码)具有至少75％、80％、85％、90％、95％、96％、97％、98％、99％或100％的同一性。
在一些实施方案中，RNA结合蛋白能够结合至少一个特定RNA序列，所述RNA序列包含四环。术语“四环”与术语“茎环”和“发夹环”可互换使用。
在一些实施方案中，所述至少一个四环是MS2 RNA四环结合序列。
在一些实施方案中，四环包含在引导RNA(gRNA)内。在某些实施方案中，gRNA与Cas9蛋白形成复合物，如上所述。
在一些实施方案中，gRNA包含至少一个MS2 RNA四环结合序列。在一些实施方案中，gRNA包含多于一个MS2 RNA四环结合序列。
在一些实施方案中，包含至少一个MS2 RNA四环结合序列的gRNA与SEQ ID NO:153(例如由SEQ ID NO:152的DNA序列编码)具有至少75％、80％、85％、90％、95％、96％、97％、98％、99％或100％的同一性。
在一些实施方案中，融合蛋白中的MCP非共价结合至gRNA本身中包含的至少一个MS2 RNA四环结合序列，而gRNA非共价结合至Cas9蛋白；特别地，融合蛋白与Cas9/gRNA复合物的结合将修饰的高活性PiggyBac转座酶的切除活性引导至Cas9/gRNA复合物特异性识别的位点。
如下文将进一步详述地，本公开的某些方面还涉及包含编码本文所述的融合蛋白的核酸构建体的载体或质粒(例如，表达载体、包装载体等)；所述载体或质粒优选地适合在宿主细胞中表达，所述宿主细胞例如是哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞。
根据本发明，组合物可以包含第一蛋白和/或第二蛋白(或包含两者的融合蛋白)，其或者是如上所述蛋白或者是如上所述编码这些蛋白的核酸构建体。
核酸序列的靶向编辑，例如，将特定修饰引入(例如，将外源核酸插入)基因组DNA中，是治疗人类遗传疾病的有前景的方法。为此，发明人旨在提供用于基因组编辑的改进的核酸构建体，其能够高效地安装所需的修饰、使脱靶活动最小以及被工程化以准确编辑人类基因组内位点的能力。
因此，本申请的某些方面涉及用于改善外源核酸例如目标基因(GOI)向基因组中的位点特异性插入的核酸构建体。在一些实施方案中，GOI是治疗基因，例如编码治疗蛋白的基因。目标治疗基因的示例包括用于治疗囊性纤维化疾病的CFTR基因(囊性纤维化跨膜传导调节因子)；用于治疗脊髓性肌萎缩症(SMA)的SMN1基因(存活运动神经元1)；用于预防骨质疏松和骨折的LRP5基因(LDL受体相关蛋白5)变体G171V；以及用于降低阿尔茨海默病易感性的APP基因(β淀粉样蛋白前体蛋白)变体A673T。
在一些实施方案中，用于插入的外源核酸(例如，GOI)的长度可以是多达约10kb、多达约15kb、多达约20kb、多达约25kb、多达约30kb、多达约35kb或多达约40kb。
在一些实施方案中，用于插入的外源核酸的长度可以是多达10kb、多达15kb、多达20kb、多达25kb、多达30kb、多达35kb或多达40kb，例如约1kb至约40kb、约1kb至约39kb、约1至约38kb、约1kb至约37kb、约1kb至约36kb、或约1kb至约35kb，并且更优选是5至25kb，通常是8至20kb。
在一个实施方案中，本发明的组合物包含以下或由以下组成：
a.编码上述第一蛋白的核酸构建体，所述第一蛋白包含上述位点特异性DNA结合蛋白或由上述位点特异性DNA结合蛋白组成；
b.编码第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由转座酶组成，所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变，如上所述。
在另一个实施方案中，本发明的组合物包含编码上述融合蛋白的核酸构建体或由其组成，上述融合蛋白包含以下或由以下组成：(i)第一蛋白，其包含位点特异性DNA结合蛋白或由其组成，和(ii)第二蛋白，其包含转座酶或由转座酶组成，所述第二蛋白是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变，如上所述。
在一个实施方案中，编码融合蛋白的核酸构建体还包含编码第一和第二蛋白之间的接头的核酸序列，如上所述；或者在三重融合蛋白的情况下，编码融合蛋白的核酸构建体还包含编码在其蛋白中的两个之间或在三个蛋白之间的接头的核酸序列。
根据本公开，第一和第二蛋白或包含所述第一和第二蛋白或由所述第一和第二蛋白组成的融合蛋白能够实现和/或促进外源核酸的位点特异性插入。
一些实施方案涉及质粒或载体(例如表达载体)，其包含以下其一：
编码第一蛋白的核酸构建体；或
编码第二蛋白的核酸构建体；或
编码第一蛋白的核酸构建体和编码第二蛋白的核酸构建体；或
编码融合蛋白或三重融合蛋白的核酸构建体。
在一些实施方案中，质粒是包装质粒。在一些实施方案中，质粒还包含编码衣壳蛋白如gag和pol的多核苷酸。在一些实施方案中，质粒与包含编码病毒包膜蛋白的多核苷酸的第二质粒(包膜质粒)以及包含含有外源核酸转基因的核酸构建体的第三质粒进行组合，其中当将所述组合引入生产细胞系(例如，真核细胞、原核细胞和/或细胞系)中时，产生的病毒颗粒包含编码外源核酸转基因的核酸构建体以及编码第一蛋白、第二蛋白、第一和第二蛋白两者或融合蛋白的其中之一的核酸构建体。
在一些实施方案中，质粒与包含编码衣壳蛋白如gag和pol的多核苷酸的第二质粒(包装质粒，其中包装质粒缺乏功能性整合酶)、包含编码病毒包膜的多核苷酸的第三质粒(包膜质粒)以及包含含有外源核酸转基因的核酸构建体的第四质粒进行组合，其中当将所述组合引入生产细胞系(例如，真核细胞、原核细胞和/或细胞系)时，产生的病毒颗粒包含含有外源核酸转基因的核酸构建体以及编码第一蛋白、第二蛋白、第一和第二蛋白两者或融合蛋白的其中之一的核酸构建体。
在一个实施方案中，使用慢病毒颗粒将第一蛋白、第二蛋白、第一蛋白和第二蛋白两者或融合蛋白和/或外源核酸转基因递送至细胞。
在一个实施方案中，核酸构建体包含以下：编码第一蛋白的第一多核苷酸序列，所述第一蛋白包含经工程化以结合靶核酸序列的位点特异性DNA结合蛋白或由其组成；编码第二蛋白的第二多核苷酸序列，所述第二蛋白包含转座酶或由转座酶组成，所述转座酶能够将外源核酸转基因插入基因组中；以及任选地，包含编码第一和第二多核苷酸之间的接头的核酸序列的第三多核苷酸序列。在一些实施方案中，第一蛋白是锌指蛋白或Cas9蛋白或其变体，如上所述；和/或第二蛋白是修饰的高活性PiggyBac转座酶，如上所述。
上文已经描述了产生融合蛋白的合适接头的实例。
在一些实施方案中，不需要接头，因为第一蛋白是从独立于第二蛋白的质粒表达的。
在一个实施方案中，不使用接头，而是第一和/或第二多核苷酸序列分别包含编码第一蛋白和第二蛋白的核酸，并且还在其至少一个末端中包含发挥接头功能的额外的核苷酸。
在一个实施方案中，核酸构建体是DNA或RNA形式。
本文还提供了包含本公开中提供的任何核酸构建体的载体。特别地，载体适合在哺乳动物细胞、酵母细胞、昆虫细胞、植物细胞、真菌细胞或藻类细胞中表达。本文还提供了包含本公开中提供的任何核酸构建体或载体的宿主细胞。
在一些实施方案中，本公开的核酸构建体在宿主细胞中表达。合适的宿主细胞包括但不限于真核细胞和原核细胞和/或细胞系。此类宿主细胞或由此类细胞产生的细胞系的非限制性实例包括COS、CHO(例如CHO-S、CHO-K1、CHO-DG44、CHO-DUXB11、CHO-DUKX、CHOK1SV)、VERO、MDCK、WI38、V79、B14AF28-G3、BHK、HaK、NS0、SP2/0-Ag14、HeLa、HEK293(例如，HEK293-F、HEK293-H、HEK293-T)和perC6细胞，以及昆虫细胞如草地贪夜蛾(Spodopterafugiperda，Sf)，或真菌细胞如酵母(Saccharomyces)、毕赤酵母(Pichia)和裂殖酵母(Schizosaccharomyces)。
在一些实施方案中，宿主细胞来自微生物。可用于本文公开的某些方法的微生物包括例如细菌(例如大肠杆菌(E.coli))、酵母(例如酿酒酵母(Saccharomycescerevisiae))和植物。宿主细胞可以是原核或真核的。在一些实施方案中，宿主细胞是真核的。合适的真核宿主细胞包括但不限于酵母细胞、昆虫细胞、植物细胞、真菌细胞和藻类细胞。
在一些实施方案中，宿主细胞是感受态宿主细胞。在一些实施方案中，宿主细胞是天然感受态的。在一些实施方案中，例如通过使用氯化钙和热休克的方法使宿主细胞具有感受态。所使用的细胞可以是任何细胞感受态细胞，特别是真核细胞，特别是哺乳动物细胞，例如人或动物细胞。它们可以是体细胞或胚胎干细胞或分化的细胞。在一些方面，细胞包括293T细胞、成纤维细胞、肝细胞、肌肉细胞(骨骼细胞、心脏细胞、平滑肌细胞、血管细胞等)、神经细胞(神经元、神经胶质细胞、星形胶质细胞)、上皮细胞、肾细胞、眼细胞等。还可以包括昆虫、植物细胞、酵母或原核细胞。另外，可分离原代细胞并离体使用以在用核酸酶(例如ZFN或TALEN)或核酸酶系统(例如CRISPR/Cas)处理后重新引入待治疗的受试者中。合适的原代细胞包括外周血单核细胞(PBMC)和其他血细胞亚群，例如但不限于T淋巴细胞，例如CD4+T细胞或CD8+T细胞。合适的细胞还包括干细胞，例如胚胎干细胞、诱导多能干细胞、造血干细胞(CD34+)、神经元干细胞和间充质干细胞。
在一些实施方案中，宿主细胞用包含本文公开的核酸构建体的质粒转染。在一些实施方案中，包含核酸构建体的质粒是包装质粒。在一些实施方案中，包含核酸构建体的质粒还包含编码衣壳蛋白例如gag和pol的多核苷酸。在一些实施方案中，宿主细胞用以下转染：(i)包含核酸构建体的质粒在宿主细胞中与(ii)包含编码病毒包膜蛋白的多核苷酸的质粒(包膜质粒)组合；以及(iii)包含外源核酸序列(例如GOI)的质粒，其中产生的病毒颗粒包含外源核酸(例如GOI)以及第一和第二蛋白(单独地或作为上述融合蛋白的一部分)。
在一些实施方案中，宿主细胞用以下转染：(i)包含核酸构建体的质粒与(ii)包含进一步包含编码衣壳蛋白如gag和pol的多核苷酸的核酸构建体的质粒(包装质粒，其中包装质粒缺乏功能性整合酶)组合；(iii)包含编码病毒包膜蛋白的多核苷酸的质粒(包膜质粒)和(iv)包含外源核酸序列(例如GOI)的质粒，其中产生的病毒颗粒包含外源核酸如GOI以及第一和第二蛋白(单独地或作为上述融合蛋白的一部分)。
在进一步的实施方案中，载体，例如本公开的慢病毒载体，可以用于递送由本公开的核酸构建体编码的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)和外源核酸至生物体，例如哺乳动物，并且更具体地，递送至目标哺乳动物靶细胞。包含第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的慢病毒载体能够转导各种细胞类型，例如肝脏细胞(例如肝细胞)、肌细胞、脑细胞、肾细胞、视网膜细胞和造血细胞。在一些实施方案中，本公开的靶细胞是“非分裂”细胞。这些细胞包括通常不分裂的细胞，例如神经元细胞。然而，本公开并不旨在限于非分裂细胞(包括但不限于肌细胞、白细胞、脾细胞、肝细胞、眼细胞、上皮细胞等)。
在某些实施方案中，将包装的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)施用于生物体，例如用于生物体DNA的基因编辑。在一些实施方案中，生物体是人。在一些实施方案中，生物体是非人哺乳动物。在一些实施方案中，生物体是非人灵长类动物。在一些实施方案中，生物体是啮齿动物。在一些实施方案中，生物体是绵羊、山羊、牛、猫或狗。在一些实施方案中，生物体是脊椎动物、两栖动物、爬行动物、鱼、昆虫、两翼昆虫或线虫。在一些实施方案中，生物体是研究动物。在一些实施方案中，生物体是基因工程化的，例如，基因工程化的非人受试者。生物体可以是任何性别，处于任何发育阶段。
已经描述了将核酸例如外源核酸插入基因组中的方法。参见，例如，Yusa等人PNAS4(108):1531-1536(2011)；Feng等人Nuc.Acid Res.4(38):1204-1216(2009)；Kettlun等人Amer.Soc.Gene and Cell Ther.9(19):1636-1644(2011)；Skipper等人20(92):1-23(2013)；Li等人PNAS 25:E2279-E2287(2013)；Mátés等人Nature Genetics 41(6):753-761(2009)；Mali等人Nat.Methods 10(10):957-963；Vargas等人J.Trans.Med.14(288):1-15(2016)；Gersbach等人Acc.Chem.Res.47:2309-2318(2014)；Chandrasegaran等人CellGene Ther.Ins.3(1):33-41(2017)；Wilson等人649:353-363(2010)；Zhao Zhang,等人MolTher Nucleic Acids.9:230–241(2017)；Naldini L.EMBO Mol Med.11(3)(2019)；和Naldini L,等人Hum Gene Ther.27(10):727-728(2016)，每一篇均通过引用并入本文。
本公开提供了编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体，用于将核酸(通常是外源核酸)插入基因组的特定位点。本发明还提供了第一和第二蛋白(单独地或作为上述融合蛋白的一部分)，用于将外源核酸插入基因组的特定位点。在一些实施方案中，用于插入的外源核酸的长度可以高达5kb、高达10kb、高达15kb、20kb、高达25kb、高达30kb、高达35kb或高达40kb，并且特别是对于长核酸，例如在5kb和25kb之间，通常在8kb和20kb之间。
在另一个实施方案中，提供了用于将位点特异性核酸插入基因组中的方法。
因此，本公开涉及用于将外源核酸序列位点特异性整合至细胞基因组中的方法，所述方法包括向细胞递送包含以下的组合物：
(i)如本文公开的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)，或如本文公开的核酸构建体，
(ii)待整合到细胞基因组中的外源核酸，以及
(iii)用于确定所述外源核酸向细胞基因组中的位点特异性整合的引导RNA。
其中所述第一和第二蛋白(单独地或作为上述融合蛋白的一部分)与细胞基因组中特定基因组DNA序列的结合导致基因组的切割和所述外源核酸序列由引导RNA确定而位点特异性整合至细胞基因组中。
在所述方法的具体的实施方案中，所述外源核酸是大小为至少5kb、至少6kb、至少7kb、至少8kb、至少9kb、通常包含在5kb与25kb之间、优选在8kb与20kb之间的核酸片段。
在所述方法的具体的实施方案中，所述外源核酸是待插入有此需要的受试者的基因组中以纠正遗传性疾病的缺陷的治疗性转基因。
在所述方法的具体的实施方案中，所述组合物在体外或离体递送，通常在哺乳动物细胞中，优选在人类细胞中，并且更优选在从患有遗传性疾病的人受试者获得的人细胞中。
在所述方法的具体的实施方案中，将所述组合物体内递送至哺乳动物，例如有此需要的人受试者中，通常用于遗传性疾病的治疗性治疗。
在一些实施方案中，所述方法包括使靶DNA与包含本文描述的Cas9和转座酶的任何融合蛋白接触。例如，在一些实施方案中，所述方法包括使DNA与包含两个连接的多肽即(i)Cas9和(ii)转座酶的融合蛋白接触，其中活性Cas9结合与DNA如基因组DNA的区域杂交的gRNA。
在一些实施方案中，所述方法包括使靶DNA与包含本文所述的ZFP和整合酶的任何融合蛋白接触。例如，在一些实施方案中，所述方法包括使DNA与包含两个连接的多肽即(i)ZFP和(ii)整合酶的融合蛋白接触，其中活性ZFP与DNA如基因组DNA的区域杂交。
在一些实施方案中，使用病毒载体，例如慢病毒颗粒，将第一和第二蛋白(单独地或作为上述融合蛋白的一部分)递送至包含靶DNA如基因组DNA的生物体和/或细胞。
慢病毒包装方法已有描述。参见Grandchamp等人9(6):1-13(2014)；Voelkel等人107(17):7805-7810(2010)；Tan等人80(4)1939-1948；Li等人9(8):1-9(2014)；Mátés等人Nature Genetics 41(6):753-761(2009)；和Robert H Kutner1等人NATURE PROTOCOLS 4(4):495(2009)，每一篇均通过引用并入本文。
通常，慢病毒递送系统使用分裂系统(split system)，该系统在多个独立的质粒上具有不同的慢病毒基因，用于产生不包含引起病毒性疾病所需的遗传组分的完整病毒。例如，一个质粒(包膜质粒)可以编码病毒包膜(env)的蛋白；另一个质粒(包装质粒)可以编码衣壳蛋白(例如，gag和pol)和酶，如逆转录酶和/或整合酶；又一个质粒包含侧翼是长末端重复序列(用于基因组整合)和psi序列(显示将基因包装到病毒中的信号)的目标基因(GOI)(转移质粒)。如果将这些质粒同时引入细胞中，将产生含有目标基因而不含引起疾病所需的病毒基因的病毒。
在本公开的某些方面，本发明的慢病毒载体(或颗粒)可通过分裂系统如反式互补系统(载体/包装系统)、通过用含有慢病毒载体基因组的某些组分的质粒以及至少一个其他质粒体外转染受纳细胞(例如293T细胞)来获得，所述其他质粒反式提供编码多肽GAG、POL和包膜蛋白或能够使逆转录病毒颗粒形成的这些多肽的一部分的gag、pol和env序列。
作为示例，宿主细胞用以下转染：a)包含慢病毒gag和pol序列的包装质粒，b)包含编码包膜蛋白(例如VSV-G)的基因的第二质粒(包膜表达质粒或假型env质粒)，c)包含在5'和3'LTR序列之间的psi衣壳化序列和转基因的质粒载体，以及d)包含编码本文公开的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体的质粒载体。在一些实施方案中，编码本文公开的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体位于包装质粒上，而不是单独的质粒上。编码gag、pol和env cDNA的核酸可以根据常规技术从现有技术和数据库中可获得的病毒基因序列进行有利地制备。
在一些实施方案中，慢病毒载体包含本文所述的核酸构建体。在一些实施方案中，慢病毒载体包含如本文所述的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)。
质粒中使用的启动子可以相同或不同。在一些实施方案中，在质粒反式互补系统中，包膜质粒和质粒载体分别促进表达外壳蛋白gag和pol，载体基因组的mRNA和转基因，启动子可以相同或不同。此类启动子可以有利地选自普遍存在的启动子或特定的启动子，例如病毒启动子CMV、TK、RSV LTR启动子和RNA聚合酶III启动子如U6或H1或编码env、gag和pol的辅助病毒(即腺病毒、杆状病毒、疱疹病毒)的启动子。
为了产生本公开的慢病毒载体，可以将本文描述的质粒引入宿主细胞中并产生和收获病毒。合适的细胞包括但不限于真核和原核细胞和/或细胞系。此类细胞或由此类细胞产生的细胞系的非限制性实例包括例如COS、CHO(例如CHO-S、CHO-K1、CHO-DG44、CHO-DUXB11、CHO-DUKX、CHOK1SV)、VERO、MDCK、WI38、V79、B14AF28-G3、BHK、HaK、NS0、SP2/0-Ag14、HeLa、HEK293(例如HEK293-F、HEK293-H、HEK293-T)和perC6细胞以及昆虫细胞如草地贪夜蛾(Sf)，或真菌细胞如酵母菌、毕赤酵母和裂殖酵母。
一旦用质粒转染宿主细胞并产生本公开的慢病毒载体(或颗粒)，则可以从细胞的上清液中纯化本公开的慢病毒载体(或颗粒)。纯化慢病毒载体以提高浓度可以通过任何合适的方法来完成，例如通过密度梯度纯化(例如，氯化铯(CsCl))、通过色谱技术(例如，柱色谱或分批色谱)、或通过超速离心。例如，本发明的载体可以经历两个或三个CsCl密度梯度纯化步骤。期望的是，利用包括裂解细胞、将裂解物施加至色谱树脂、从色谱树脂洗脱病毒以及收集含有本公开的慢病毒载体的级分的方法，将载体从感染的细胞中纯化。
已经描述了递送慢病毒载体的方法。参见，例如，Vargas等人J.Trans.Med.14(288):1-15(2016)；Mali等人Nat.Methods 10(10):957-963；Mátés等人Nature Genetics41(6):753-761(2009)；Skipper等人20(92):1-23(2013)。
包含第一和第二蛋白(单独地或作为上述融合蛋白的一部分)或编码其的核酸构建体的慢病毒载体可以通过任何途径施用于受试者。在一些实施方案中，本公开的慢病毒载体可以体内或离体递送至受试者的细胞。
在一些实施方案中，本公开的慢病毒载体可以体内递送。在一些实施方案中，包含由本公开的核酸构建体编码的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的慢病毒载体可用于递送目标基因和/或靶向受试者DNA中的遗传缺陷。在一些实施方案中，将慢病毒载体胃肠外、优选血管内(包括静脉内)施用给受试者。当胃肠外施用时，优选地，载体在适合注射的药物媒介物中给予，例如无菌水溶液或分散液。
在一些实施方案中，本公开的慢病毒载体可以离体使用。
在一些实施方案中，包含由本公开的核酸构建体编码的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的慢病毒载体可用于递送目标基因和/或靶向受试者DNA中的遗传缺陷。在一些实施方案中，从受试者中取出细胞，并将包含由本公开的核酸构建体编码的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的慢病毒载体离体施用至所述细胞以修饰细胞的DNA。然后，将携带修饰的DNA的所述细胞扩增并重新输注回受试者体内。在某些实施方案中，包含由本公开的核酸构建体编码的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的慢病毒载体可用于嵌合抗原受体(CAR)T细胞治疗，以对患者的自体T细胞进行基因修饰，使其表达对肿瘤抗原具有特异性的CAR。在进一步的实施方案中，将修饰的CAR-T细胞离体扩增并重新输注回患者体内。在一些实施方案中，改变的T细胞更特异性地靶向癌细胞。与抗体治疗不同，CAR-T细胞能够在体内复制，从而实现长期持续性。
在施用本公开的慢病毒载体或使用本公开的慢病毒载体离体修饰的细胞后，可以监测受试者以检测转基因的表达。治疗的剂量和持续时间根据待治疗的病况或疾病单独确定。基于通过施用本发明载体中的目标基因产生的基因表达，可以治疗多种病况或疾病。使用本发明的方法递送的载体的剂量将根据期望的宿主响应和所用载体而变化。
在一些基因治疗应用中，期望基因治疗载体以高度特异性递送至特定组织类型。因此，通过将配体表达为与病毒外表面的病毒外壳蛋白融合的蛋白，可以修饰病毒载体以对给定细胞类型具有特异性。选择对已知存在于目标细胞类型上的受体具有亲和力的配体。
本公开的某些方面涉及一种将外源核酸序列插入生物体基因组DNA中的方法，所述方法包括：鉴定生物体基因组中的特定基因组DNA序列；将包含本公开的核酸构建体的慢病毒颗粒施用于生物体以结合特定的基因组DNA序列和将外源核酸插入基因组DNA中；其中外源核酸被整合在特定基因组DNA序列处。
本公开的某些方面涉及用于将外源核酸序列的单拷贝或多拷贝受控地位点特异性整合至细胞中的方法，该方法包括：a)将本公开的核酸构建体、载体或第一和第二蛋白(单独地或作为上述融合蛋白的一部分)递送至细胞，以及b)将外源核酸递送至细胞；其中第一和第二蛋白(单独地或作为上述融合蛋白的一部分)与细胞基因组中的特定基因组DNA序列的结合导致基因组的切割和外源核酸的一个或多个拷贝整合进细胞基因组。在一些方面，通过慢病毒颗粒递送至细胞。
可以使用多种策略来检测整合站点，并筛选引导整合的最佳机制。
为了分析本文公开的修饰的转座子，可以使用具有启动子、一半GFP编码序列和基因组中靶向插入位点下游的剪接位点供体的报告细胞系。例如，慢病毒负载(payload)可以具有融合整合酶变体，然后是反向剪接位点受体和GPF的另一半。当发生直接插入并且从插入位点生成包含mRNA的GFP的剪接，并且整合的负载产生了完整的GFPCDS时，发生GFP表达。
VPR反式互补系统还可用于筛选和比较整合突变体。反式互补系统可用于含有融合整合酶变体的慢病毒负载的靶向插入，所述融合整合酶当被表达并被加载到颗粒中时促进其自身整合，使用VPR融合将其加载到病毒颗粒中。这将反式补充用于颗粒生产的包装载体中编码的整合缺陷IN。其他可用于整合定位分析的方法包括IC或FISH探针。还可以通过TCRa或RFP靶向破坏，或者通过利用靶向剪接位点整合的GFP激活，来筛选靶向插入。
对于将染色质中的插入和靶区域共染色的FISH方法，可以进行荧光原位杂交以在Hek293T基因组中定位目标基因转座子。Hek293T可以用1)GOI-转座子2)可编程转座酶和3)针对PPP1R12的gRNA转染。将探针设计为用于靶向PPP1R12基因、CD46基因(作为阴性对照)和GOI，并且可以使用Nick Translation Mix(Sigma)从PCR扩增的DNA中合成探针。
在一些实施方案中，与野生型转座酶(或含有相应野生型转座酶的融合蛋白)相比，包含本文所公开的修饰的转座酶的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)提高了外源核酸插入基因组的特异性，如通过Genetrap测定法所确定的。在一些实施方案中，HEK293T细胞或任何其他允许的细胞用具有以下质粒或负载的慢病毒颗粒转染或转导：(i)包含靶向DNA特定区域的gRNA的质粒，(ii)本公开的包含编码第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的核酸构建体的质粒，其中第二蛋白是修饰的转座酶，以及(iii)包含编码报告蛋白如GFP的缺少启动子的核酸序列的基因捕获质粒。在一些实施方案中，基因捕获质粒还包含具有反向重复的转座子。
在一些实施方案中，含有GFP插入的细胞的百分比可以通过流式细胞术测定。在一些实施方案中，与相应的野生型蛋白相比，第一和第二蛋白(单独地或作为上述融合蛋白的一部分，第二蛋白是修饰的转座酶)使含有GFP插入的细胞的百分比增加至少5％、至少10％、至少15％、至少20％、至少25％或至少30％。在一些实施方案中，第一和第二蛋白(单独地或作为上述融合蛋白的一部分，第二蛋白是修饰的转座酶)使含有GFP插入的细胞的百分比增加约15-30％。
在一些实施方案中，靶位点处的插入百分比和靶位点处的覆盖百分比(每个插入位点的读段数量)可以通过基因组DNA提取和使用对病毒LTR具有特异性的寡核苷酸的靶向测序来确定。在一些实施方案中，与相应的野生型蛋白相比，第一和第二蛋白(单独地或作为上述融合蛋白的一部分，第二蛋白是修饰的转座酶)使靶位点处的插入百分比增加至少10倍、至少20倍、至少30倍、至少40倍、至少50倍、至少60倍、至少70倍、至少80倍、至少90倍或至少100倍。在一些实施方案中，靶位点处的插入百分比增加约10-100倍。在一些实施方案中，与相应的野生型蛋白相比，第一和第二蛋白(单独地或作为上述融合蛋白的一部分，第二蛋白是修饰的转座酶)使靶位点处的覆盖百分比(每个插入位点的读段数量)增加至少10倍、至少20倍、至少30倍、至少40倍、至少50倍、至少60倍、至少70倍、至少80倍、至少90倍、至少100倍、至少110倍、至少120倍、至少130倍、至少140倍、至少150倍、至少160倍、至少170倍、至少180倍、至少190倍或至少200倍。在一些实施方案中，靶位点处的覆盖百分比(每个插入位点的读段数量)增加至少100倍。
在一些实施方案中，靶位点处的插入百分比和靶位点处的覆盖百分比(每个插入位点的读段数量)可以通过基因组DNA提取和使用对病毒插入的LTR具有特异性的寡核苷酸进行靶向测序来确定。
包含本公开的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)的慢病毒载体的可能应用包括基因治疗，即在任何哺乳动物细胞中、特别是在人细胞中的基因转移。它可以是分裂细胞或静止细胞，属于中枢器官或外周器官如肝脏、胰腺、肌肉、心脏等的细胞。基因治疗可以允许蛋白如神经营养因子、酶、转录因子、受体等的表达。本发明的慢病毒载体还可以特别适合于研究目的。
在一些实施方案中，将本公开的核酸构建体、第一和第二蛋白(单独地或作为上述融合蛋白的一部分)和/或慢病毒载体施用于受试者以治疗疾病。在一些实施方案中，疾病是可以受益于基因治疗的遗传性疾病。
在一些实施方案中，第一和第二蛋白(单独地或作为上述融合蛋白的一部分)、或编码其的核酸构建体、或下文公开的试剂盒或组合物、或包含本公开的第一和第二蛋白(单独地或作为上述融合蛋白的一部分)或核酸构建体的慢病毒载体可以用作药物。
慢病毒载体可以特别适合于治疗受试者中的遗传性疾病。
本发明还涉及一种组合物，其包含
(i)RNA引导的核酸酶或锌指核酸酶，
(ii)转座酶，
(iii)引导RNA，以及
(iv)目标核酸或基因，例如用于插入基因组中的外源核酸，
其中所述转座酶是修饰的高活性Piggybac，其与SEQ ID NO:9的高活性Piggybac相比包含一个或多个氨基酸突变。
在一个实施方案中，修饰的高活性PiggyBac突变包含氨基酸取代R372A/K375A/D450N。
在优选的实施方案中，修饰的高活性PiggyBac突变不包含氨基酸取代R372A/K375A/D450N。
在一些实施方案中，修饰的高活性PiggyBac突变包含以下氨基酸取代或氨基酸取代的组合：S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、T43I/R372A/K375A/A411T/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G，位置编号对应于高活性PiggyBac序列(SEQ ID NO:9)的氨基酸编号。
在优选的实施方案中，修饰的高活性PiggyBac突变包含以下氨基酸取代或氨基酸取代的组合：R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G，位置编号对应于高活性PiggyBac序列(SEQ IDNO:9)的氨基酸编号。
在一些实施方案中，RNA引导的核酸酶是Cas9蛋白。在一些实施方案中，RNA引导的核酸酶是SpCas9蛋白。在一些实施方案中，RNA引导的核酸酶是SaCas9蛋白。
本发明还涉及包含编码以下的核酸的组合物：
(i)RNA引导的核酸酶或锌指核酸酶，如上文所述，
(ii)转座酶，如上文所述，
(iii)引导RNA，以及
(iv)目标核酸或基因，例如用于插入基因组中的外源核酸。
在一些实施方案中，所述组合物的核酸通过合适的表达载体在细胞中表达。如本文所用，术语“表达载体”是指包含多核苷酸的载体，所述多核苷酸包含与待表达的核苷酸序列有效连接的表达控制序列。表达载体包含对于表达足够的顺式作用元件；用于表达的其他元件可以由宿主细胞提供或在体外表达系统中提供。表达载体包括本领域已知的所有那些，包括引入重组多核苷酸的粘粒、质粒(例如，裸的或包含在脂质体中)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
在一个优选的实施方案中，两个核酸在相同的细胞或细胞群中共表达。在一些实施方案中，两个核酸同时共表达。在另一个实施方案中，首先表达编码RNA引导的核酸酶或锌指核酸酶的核酸。在另一个实施方案中，首先表达编码转座酶的核酸。
本发明还涉及一种组合物，其包含
(i)本文公开的包含RNA引导的核酸酶和转座酶的融合蛋白，或编码其的核酸，
(ii)本文所公开的转座酶，或编码其的核酸，
(iii)引导RNA，以及
(iv)目标核酸或基因，例如外源核酸，用于插入基因组中。
本发明还涉及一种组合物，其包含：
(i)本文公开的包含RNA引导的核酸酶和转座酶的第一融合蛋白，或编码其的核酸，
(ii)本文公开的包含经工程化以结合至少一个特定RNA序列的RNA结合蛋白和转座酶的第二融合蛋白，或编码其的核酸，
(iii)引导RNA，以及
(iv)目标核酸或基因，例如外源核酸，用于插入基因组中。
本公开还提供了用于实施本文所述的公开方法的组合物。在一些实施方案中，组合物包含本公开中定义的核酸构建体或载体，以及编码用于插入基因组中的包含在包装载体中或结合至包装载体的外源核酸的多核苷酸序列。
本发明还涉及一种组合物，其包含：
(i)本文公开的包含RNA引导的核酸酶和转座酶的融合蛋白，或编码所述融合蛋白的核酸，
(ii)引导RNA，和
(iii)目标核酸或基因，例如外源核酸，用于插入基因组中。
在具体的实施方案中，所述目标核酸或基因是大的DNA片段，通常具有5kb至25kb之间的大小，并且更优选8kb至20kb。
本公开还提供了用于实施本文所述的公开方法的试剂盒。所述试剂盒可含有本文所述的核酸构建体或融合蛋白。在一些方面，所述试剂盒可含有慢病毒颗粒，所述慢病毒颗粒含有本文所述的核酸构建体或融合蛋白。
本发明的试剂盒还可以包含关于使用试剂盒的组分来实施本发明方法的说明。用于实施本发明方法的说明通常记录在合适的记录介质上。例如，说明可以印刷在基材上，例如纸或塑料等。因此，说明可以作为包装插页存在于试剂盒中、存在于试剂盒的容器的标签中或作为其组分(即，与包装或分包装关联)。在其他实施方案中，说明作为存在于合适的计算机可读存储介质上的电子存储数据文件而存在。在其他实施方案中，实际的说明不存在于试剂盒中，但提供了用于从远程源例如经由互联网获取说明书的手段。该实施方案的一个示例是试剂盒包括可以查看说明和/或可以从其下载说明的网址。与说明一样，这种用于获得说明的手段被记录在合适的基材上。
本公开通常涉及一种试剂盒，其包含：
第一组合物，其包含：
(i)本文定义的第一融合蛋白，或编码所述第一融合蛋白的核酸，其中所述第一融合蛋白包含与修饰的高活性Piggybac融合的第一引导RNA切口酶Cas9(通常为SEQ ID NO:70的SpCas9切口酶)的氨基酸序列，和
(ii)第一引导RNA核酸(first guided RNA nucleic acid)；
第二组合物，其包含：
(iii)如本文定义的第二融合蛋白，或编码所述第二融合蛋白的核酸，其中所述第二融合蛋白包含与修饰的高活性Piggybac融合的第二引导RNA切口酶Cas9(通常为SEQ IDNO:76的SaCas9切口酶)的氨基酸序列，
(iv)第二引导RNA核酸(second guided RNA nucleic acid)，
任选地，用于插入基因组中的核酸，例如具有5kb至25kb、更优选8kb至20kb大小的核酸。
其中所述第一和第二融合蛋白能够形成异二聚化并在基因组DNA区域的相邻位点处产生由所述第一和第二引导RNA确定的双切割，并且任选地将所述核酸插入所述相邻位点之间。
在具体的实施方案中，所述组合物或试剂盒包含微环、质粒或病毒载体中的外源核酸，特别是非整合病毒载体中，例如非整合慢病毒载体中。
在具体的实施方案中，本文公开的组合物或试剂盒包含在纳米颗粒中。
在具体的实施方案中，所述组合物是核酸组合物，其包含：
(i)编码本文公开的融合蛋白的核酸构建体，
(ii)引导RNA，和
(iii)目标核酸或基因，例如外源核酸，用于插入基因组中。
在具体的实施方案中，所述试剂盒包含：
第一组合物，其包含：
(i)编码本文公开的第一融合蛋白的核酸构建体，其中所述第一融合蛋白包含与修饰的高活性Piggybac融合的第一引导RNA切口酶Cas9(通常是SEQ ID NO:70的SpCas9切口酶)的氨基酸序列，和，
(ii)第一引导RNA核酸；
第二组合物，其包含：
(iii)编码本文公开的所述第二融合蛋白的核酸构建体，其中所述第二融合蛋白包含与修饰的高活性Piggybac融合的第二引导RNA切口酶Cas9(通常是SEQ ID NO:76的SaCas9切口酶)的氨基酸序列，和
(iv)第二引导的RNA核酸。
在具体的实施方案中，所述试剂盒或组合物用作药物，特别是用于治疗人病症，例如用于治疗有此需要的人受试者的遗传缺陷。
在一些实施方案中，核酸构建体是RNA、DNA或蛋白的形式，并且编码外源核酸的多核苷酸序列是RNA或DNA的形式，这取决于递送方法。具体地，编码外源核酸的多核苷酸序列是RNA的形式。
在一些实施方案中，所述组合物或试剂盒是无病毒的并且包装载体是纳米颗粒，例如聚合物或脂质纳米颗粒。包装载体还可以是与组合物的成分结合的载体。在一些实施方案中，组合物包含在病毒载体中，特别是慢病毒颗粒中。
在一些实施方案中，所述组合物或试剂盒包含(a)RNA形式的本文所述的核酸构建体(例如包含Cas9和转座酶)，(b)如果需要的话，引导RNA(例如作为单独的线性单链RNA分子)，和(c)包含用于以DNA形式插入(例如在载体中)的外源基因的多核苷酸，其包含在包装载体中或结合至包装载体。
在一些实施方案中，所述组合物包含(a)蛋白形式的本文所述的融合蛋白(例如包含Cas9和转座酶)，(b)如果需要的话，引导RNA(例如作为单独的线性单链RNA分子)，其中融合蛋白和引导RNA形成核糖核酸蛋白复合物(RNP)，和(c)包含用于以DNA形式插入(例如在载体中)的外源基因的多核苷酸，其包含在包装载体中或结合至包装载体。
在一些实施方案中，所述组合物包含(a)DNA形式的本文所述的核酸构建体(例如包含Cas9和转座酶)，(b)如果需要的话，引导RNA(例如作为单独的线性RNA分子或作为载体中的DNA))，和(c)包含用于以DNA形式插入(例如在载体中)的外源基因的多核苷酸，其包含在包装载体中或结合至包装载体。
在一些实施方案中，所述组合物包含(a)蛋白形式的本文所述的融合蛋白(例如包含Cas9和整合酶)，(b)如果需要的话，引导RNA(例如作为与融合蛋白复合的单独的RNA分子)，和(c)包含用于插入的外源基因的多核苷酸，其包含在包装载体中或结合至包装载体。在一个具体的实施方案中，包装载体是慢病毒颗粒。在一些实施方案中，(a)融合蛋白与慢病毒衣壳通过gag-pol或VPR(病毒蛋白R)的方法进行结合。在一些实施方案中，(c)多核苷酸呈RNA形式，作为整合酶的负载。
在具体的实施方案中，当使用ZFP时，(b)引导RNA可以是不需要的。
本发明还涉及一种组合物，其包含：
(i)融合蛋白，其包含经工程化以结合至少一个特定RNA序列的RNA结合蛋白、能够将外源核酸插入基因组中的DNA结合蛋白、以及连接第一和第二蛋白的接头，
(ii)Cas9蛋白，和
(iii)引导RNA，其包含所述至少一个用于结合融合蛋白的特定RNA序列，
(iv)目标核酸或基因，例如外源核酸，用于插入基因组中，
其中DNA结合蛋白是本公开的修饰的转座酶，通常是修饰的高活性Piggybac，其与未修饰的高活性Piggybac相比包含增加切除活性的一个或多个氨基酸突变，以及与SEQ IDNO:9的未修饰的高活性Piggybac相比包含降低DNA结合活性的一个或多个氨基酸突变。
在一些实施方案中，RNA结合蛋白是MS2噬菌体外壳蛋白(MCP)。
在一些实施方案中，由融合蛋白的MCP识别的所述至少一个RNA序列是四环。如本文所用，术语“四环(tetraloop)”与术语“茎环”和“发夹环”可互换使用。在一些实施方案中，所述至少一个RNA四环是MS2 RNA四环结合序列。
在一些实施方案中，引导RNA包含至少一个MS2 RNA四环结合序列。在一些实施方案中，gRNA包含多于一个MS2 RNA四环结合序列。如本文所用，术语“多于一个”是指2、3、4、5、6、7、8、9、10、11或更多个。
权利要求中描述了多种实施方案。下面公开一些另外的实施方案：
E1.一种融合蛋白，包含
(i)由RNA引导的核酸酶或锌指核酸酶组成的第一蛋白，
(ii)由转座酶组成的第二种蛋白，以及
(iii)任选地，连接第一和第二蛋白的接头，
其中所述转座酶是修饰的高活性Piggybac，其与SEQ ID NO:9的高活性Piggybac相比包含一个或多个氨基酸突变，
并且其中所述第一蛋白直接或通过接头间接融合在第二蛋白的C末端。
E2.实施方案1的融合蛋白，其中所述转座酶是修饰的高活性Piggybac，其与未修饰的高活性Piggybac相比包含增加切除活性的一个或多个氨基酸突变，并且与未修饰的高活性Piggybac相比包含降低DNA结合活性的一个或多个氨基酸突变。
E3.实施方案2的融合蛋白，其中所述增加切除活性的一个或多个氨基酸突变选自氨基酸位置编号[194-200]、[214-222]、[434-442]或[446-456]定义的区域内的氨基酸突变，例如位置D198、D201、R202、M212或S213处的氨基酸取代；所述位置编号对应于SEQ IDNO:9的未修饰的高活性Piggybac的氨基酸编号。
E4.实施方案1-3中任一项的融合蛋白，其中所述一个或多个氨基酸突变选自M194或D450位置处的增加切除活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号，优选选自氨基酸取代M194V和/或D450N。
E5.实施方案1至4中任一项的融合蛋白，其中所述一个或多个氨基酸突变选自位置R372、K375、R376、E377和/或E380处的降低DNA结合活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号，优选选自氨基酸取代R372A、K375A、R376A、E377A和/或E380A。
E6.实施方案1至5中任一项的融合蛋白，其中修饰的高活性Piggybac包括位置D450处的增加切除活性的至少一个氨基酸取代，和位置R372和K375处的降低DNA结合活性的至少两个氨基酸取代，优选地，所述修饰的高活性Piggybac转座酶包含三重突变D450N、R372A和K375A，所述位置编号对应于SEQ ID NO:9的未修饰的高活性Piggybac的氨基酸编号。
E7.实施方案1至6中任一项所述的融合蛋白，其中所述修饰的高活性Piggybac在氨基酸位置编号[158-169]定义的区域中进一步包含至少一个突变，例如A166S；和/或在位置Y527、R518、K525、N463处包含至少一个突变。
E8.实施方案1至7中任一项的融合蛋白，其中所述修饰的高活性Piggybac包含与SEQ ID NO:1的修饰的高活性Piggybac具有至少85％、至少90％、至少95％同一性或100％同一性的氨基酸序列。
E9.实施方案1至8中任一项的融合蛋白，其中所述修饰的高活性Piggybac是SEQID NO:1的高活性Piggybac的变体，具有一个或多个氨基酸取代，通常具有不超过1、2、3、4、5、6、7、8、9或10个氨基酸取代。
E10.实施方案1至9中任一项的融合蛋白，其中所述修饰的高活性Piggybac进一步包含在位置编号245、268、275、277、287、290、315、325、341、346、347、350、351、356、357、388、409、412、432、447、460、461、465、517、560、564、571、573、576、586、587、589、592和/或594处的一个或多个氨基酸取代，所述位置编号对应于高活性PiggyBac序列(SEQ ID NO:9)的氨基酸编号。
E11.实施方案10的融合蛋白，其中修饰的高活性PiggyBac突变包含以下氨基酸取代或氨基酸取代的组合：R245A、D268N、R275A、R277A、K287A、K290A、K287A/K290A、R315A、G325A、R341A、D346N、N347A、N347S、T350A、S351E、S351P、S351A、K356E、N357A、R388A、K409A、K412A、K432A、D447A、D447N、D450N、R460A、K461A、W465A、S517A、T560A、S564P、S571N、S573A、K576A、H586A、I587A、M589V、S592G或F594L、D450N/R372A/K375A、R275A/R277A、K409A/K412A、R460A/K461A、R275A/R277A/N347S/K375A/T560A/S573A/M589V/S592G和R245A/R275A/R277A/R372A/W465A，所述位置编号对应于高活性PiggyBac序列(SEQ IDNO:9)的氨基酸编号。
E12.实施方案10的融合蛋白，其中修饰的高活性PiggyBac突变包含以下氨基酸取代或氨基酸取代的组合：R372A/K375A/D450N、R372A/K375A/R376A/D450N、K375A/R376A/E377A/E380A/D450N、R372A/K375A/R376A/E377A/E380A/D450N、M194V、R376A、E377A、E380AM194V/R372A/K375A、S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L；所述位置编号对应于高活性PiggyBac序列(SEQ ID NO:9)的氨基酸编号，通常所述修饰的转座酶具有选自SEQ ID NO:1-8和10-18中任一个的氨基酸序列。
E13.实施方案1-12中任一项的融合蛋白，其中所述接头是包含XTEN序列或GGS序列的肽接头，优选包含XTEN序列的肽接头。
E14.实施方案1-13中任一项的融合蛋白，其中所述接头是长度为3至50个氨基酸的肽接头，通常选自SEQ ID NO:49、51、53、55、57、59、61中的任一个。
E15.实施方案1-14中任一项的融合蛋白，其中所述第一蛋白是包含活性DNA切割结构域和引导RNA结合结构域的Cas9蛋白。
E16.实施方案1-15中任一项的融合蛋白，其中所述第一蛋白是包含活性DNA切割结构域和引导RNA结合结构域的核酸酶蛋白，并且与SEQ ID NO:31的酿脓链球菌Cas9、SEQID NO:72的SaCas9、SEQ ID NO:74的Cpf1、SEQ ID NO:29的CjCas9、SEQ ID NO:70的SpCas9切口酶、SEQ ID NO:75的CasX或SEQ ID NO:76的SaCas9切口酶具有至少80％、90％、95％、99％或至少100％的同一性。
E17.实施方案1-16中任一项的融合蛋白，其中所述第一蛋白是选自SEQ ID NO:72的SaCas9或SEQ ID NO:31的酿脓链球菌Cas9的Cas9蛋白。
E18.实施方案1至17中任一项所述的融合蛋白，其是三重融合蛋白，包含：
(i)由RNA引导的核酸酶或切口酶组成的第一蛋白，
(ii)由第一转座酶组成的第二蛋白，
(iii)由第二转座酶组成的第三蛋白，和
(iv)任选地，第一和第二蛋白以及第二和第三蛋白之间的肽接头，
并且其中第一和第二转座酶具有修饰的Piggybac转座酶的相同或不同序列，例如实施方案3-12中任一个所定义的。
E19.实施方案1至14中任一项的融合蛋白，其中所述第一蛋白是SEQ ID NO:33的锌指蛋白。
E20.一种组合物，包含：
(i)实施方案1-19中任一项所述的融合蛋白，或编码所述融合蛋白的核酸，
(ii)引导RNA，和
(iii)用于插入基因组中的外源核酸。
E21.实施方案20的组合物，其中所述外源核酸是大的DNA片段，通常具有5kb至25kb且更优选8kb至20kb的大小。
E22.一种试剂盒，包含：
(i)第一组合物，其包含
实施方案1和18中任一项所定义的第一融合蛋白或编码所述第一融合蛋白的核酸，并且其中所述第一融合蛋白包含与修饰的高活性Piggybac融合的第一引导RNA切口酶(first guided RNA nickase)Cas9(通常为SEQ ID NO:70的SpCas9切口酶)的氨基酸序列，
第一引导RNA核酸(first guided RNA nucleic acid)，
(ii)第二组合物，其包含
实施方案1至18中任一项所定义的第二融合蛋白或编码所述第二融合蛋白的核酸，并且其中所述第二融合蛋白包含与修饰的高活性Piggybac融合的第二引导RNA切口酶Cas9(通常SEQ ID NO:76的SaCas9切口酶)的氨基酸序列，
第二引导RNA核酸，
(iii)任选地，用于插入基因组中的外源核酸，例如大小为5kb至25kb、更优选8kb至20kb的外源核酸。
其中所述第一和第二融合蛋白能够形成异二聚化并在基因组DNA区域的相邻位点处进行由所述第一和第二引导RNA确定的双切割，并且任选地在相邻位点之间插入所述外源核酸。
E23.实施方案20-22的组合物或试剂盒，其中所述外源核酸包含在微环、质粒或病毒载体中，特别是非整合病毒载体中，例如非整合慢病毒载体中。
E24.实施方案20-23的组合物或试剂盒，其中所述组合物包含在纳米颗粒中。
E25.修饰的高活性Piggybac转座酶，其与未修饰的高活性Piggybac相比包含增加切除活性的至少一个氨基酸突变，和/或与未修饰的高活性Piggybac相比包含降低DNA结合活性的至少一个氨基酸突变，
其中，增加切除活性的至少一个氨基酸突变是在位置194处的M的氨基酸取代，通常为M194V，和/或其中降低DNA结合活性的至少一个氨基酸突变选自位置R376、E377和E380处的氨基酸取代，通常为R376A、E377A和/或E380A。
E26.修饰的高活性Piggybac转座酶，其与SEQ ID NO:9的未修饰的高活性Piggybac相比包含以下突变组合：R372A/K375A/D450N、R372A/K375A/R376A/D450N、K375A/R376A/E377A/E380A/D450N、R372A/K375A/R376A/E377A/E380A/D450N、M194V、R376A、E377A、E380A、M194V/R372A/K375A、S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L。
E27.实施方案25或26的修饰的高活性Piggybac转座酶，其进一步包含选自以下组成的组的一个或多个氨基酸突变：位置245处的氨基酸是A、位置275处的氨基酸是R或A、位置277处的氨基酸是R或A、位置325处的氨基酸是A或G、位置347处的氨基酸是N或A、位置351处的氨基酸是E、P或A、位置372处的氨基酸是R、位置375处的氨基酸是A、位置450处的氨基酸是D或N、位置465处的氨基酸是W或A、位置560处的氨基酸是T或A、位置564处的氨基酸是P或S、位置573处的氨基酸是S或A、位置592处的氨基酸是G或S、位置594处的氨基酸是L或F。
E28.实施方案25或26的修饰的高活性PiggyBac转座酶，其与SEQ ID NO:9的未修饰的高活性PiggyBac相比包含1、2、3、4、5、6、7、8、9或10个额外的突变，其中所述修饰的高活性PiggyBac与SEQ ID NO:9的高活性PiggyBac相比显示出降低的DNA结合活性和/或增加的切除活性。
E29.编码实施方案1至18中任一项的融合蛋白的核酸，通常为信使RNA(mRNA)。
E30.编码实施方案28的融合蛋白的核酸，其包含选自由SEQ ID NO:110-112组成的组或其相应的mRNA序列的序列。
E31.核酸，其编码实施方案25-28中任一项的修饰的高活性Piggybac。
E32.表达载体，其包含实施方案29-31中任一项的核酸。
E33.宿主细胞，其包含实施方案29-31中任一项的核酸或实施方案32的表达载体。
E34.用于将外源核酸序列位点特异性整合至细胞基因组中的方法，所述方法包括向细胞递送组合物，所述组合物包含：
(i)实施方案1至18中任一项的融合蛋白，或实施方案29-31中任一项的核酸，
(ii)待整合到细胞基因组中的外源核酸，以及
(iii)用于确定所述外源核酸向细胞基因组中的位点特异性整合的引导RNA。
其中所述融合蛋白与细胞基因组中的特定基因组DNA序列的结合导致基因组的切割和所述外源核酸序列位点特异性整合至引导RNA所确定的细胞基因组中。
E35.实施方案34的方法，其中所述外源核酸是大小为至少5kb、至少6kb、至少7kb、至少8kb、至少9kb、通常包含在5kb和25kb、优选8到20kb的核酸片段。
E36.实施方案34或35的方法，其中所述外源核酸是待插入有此需要的受试者的基因组中以纠正遗传性疾病的缺陷的治疗性转基因。
E37.实施方案34-36中任一项的方法，其中所述组合物在体外或离体递送，通常在哺乳动物细胞中，优选在人细胞中，更优选在从患有遗传性疾病的人受试者获得的人细胞中。
E38.实施方案34-37中任一项的方法，其中将所述组合物体内递送至哺乳动物，例如有此需要的人受试者中，通常用于遗传性疾病的治疗性治疗。
E39.将外源核酸序列插入生物体的基因组DNA中的方法，包括：向生物体施用一种或多种如实施方案20-24中任一项所定义的组合物或试剂盒，使得所述一种或多种组合物或试剂盒中包含的融合蛋白与特定的基因组DNA序列结合，并且使包含在所述组合物中的外源核酸能够插入到基因组DNA中；
其中所述外源核酸整合在所述生物体(例如非人生物体或有此需要的人受试者)的细胞的基因组的特定位点处。
实施例
为了实现可编程转座酶，我们的设计原则尝试将CRISPR系统的准确基因组靶向与表现出增强的插入和切除活性以及较低的靶DNA结合活性的PB变体结合起来。我们首先将核酸酶SpCas9与PB转座酶融合(图1)。我们构建了一个多样化的PB和SpCas9变体文库(图2a、b、c)，其中我们测试了PB变体文库、3个SpCas9变体(核酸酶SpCas9(cas9)、死SpCas9(dcas9)、切口酶SpCas9(ncas9))和6个接头(4xGGS、5xGGS、7xGGS、8xGGS、XTEN、FOKI)。
PB转座酶的不同部分具有多样化。特别重点关注催化核心结构域，该结构域由3个天冬氨酸残基(D268、D346、D447)的催化三联体形成，周围环绕着可能参与转座酶-靶标DNA相互作用的15个精氨酸和赖氨酸残基14。除了过去探索的高活性版本之外9，我们还对可能影响PB切除和整合活性的其他残基进行了多样化15(SEQ 1-N)。为了分离出表现最好的突变体，我们开发了一种用于靶向基因插入的灵敏的报告系统，该系统基于中靶整合后的Emerald GFP(emGFP)重构。将无启动子的emGFP的C末端(C-t)一半(前面有剪接接受体)插入Hek293T细胞的基因组中，以构建报告细胞系(称为Hershey)。构建了一个互补的“插入陷阱报告子”，编码emGFP的N末端(N-t)前半部分，带有上游启动子，后跟剪接供体，并且两侧都是PB反向重复序列。特异性的gRNA引导的cas9指导PB以将N-t半部插入到C-t半部附近，其在所得转录本剪接时产生绿色荧光(图3)。组装了cas9-PB嵌合蛋白文库，并与“插入陷阱报告子”和引导RNA(gRNA)一起转染至含有Hershey报告子的细胞中。使用相同的报告细胞系分别测试呈现较高可编程插入的变体(图2a、2b；4a、4b)。该测定使用含有N-t半部的emGFP和上游完整RFP序列的双转座子测试了中靶活性(emGFP阳性细胞)和总转座活性(RFP阳性细胞)。通过将emGFP阳性细胞的百分比(中靶活性)除以RFP阳性细胞的总百分比(总插入活性)，来计算中靶与脱靶的比率。
我们测试了与PB变体融合的cas9变体的各种组合在Hershey细胞系中的中靶和脱靶转座。我们观察到Nter-cas9-PB-Cter融合物中可编程插入水平最高，该融合物包含i)具有完整核酸酶活性的cas9(图2A)和ii)具有增加的切除活性的PB变体和iii)降低的t-DNA结合活性(图2B；4A，4B)。我们观察到，所有三个参数对于实现准确且高效的靶向插入都很重要。首先，与PB融合的ncas9(D10A)和dcas9(D10A和H840A)的靶向插入效率显著较低，这证明了对cas9核酸酶活性的需求(图2A)。为了进一步探究cas9的双链断裂(DSB)活性在促进靶向整合中的作用，我们使用用于转座子的引导定位的锌指-PB融合物将在位靶向(on-site targeting)和DSB活性分开，并通过独立的Cas9核酸酶对其补充了在位DSB。Znf-PB融合物没有表现出或表现出非常低的靶向插入活性，当与使用gRNA引导的cas9在Znf结合位点附近引入DSB相结合时，这种活性得以恢复(图5)。这些结果与cas9在PB附近产生的DSB促进PB的插入活性并绕过其对插入位点的规范TTAA的要求的机制相一致。事实上，我们对cas9-PB插入位点的分析表明，反向末端重复(ITR)序列因遭受破坏，在靶向位点附近存在小的插入缺失(图6)。这种破坏的一个重要后果是PB介导的整合机制的不可逆性。ITR或TTAA被破坏的转座子的移动要么消除，要么减少16。这种机制可能促成了cas9-PB的可编程插入的效率，并且cas9的“查找”和“切割”活性与修饰的PB的“粘贴(pasting)”活性的偶联促成了高准确水平。其次，高切除PB突变体似乎促成了更高的可编程插入(D450N、M194V；图2B、4A、4B)。这种增强的切除可能是为整合而准备的供体DNA底物增加的结果。此外，优选切除底物的破坏可能会阻止充分的切除，即使是具有较高切除活性的PB突变体(图6)。第三，t-DNA结合减少的PB突变体导致最低的脱靶水平(图2B、4A、4B)。这一结果与PB的内在非特异性DNA结合的减少相一致，这种减少通过使PB单独插入活性失活的cas9-PB融合物中的cas9的序列特异性DNA结合进行补充(图7)。
使用基因组引导方法，我们能够使用基于Guide-seq17的方法的修改版本来表征可编程转座酶插入位点和脱靶水平(图8A)。在TTAA位点上未发现任何中靶插入，这进一步证明了cas9生成的DSB位点上的整合并导致优选切除底物的丢失(图8B)。此外，我们对使用靶向TRAC基因座的可编程转座酶技术进行修饰的细胞进行Guide-seq分析。我们检测了中靶的所有插入，灵敏度低至1-10％(图9)。
我们用当前的用于准确基因传递的方法(例如基于cas9的HDR)对可编程转座酶技术进行了基准测试(图10)，可编程转座酶显示出较高的效率，在大负载中差距会扩大。最好的突变体以比HDR高2倍的效率且高准确性地实现了插入(高达8kb)。我们还将可编程转座酶与HITI变体进行了比较，其中我们将Cas9与PB的催化性死版本融合，这可能有助于将DNA募集至插入位点，因为最近使用SB100转座酶的DNA结合结构域的类似方法提出了这一点。与其他辅助HITI方法相比，可编程转座酶显示2倍的更高效率(图18)。为了在体内小鼠模型中证明可编程转座酶活性，我们构建了可编程转座酶的mRNA版本。我们使用体内JetPEI试剂将可编程转座酶递送至小鼠肝脏，靶向Rosa26基因组安全港，并观察到与内源基因相比转基因的高拷贝数(图11)，以及转基因表达随着时间推移维持(图20)。
综上所述，我们将CRISPR分子识别和切割与修饰的PB的DNA切割-粘贴活性偶联起来，生成了一种有效的工具来执行准确和高效的基因递送。该技术可以很好地根据负载大小进行缩放。我们证明了它在Hek293T和小鼠肝脏中的功效。我们想到了可编程转座酶技术作为先进疗法和其他应用的治疗基因传递的通用平台。
与hyPB融合的Cas变体的结果
为了进一步表征工程化hyPB执行可编程转座的能力，我们用来自不同生物体的具有核酸酶活性的其他Cas蛋白(即SEQ ID NO:72的SaCas9、SEQ ID NO:74的cpf1、SEQ IDNO:75的CasX和SEQ ID NO:29的CjCas9)，替换了所测试的可编程转座酶的SpCas9模块。设计了靶向分裂GFP报告子上游区域的特异性gRNA，并进行克隆以用于Hershey细胞系转染(参见下表2)。通过GFP表达的方法来测量靶向转座(图12)。
这些结果在另一组实验中得到了证实：我们获得了CjCas9和LbCpf1的良好的可编程插入活性，而CasX在我们的测定中没有实现任何可编程整合。值得注意的是，SaCas9在测试的Cas蛋白中具有最高水平的可编程插入，与融合至修饰的hyPB的SpCas9水平相似(图19)。通过Ilumina NGS测定了所使用的不同Cas蛋白和为每种蛋白设计的三种不同gRNA的插入缺失(图21)，以标准化目的而显示。
这些积极的结果验证了用于可编程转座的工程化hyPB可用于任何序列特异性核酸酶模块。
与PB突变体二聚体融合的Cas9的进一步结果
鉴于PB在进行转座时以二聚体发挥作用的性质，我们尝试生成Cas9和hyPBR372A-K375A-D450N突变体的融合蛋白。我们将这些融合物与单独的Cas9-PB突变体的中靶活性进行了比较。我们观察到，构型Cas9-PB-PB具有更好的性能；而构型PB-Cas9-PB并未优于Cas9-PB单体融合物(图14)。对于与Cas9的二聚融合物，我们使用已记录版本的hyPBR372A-K375A-D450N突变体以促进克隆和表达。
有趣的是，如果与二聚hyPB R372A-K375A-D450N融合的Cas9是SaCas9而不是SpCas9，则活性进一步增加(图24)。与二聚hyPB一起时SaCas9与SpCas9相比性能的提高与使用单体hyPB获得的结果相一致(图12和19)。
ZNF-PB突变体恢复的结果
我们想进一步探究两个gRNA在靶位点(4个核苷酸)邻近诱导的双链断裂(DSB)活性(通过SpCas9切口酶变体(D10A)促进单链切割)在促进靶向整合、同时通过在脱靶位点(off target site)的非诱导DSB的方式降低脱靶活性(off target activity)方面的作用。我们使用用于转座子的引导定位的锌指-PB融合物(通过与D450N突变体和R372A-K375A-D450N突变体融合)，并用由独立的切口酶Cas9产生的两个位点上单链断裂或由Cas9核酸酶产生的单个DSB对其进行补充。
Znf-PB融合物没有表现出或表现出非常低的靶向插入活性，当与使用单或双gRNA引导的cas9核酸酶或切口酶在Znf结合位点附近引入DSB相组合时，这种活性得以恢复(图13)。
Cas9-hyPB突变变体的结果
为了进一步探索能够以更高的效率进行可编程转座的突变组合，进行了几轮细胞选择，其中通过可编程插入分裂GFP报告系统来重构GFP。有趣的是，我们观察到几种组合的性能优于Cas9-hyPB R372A-K375A-D450N(图15)。特别值得一提的是，与Cas9融合的在hyPB的以下AA处发生突变的hyPB变体：A351-A372-A375-A388-N450-A465-A573-V589-G592-L594(也被鉴定为SEQ ID NO:2)，与R372A-K375A-D450N(SEQ ID NO:1)相比其在阳性细胞群中丰富数倍；以及A245-A275-A277-A372-A465-V589(SEQ ID NO:3)和A275-A325-A372-A560(SEQ ID NO:4)，其程度较低。
在另一系列实验中，PiggyBac DNA文库由Twist Bioscience产生，与cas9融合克隆到慢病毒载体中，并转化到stb4感受态细胞中，确保x100变体复杂性(variantcomplexity)。通过maxiprep纯化质粒并与慢病毒包装质粒共转染至Hek293T细胞中。慢病毒用于感染1/2GFP报告细胞系。感染的细胞用1/2GFP转座子和靶向AAVS1序列的gRNA转染。通过流式细胞仪分选选择GFP阳性细胞并提取基因组DNA。从提取的gDNA中扩增PB，将其重新克隆到慢病毒载体中以重新开始新的循环。选择表现最佳的可编程转座酶变体，并分别用AAVS1 gRNA和MC1/2GFP转染。
首先，随机选择96个变体，并单独筛选表现最佳的变体(图16)。对高中靶插入的最佳PB氨基酸变体的总结证实了突变D450N、R372A和K375A的重要性；但也凸显了促进靶向效率提高的其他重要残基(图17B)。选择了具有最佳中靶效率的六种PB变体(图17A)。与FiCAT(Cas9-hyPBR372A-K375A-D450N)相比，以下变体的单独中靶活性显著提高：N347A-D450N；N347S-D450N-T560A-S573A-F594L；R202K-R275A-N347S-R372A-D450N-T560A-F594L；R275A-N347S-K375A-D450N-S592G；R275A-N347S-R372A-D450N-T560A-F594L；和R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L(双向t检验)。
对该实验进行重复并得到了证实(图22A)。我们还产生了表达每轮的大量变体的慢病毒和感染的报告细胞系，通过PB变体CN校正其滴度，证明了中靶效率随轮次呈类似增加(图22B)。经过4和5轮cas9_PB文库富集后，从大量变体中分离出单突变。通过用FiCAT突变体、gRNA tcr1和1/2GFPMC转座子转染中靶报告细胞系，单独对突变体进行了测试。与FiCATR372A_K375A_D450N对比显示了最佳FiCAT突变体(图23)。与FiCAT(Cas9-hyPBR372A-K375A-D450N)相比，以下变体的单独的中靶活性显著提高：R202K-R275A-N347S-R372A-D450N-T560A-F594L；R245A-N347S-R372A-D450N-T560A-S564P-S573A-S592G；R275A-N347S-R372A-D450N-T560A-F594L；N347A-D450N；R277A-G325A-N347A-K375A-D450N-T560A-S564P-S573A-S592G-F594L；N347S-D450N-T560A-S573A-F594L；V34M-R275A-G325A-N347S-S351A-R372A-K375A-D450N-T560A-S564P；G325A-N347S-K375A-D450N-S573A-M589V-S592G；S230N-R277A-N347S-K375A-D450N；T43I-R372A-K375A-A411T-D450N；G325A-N347S-S351A-K375A-D450N-S573A-M589V-S592G；Y177H-R275A-G325A-K375A-D450N-T560A-S564P-S592G。
与突变R372A-K375A-D450N相比，突变R202K-R275A-N347S-R372A-D450N-T560A-F594L、R275A-R277A-N347S-R372A-D450N-T560A-S564P-F594L和R275A-N347S-R372A-D450N-T560A-F594L的优越性在包含SpCas9和两个hyPB的三重融合蛋白中进一步得到证明(图29)。
Cas9与hyPB非共价连接的结果
除了Cas9与hyPB R372A-K375A-D450N通过接头共价结合外，我们还利用MS2-MCP系统，通过含有结合MCP蛋白的MS2序列的四环的修饰gRNA，将Cas9和由MCP蛋白和hyPBR372A-K375A-D450N组成的融合蛋白进行连接。
与Cas9-hyPB R372A-K375A-D450N融合蛋白相比，MCP-hyPB R372A-K375A-D450N融合蛋白与Cas9的组合具有增加的可编程插入活性(图25A)。此外，我们将MCP蛋白与hyPB的其他突变体融合，从而与SpCas9结合进行可编程转座。使用的两种变体(R202K-R275A-N347S-R372A-D450N-T560A-F594L和R275A-N347S-R372A-D450N-T560A-F594L)均优于R372A-K375A-D450N(图25B)。
Cas9和hyPB解偶联的可编程转座的结果
我们还尝试了SpCas9与hyPB R372A-K375A-D450N在不使用接头或MS2-MCP系统时的性能。我们在同一细胞中共表达SpCas9和hyPB R372A-K375A-D450N，并且记录了与Cas9-hyPB R372A-K375A-D450N融合蛋白相比增加的可编程插入活性(图26A)。我们扩展了测试的hyPB突变变体的数量，它们不与Cas9融合但同时表达并且共同作用以实现可编程转座的活性(图26B)。
Cas9-hyPB与MCP-hyPB融合蛋白的共表达的结果
我们共转染了与MCP蛋白融合的hyPB R372A-K375A-D450N突变体以及与SpCas9融合的hyPB突变体，以获得其中一个单体非共价连接的融合物的二聚体版本。比较了与SpCas9融合的几种hyPB突变体的特异性靶标整合(图27)。
Cas9-hyPB与hyPB变体的共表达的结果
以类似的方式，我们独立地共转染了SpCas9 hyPB R372A-K375A-D450N融合蛋白和hyPB突变体，以获得其中一个单体未连接的融合物的二聚体版本(图28)。
方法
克隆和质粒。用于监测随机插入的RFP转座子PB512-B购自System BiosciencesInc。hyPB载体获自Wellcome Trust Sanger Institute(pCMV_hyPBase)9。质粒载体来自Invitrogen，cas9、ncas9和SP-dcas9-VPR获自Addgene(Addgene质粒#41815，#41816，#63798)。最后，SB100X和pT4-HB是来自Zsuzsana Zizsvak博士的馈赠。使用The Zero Blunt TOPO PCR克隆试剂盒(Invitrogen)以及gBlock GeneFragment(Integrated DNA Technologies)来产生gRNA，其含有U6启动子、20nt靶位点、gRNA支架和终止子。gRNA TRAC在实验室中设计和验证，gRNA aavs1 3序列先前已有描述18。
使用BspQI酶和标准方法，通过Golden Gate组装来进行核酸酶(切口酶和死cas9)与hyPB和PB RFP1/2emGFP SMN1转座子的融合。
按照Quickchange Lightning诱变试剂盒(Agilent)的说明，通过定点诱变将不同的突变引入与cas9融合的hyPB序列(cas9_PB质粒)中。使用QuickChange Primer Design设计引物，以获得对hyPB序列的以下突变：M194V、R245A、G325A、R372A、K375A、R376A、E377A、E380A、D450N、S564P(参见SEQ ID NO:90-99)。所有质粒均可根据需求提供。PB1/2emGFPSMN1是通过将emGFP序列的前半部分和SMN1内含子6序列引入piggyBac接受体载体获得的。pT4 SMN1 2/2emGFP是通过在SB100X转座子载体中添加后半部分SMN1内含子6和部分emGFP获得的。含有SMN1的emGFP序列是从DYP004reporter19获得的，这是来自Sri Kosuri的馈赠。
通过对分裂emGFP报告系统上游的部分cDNA(NC_000006.12)片段进行克隆，来生成不同大小的转座子和HDR模板。
细胞培养、转染和电穿孔。Hek293T细胞系(Thermo Fisher Scientific)和C2C12细胞系(ATCC)在5％ CO2培养箱中于37℃下培养，采用补充有高葡萄糖(Gibco，ThermFisher)、10％胎牛血清(FBS)、2mM谷氨酰胺和100U青霉素/0.1mg/mL链霉素的达尔伯克改良伊格尔氏培养基(DMEM)。Jurkat细胞系在5％ CO2培养箱中于37℃下培养，使用补充有Glutamax和HEPES(Gibco，Thermo Fisher)和10％ FBS的Roswell Park MemorialInstitute1640培养基(RPMI)。该细胞系是Manel Juan(Hospital Clinic,Barcelona)的惠赠。Hek293T细胞的转染实验按照制造商的说明使用lipofectamine 3000试剂或在OptiMem中以1:3DNA-PEI比例使用聚乙烯亚胺(PEI，Thermo Fisher Scientific)进行。在前一天接种细胞，以在转染当天达到70％汇合度(贴壁p12孔板中通常有290.000个细胞)。质粒DNA比例为1转座酶：2.5gRNA：2.5转座子，或1Cas9：2.5gRNA：2.5HDR模板，使用0.076pmol可编程转座酶或Cas9用于p12孔板。
基于emGFP剪接的重构测定。通过PEI介导的SB100X和pT4 SMN1 2/2emGFP DNA构建体转染，然后进行单克隆扩增和PCR基因分型，产生含有pT4 SMN1 2/2emGFP的Hek293T细胞系(补充表3)。选择并扩增阳性克隆并用于后续测定。对于emGFP重构测定，转染可编程转座酶、gRNA和转座子质粒，比例为1可编程转座酶：2.5gRNA：2.5转座子，使用0.076pmol可编程转座酶或hyPB和0.19pmol转座子和gRNA用于12孔板。在转染后5天通过emGFP荧光测量中靶插入。在转染后15天通过RFP荧光测量脱靶转座。通过共转染PB变体、gRNA和PB1/2SMN1RFP emGFP(插入确定性构建体名称)表达质粒，并在14天后(针对游离体衰减，设定平均n天)通过在(BD LSR Fortessa；BD Biosciences)带有530/30滤光片的蓝色488nm激光和带有610/20滤光片的黄绿561nm激光下测量的emGFP表达来确定emGFP和RFP荧光，以测量中靶插入。
用于插入位点测序的连接PCR(junction PCR)。使用BD FACSAria(Biosciences)对emGFP分选的细胞进行连接PCR。所选的细胞具有靶向报告细胞系上的TRAC靶位点的PB1/2emGFP SMN1转座子的中靶插入。使用DNeasy血液和组织试剂盒(Qiagen)提取基因组DNA。引物通过转座子的3'ITR设计(正向)，并靶向报告细胞系的2/2emGFP的内含子或内源T细胞受体(TRAC)(反向)(补充表4)。
Guide-SEQ实验的生物信息学分析。将Illumina读段与usearch 20聚类，并使用bwa-mem 21定位至参照。对于中靶插入表征，使用Python脚本和Samtools22选择覆盖靶插入位点的5'和3'连接的读段。使用CRISPR-GA23获得插入缺失的数量。对于中靶和脱靶实验，选择针对载体定位的聚类读段，并使用bwa-mem定位至参照基因组。使用macs224算法评估插入峰的显著性。
针对靶向插入改造的Guide-seq文库制备。通过使用DNeasy血液和组织试剂盒(Qiagen)提取基因组DNA并使用Q800R3超声仪将其片段化为500bp片段，进行改造的Guide-seq15方案。使用KAPA Hyper Prep Kit(KR0961–v5.16)和3ug片段化基因组DNA进行末端修复、A加尾和Y-适体连接，然后以1X比例进行AMPure XP SPRI珠子纯化。连接适体后，将每个样品一分为二，并用GSP5'或GSP3'进行扩增以分别捕获5'和3'连接。为了捕获5'和3'转座子-基因组连接，使用KAPA HiFi DNA聚合酶根据制造商的方案进行两个巢式PCR：PCR1使用P5_1和PB_5_GSP1或PB_3_GSP1，最终体积为25ul；PCR2使用P5_2PB_5_GSP2或PB_3_GSP2，最终体积为25ul。使用AMPure XP SPRI珠子纯化法以1X比例纯化5'和3'PCR产物，以等摩尔比例混合并使用Illumina Miseq Reagent试剂盒V2进行测序-500个循环(2x250bp配对末端)。将3ul 100μM定制引物index 1和Read 2添加到测序反应中。
小鼠肝脏中的体内靶向插入。动物实验程序经巴塞罗那生物医学研究园动物实验伦理委员会(Animal Experimentation Ethic Committee of Barcelona BiomedicalResearch Park)批准。本研究使用8-10周龄的C57BL/6J。动物购自Jackson Laboratories，使用时不区分雄性和雌性。可编程转座酶mRNA按照制造商的说明使用RiboMAX大规模RNA生产系统-T7(Promega)生产。Rosa26 gRNA25购自IDT。将可编程转座酶mRNA、靶向Rosa26的gRNA和PB512-B转座子以1:1:2.5的比例通过眼眶后注射。将总共60ug核酸与in vivo-JetPEI(Polyplus转染)复合，NP比例为7。注射后10天对动物实施安乐死，分离肝脏并匀浆。使用DNeasy血液和组织试剂盒(Qiagen)从肝脏样品中提取基因组DNA。通过qPCR获得相对于Tfrc内源基因的转座子相对拷贝数(引物列于补充表1)。使用IVIS光谱成像系统(Caliper Life Sciences)，在FiCAT-gRNA-转座子或转座子对照施用后的不同时间点，进行荧光素酶表达的成像。根据制造商的说明，在腹腔注射D-荧光素钾盐(GoldBiotechnology)后5分钟拍摄图像。
PB结构建模。粉纹夜蛾(trichoplusia ni)PiggyBac转座酶蛋白的3D结构通过Robetta Web蛋白结构预测服务器(http://robetta.bakerlab.org)获得。核心结构域(131-550aa)是通过Rosetta比较建模方法预测的，该方法基于蒙特卡罗算法并具有嵌入的笛卡尔空间最小化和全原子优化26。使用基于SPServer和ProSa-Web知识的方法，对三级结构折叠进行了分析和验证(补充图2)。使用基于PSIPRED和HHPred机器学习的方法，分析二级结构。然后通过比较蛋白建模方法，使用PyMOL对PB的核心进行建模以进行精细化。精细化过程通过piggyBac模型与Cryo-EM HIV-1Strand Transfer Complex Intasome(PDB ID:5U1C)的叠加进行指导，后者由与病毒DNA和靶宿主DNA结合的HIV整合酶四聚体和X射线衍射Tn5转座酶复合体结构(PDB ID：1MUS27)组成。链转移DNA和供体DNA分别从HIV-1Intasome和Tn5的叠加中推导。用X3DNA作为双链DNA，分析与蛋白接触的界面中的核苷酸。我们使用统计势能对蛋白和DNA之间的相互作用进行评分，并生成理论PWM28。通过测试界面中所有潜在的双链DNA序列，根据统计势能对它们进行排序，并选择前列的进行多序列比对，来获得理论PWM。在该手稿提交期间，冷冻电镜结构变得可用，其显示了与所进行的建模的重要一致性29。PiggyBac转座酶链转移复合物(PDB ID:6X67)的冷冻电镜结构证实了模型的总体折叠以及我们假设的负责与供体和靶DNA接触的结构域。
表2：Cas变体gRNA
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Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 5
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
<400> 5
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ala Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Ala Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Arg
580 585 590
Cys Leu
<210> 6
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
<400> 6
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Ala Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Ser
580 585 590
Cys Leu
<210> 7
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
<400> 7
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Ala Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 8
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
<400> 8
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Ala Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Ala Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Arg
580 585 590
Cys Leu
<210> 9
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 超活性PiggyBac aa 序列
<400> 9
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 10
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
<220>
<221>位点
<222> 245..245
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 268..268
<223> 其中氨基酸可以是Asp, Asn
<220>
<221>位点
<222> 275..275
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 277..277
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 287..287
<223> 其中氨基酸可以是Ala, Lys
<220>
<221>位点
<222> 290..290
<223> 其中氨基酸可以是Ala, Lys
<220>
<221>位点
<222> 315..315
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 325..325
<223> 其中氨基酸可以是Gly, Ala
<220>
<221>位点
<222> 341..341
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 346..346
<223> 其中氨基酸可以是Asp, Asn
<220>
<221>位点
<222> 347..347
<223> 其中氨基酸可以是Asn, Ala, Ser
<220>
<221>位点
<222> 350..350
<223> 其中氨基酸可以是Thr, Ala
<220>
<221>位点
<222> 351..351
<223> Xaa可以是Ser, Glu, Pro, Ala
<220>
<221>位点
<222> 356..356
<223> 其中氨基酸可以是Lys, Glu
<220>
<221>位点
<222> 357..357
<223> 其中氨基酸可以是Asn, Ala
<220>
<221>位点
<222> 372..372
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 375..375
<223> 其中氨基酸可以是Lys, Ala
<220>
<221>位点
<222> 388..388
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 409..409
<223> 其中氨基酸可以是Lys, Ala
<220>
<221>位点
<222> 412..412
<223> 其中氨基酸可以是Lys, Ala
<220>
<221>位点
<222> 432..432
<223> 其中氨基酸可以是Lys, Ala
<220>
<221>位点
<222> 460..460
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 461..461
<223> 其中氨基酸可以是Ala, Lys
<220>
<221>位点
<222> 465..465
<223> 其中氨基酸可以是Trp, Ala
<220>
<221>位点
<222> 560..560
<223> 其中氨基酸可以是Thr, Ala
<220>
<221>位点
<222> 564..564
<223> 其中氨基酸可以是Ser, Pro
<220>
<221>位点
<222> 571..571
<223> 其中氨基酸可以是Asn, Ser
<220>
<221>位点
<222> 573..573
<223> 其中氨基酸可以是Ser, Ala
<220>
<221>位点
<222> 576..576
<223> 其中氨基酸可以是Lys, Ala
<220>
<221>位点
<222> 586..586
<223> 其中氨基酸可以是His, 任何天然存在的氨基酸
<220>
<221>位点
<222> 587..587
<223> 其中氨基酸可以是Ile, Ala
<220>
<221>位点
<222> 589..589
<223> 其中氨基酸可以是Met, Val
<220>
<221>位点
<222> 592..592
<223> 其中氨基酸可以是Ser, Gly
<220>
<221>位点
<222> 594..594
<223> 其中氨基酸可以是Phe, Leu
<400> 10
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Xaa Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Xaa Glu Gln Leu Leu
260 265 270
Gly Phe Xaa Gly Xaa Cys Pro Phe Arg Val Tyr Ile Pro Asn Xaa Pro
275 280 285
Ser Xaa Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Xaa Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Xaa Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Xaa Asn Ile Thr Cys Xaa Xaa Trp Phe Xaa Xaa Ile
340 345 350
Pro Leu Ala Xaa Xaa Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Xaa Ser Asn Xaa Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Xaa Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Xaa Pro Ala Xaa Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Xaa
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Xaa Xaa Thr Asn Arg
450 455 460
Xaa Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Xaa
545 550 555 560
Tyr Cys Pro Xaa Lys Ile Arg Arg Lys Ala Xaa Ala Xaa Cys Lys Xaa
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Xaa Xaa Asp Xaa Cys Gln Xaa
580 585 590
Cys Xaa
<210> 11
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，催化三联体中具有突变
<220>
<221>位点
<222> 268..268
<223> Xaa可以是Asp, Asn
<220>
<221>位点
<222> 346..346
<223> Xaa可以是Asp, Asn
<400> 11
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Xaa Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Xaa Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 12
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，氨基酸中具有对切除重要的突变
<220>
<221>位点
<222> 287..287
<223> Xaa可以是Lys, Ala
<220>
<221>位点
<222> 290..290
<223> Xaa可以是Lys, Ala
<220>
<221>位点
<222> 460..460
<223> Xaa可以是Arg, Ala
<220>
<221>位点
<222> 461..461
<223> Xaa可以是Lys, Ala
<400> 12
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Xaa Pro
275 280 285
Ser Xaa Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Xaa Xaa Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 13
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，具有参与靶连接的突变
<220>
<221>位点
<222> 351..351
<223> Xaa可以是Ser, Glu, Pro, Ala
<220>
<221>位点
<222> 356..356
<223> Xaa可以是Lys, Glu
<400> 13
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Xaa Ile
340 345 350
Pro Leu Ala Xaa Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 14
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，具有对整合重要的突变
<220>
<221>位点
<222> 560..560
<223> 其中氨基酸可以是Thr, Ala,
<220>
<221>位点
<222> 564..564
<223> 其中氨基酸可以是Ser, Pro
<220>
<221>位点
<222> 571..571
<223> 其中氨基酸可以是Asn, Ser
<220>
<221>位点
<222> 573..573
<223> 其中氨基酸可以是Ser, Ala
<220>
<221>位点
<222> 589..589
<223> 其中氨基酸可以是Met, Val
<220>
<221>位点
<222> 592..592
<223> 其中氨基酸可以是Ser, Gly
<220>
<221>位点
<222> 594..594
<223> 其中氨基酸可以是Phe, Leu
<400> 14
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Xaa
545 550 555 560
Tyr Cys Pro Xaa Lys Ile Arg Arg Lys Ala Xaa Ala Xaa Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Xaa Cys Gln Xaa
580 585 590
Cys Xaa
<210> 15
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，具有参与对齐的突变
<220>
<221>位点
<222> 325..325
<223> Xaa可以是Gly, Ala
<220>
<221>位点
<222> 347..347
<223> Xaa可以是Asn, Ala, Ser
<220>
<221>位点
<222> 350..350
<223> Xaa可以是Thr, Ala
<220>
<221>位点
<222> 465..465
<223> Xaa可以是Trp, Ala
<400> 15
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Xaa Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Xaa Trp Phe Xaa Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Xaa Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 16
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，在高度保守的氨基酸处具有突变
<220>
<221>位点
<222> 576..576
<223> Xaa可以是lys, Ala
<220>
<221>位点
<222> 587..587
<223> Xaa可以是Ile, Ala
<400> 16
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Xaa
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Xaa Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 17
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，具有参与Zn2+结合的突变
<220>
<221>位点
<222> 586..586
<223> 其中氨基酸可以是His, 任何天然存在的氨基酸
<400> 17
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Xaa Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 18
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac，具有参与整合的突变
<220>
<221>位点
<222> 315..315
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 341..341
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 372..372
<223> 其中氨基酸可以是Arg, Ala
<220>
<221>位点
<222> 375..375
<223> 其中氨基酸可以是Lys, Ala
<400> 18
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Xaa Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Xaa Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Xaa Ser Asn Xaa Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 19
<211> 897
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自溃疡棒状杆菌（Corynebacterium ulcerans）
<400> 19
Met Thr Asn Ala Val Ala Asn His His Val Leu Trp Ala Lys Phe Asp
1 5 10 15
Asn Val Ser Glu Pro Tyr Pro Leu Leu Ala His Leu Leu Asp Thr Ala
20 25 30
Thr Ala Ala Thr Cys Leu Phe Asn His Trp Leu Arg Lys Gly Leu Arg
35 40 45
Asp Arg Leu Ser Thr Glu Leu Gly Pro Asp Ala Glu Lys Ile Leu Gly
50 55 60
Phe Val Ala Gly Ile His Asp Leu Gly Lys Ala Asn Pro Tyr Phe Gln
65 70 75 80
Ala Gln Arg Arg Asn Lys Lys Glu Glu Trp Ile Thr Leu Arg Asp Ala
85 90 95
Ile Gln Lys Ala Gly Phe Pro Leu Ser Asn Gly Thr Ser Ala Leu Phe
100 105 110
Glu Glu Thr Lys Glu Lys Arg Arg His Glu Asn Ile Thr Leu Ser Ile
115 120 125
Leu Gly Trp Glu Ile Thr Lys Phe Leu Gln Val Lys Asp Val Trp Pro
130 135 140
Gln Leu Ala Ile Ile Gly His His Gly Asn Phe Ser Ala Pro Gly Phe
145 150 155 160
Leu Ser Asp Glu Asp Asp Leu Glu Asp Ile Glu Asp Ile Phe Asp Asp
165 170 175
Asn Gly Trp Ser Pro Thr His Glu Leu Leu Val Ser Ser Leu Leu Gln
180 185 190
Ala Val Gly Leu Glu Lys Gln Pro Glu Ile Lys His Ile Ser Pro Ala
195 200 205
Ser Ala Ile Leu Ile Ser Gly Leu Val Val Leu Ala Asp Arg Ile Ala
210 215 220
Ser Gln Ser Glu Met Ala Ser Asp Gly Leu Gln Ala Leu Gln Lys Glu
225 230 235 240
Glu Leu Phe Phe His Gln Pro Glu Lys Trp Ile Ala Asn Arg Lys Ala
245 250 255
Phe Cys Arg Glu Ile Ile Glu Asn Thr Val Gly Thr Tyr His Pro Trp
260 265 270
Glu Ser Glu Ala Ala Gly Ile Arg Ala Val Leu Gly Asp Tyr Glu Pro
275 280 285
Arg Phe Thr Gln Lys Ala Ala Leu Asn Ala Gly Asp Gly Leu Phe Asn
290 295 300
Val Met Glu Thr Thr Gly Ala Gly Lys Thr Glu Ala Ala Leu Leu Arg
305 310 315 320
His Val Lys Arg Lys Glu Arg Leu Leu Phe Phe Leu Pro Thr Gln Ala
325 330 335
Thr Thr Asn Ala Ile Met Asp Arg Ile Gly Lys Ile Phe Asp Gly Thr
340 345 350
Pro Asn Val Ala Ser Leu Ala His Gly Leu Ala Val Thr Glu Asp Phe
355 360 365
Tyr Ala His Pro Ile Leu Pro Val Gln Gly Ser Ser Asp Asp Ala Asn
370 375 380
Tyr Lys Asp Asn Gly Gly Leu Tyr Pro Thr Glu Phe Val Arg Ser Ala
385 390 395 400
Gly Thr Pro Arg Leu Leu Ala Pro Val Cys Val Gly Thr Ile Asp Gln
405 410 415
Ala Leu Met Gly Ala Leu Pro Ser Lys Phe Asn His Leu Arg Leu Leu
420 425 430
Ala Leu Ala Asn Ala His Val Val Val Asp Glu Val His Thr Met Asp
435 440 445
Gln Tyr Gln Ser Glu Leu Met Ser Gly Leu Leu Glu Trp Trp Ser Ala
450 455 460
Thr Asp Thr Pro Val Thr Leu Leu Thr Ala Thr Met Pro Ala Trp Gln
465 470 475 480
Arg Glu Lys Phe His Leu Ser Tyr Thr Gly Lys Asp Pro His Phe Lys
485 490 495
Gly Val Phe Pro Ser Leu Glu Asp Trp Ser Thr Pro Ser Lys Asn Thr
500 505 510
Glu Thr Ser Gln Glu Asn Ile Pro Thr Glu Ala Phe Thr Ile Pro Ile
515 520 525
Asn Ile Asp Lys Ile Ala His Asn Glu Ile Val Asp Ser His Val Gln
530 535 540
Trp Val Ile Glu Gln Arg Lys Leu Phe Pro Gln Ala Arg Ile Gly Ile
545 550 555 560
Ile Cys Asn Thr Val Gly Arg Ala Gln Ser Ile Ala Glu Ala Leu Ala
565 570 575
His Glu Ser Pro Ile Val Leu His Ser Arg Met Thr Ala Gly His Arg
580 585 590
Lys Glu Ala Ala Thr Lys Leu Glu Gln Ala Ile Gly Lys Lys Gly Thr
595 600 605
Ala Asn Ala Thr Leu Val Ile Gly Thr Gln Ala Ile Glu Ala Ser Leu
610 615 620
Asp Ile Asp Leu Asp Leu Leu Arg Thr Glu Leu Cys Pro Ala Pro Ser
625 630 635 640
Leu Ile Gln Arg Ala Gly Arg Leu Trp Arg Arg Leu Asp Pro Gln Arg
645 650 655
Glu Val Arg Val Pro Gly Met Val Gly Lys Lys Leu Thr Ile Ala Val
660 665 670
Val Asp Ser Pro Ser Thr Gly Gln Thr Leu Pro Tyr Leu Arg Ser Gln
675 680 685
Leu Tyr Arg Val Glu Ser Trp Leu Lys Gln Arg Asp Arg Ile Glu Phe
690 695 700
Pro Ala Asp Ile Gln Asp Phe Ile Asp Ala Thr Thr Pro Gly Leu Gln
705 710 715 720
Glu Leu Phe Gln Lys Val Ser Leu Pro Glu Asp Cys Gly Ser Ala Glu
725 730 735
Glu Arg Glu Ala Leu Ala Asp Asp Tyr Leu Asn Glu Val Ala Ser Trp
740 745 750
Val Thr Lys Gln Arg Gln Ala Gly Thr Ser Arg Ile Asp Phe Ala Lys
755 760 765
His Gly Lys Pro Arg Gln Val Leu Ala Ser Asp Cys Val Val Glu Asp
770 775 780
Phe Leu Gln Ile Thr Ser Ala Asn Asn Leu Glu Glu Ser Ala Thr Arg
785 790 795 800
Leu Ile Asp Tyr Pro Ser Ile Ser Ala Ile Leu Cys Asp Pro Thr Gly
805 810 815
Thr Ile Pro Gly Ala Trp Thr Asp Ser Val Glu Lys Leu Ile Ala Ile
820 825 830
Ser Ala Lys Asp Ser Glu Ser Leu Arg Arg Ala Leu Arg Ala Ser Ile
835 840 845
Ser Ile Pro His Ser Lys Lys Phe Leu Pro Ile Thr Ser Arg Glu Ile
850 855 860
Pro Leu Ser Glu Ala Lys Thr Leu Leu Ser Gly Tyr Ser Ala Val His
865 870 875 880
Ile Gln Pro Asp Glu Tyr Asp Leu Gln Ser Gly Leu Lys Gly Pro Gln
885 890 895
Lys
<210> 20
<211> 876
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自白喉棒状杆菌(Corynebacterium diphtheria)
<400> 20
Met Asn Pro His Glu Glu Leu Trp Ala Lys Gln Lys Gly Leu Ala Lys
1 5 10 15
Pro Tyr Pro Leu Leu Ala His Leu Leu Asp Ser Ala Ala Val Ala Gly
20 25 30
Ala Leu Trp Asp His Trp Leu Arg Gln Asp Leu Arg Gln Met Phe Ile
35 40 45
Glu Glu Leu Gly Ser Asn Ala Arg Glu Ile Ile Gln Phe Val Val Gly
50 55 60
Ser His Asp Ile Gly Lys Ala Thr Pro Leu Phe Gln Tyr Gln Lys Ala
65 70 75 80
Gln Lys Gly Glu Val Trp Asp Ser Ile Arg Tyr Ala Ile Asp Arg Thr
85 90 95
Gly Arg Tyr Gln Lys Pro Leu Pro Ser Ser Tyr Leu Val Lys Lys Thr
100 105 110
Ser Gly Gly Pro Asn Arg His Glu Gln Trp Ser Ser Phe Ala Ser Lys
115 120 125
Asn Glu Tyr Leu Lys Pro Ser Ala Ala Ala Lys Glu Asn Trp Ile Gly
130 135 140
Leu Ala Ile Gly Gly His His Gly Arg Phe Glu Pro Val Gly Tyr Gly
145 150 155 160
Arg His Gln Arg Lys Ala Ala Glu Asp Leu Ala Lys Ser Gly Trp Ser
165 170 175
Ala Ala Gln Gln Asp Leu Leu Arg Ala Leu Glu Lys Ala Ser Gly Ile
180 185 190
Thr Arg Ala Ser Leu Pro Ser Glu Leu Ser Pro Glu Leu Thr Leu Val
195 200 205
Leu Ser Gly Leu Thr Ile Leu Ala Asp Arg Ile Ser Ser Thr Glu Ser
210 215 220
Phe Val Ile Thr Gly Ala Arg Met Ile Asp Asp Gly Thr Leu His Leu
225 230 235 240
Ala Thr Pro Ile Asp Trp Leu Lys Thr Arg Lys Leu Asp Ser Glu Lys
245 250 255
His Val Ala Lys Thr Val Gly Ile Tyr His Gly Trp Asn Asn His Glu
260 265 270
Ser Ala Ile His Ser Ile Leu Lys Gly Tyr Asp Pro Arg Pro Leu Gln
275 280 285
Thr Ile Ala Leu Gln Asn Gln Val Gly Leu Leu Asn Leu Met Ala Pro
290 295 300
Thr Gly Asn Gly Lys Thr Glu Ala Ala Ile Leu Arg His Ser Leu Lys
305 310 315 320
Glu Asn Asp Arg Leu Ile Phe Leu Leu Pro Thr Gln Ala Thr Ser Asn
325 330 335
Ala Ile Met Arg Arg Val Gln Gly Ile Tyr Ser Asp Thr Pro Asn Ala
340 345 350
Ala Ala Leu Ala His Ser Leu Ala Ser Val Glu Asp Phe Tyr Gln Thr
355 360 365
Pro Leu Ser Val Phe Asp Asp His Tyr Asp Pro Ser Lys Glu Gln Phe
370 375 380
Glu Ser Ser Met Ser Gly Gly Leu Tyr Pro Ser Ser Phe Val Cys Ser
385 390 395 400
Gly Ala Ala Arg Leu Leu Ala Pro Ile Cys Ile Gly Thr Val Asp Gln
405 410 415
Ala Leu Ala Thr Ala Leu Pro Gly Lys Trp Ile His Leu Arg Ile Leu
420 425 430
Ala Leu Ala Asn Ala His Ile Val Ile Asp Glu Val His Thr Leu Asp
435 440 445
His Tyr Gln Thr Ala Leu Leu Glu Asn Ile Leu Pro Ile Leu Ala Lys
450 455 460
Leu Lys Thr Lys Ile Thr Phe Leu Thr Ala Thr Met Pro Ser Trp Gln
465 470 475 480
Arg Thr Lys Leu Leu Thr Ala Tyr Gly Gly Glu Asp Leu Gln Ile Pro
485 490 495
Pro Thr Val Phe Pro Ala Ala Glu Thr Val Leu Pro Gly Gln Phe Asn
500 505 510
Arg Thr Leu Ile Asp Ser Asp Ser Thr Thr Ile Asp Phe Thr Met Glu
515 520 525
Glu Thr Ser Tyr Asp His Leu Val Glu Ser His Val Lys Trp His Gln
530 535 540
Thr Thr Arg Leu Asn Ala Pro His Ala Arg Ile Gly Leu Ile Cys Asn
545 550 555 560
Thr Val Lys Arg Ala Gln Glu Ile Ala Ala Ala Leu Glu Lys Thr Asn
565 570 575
Asp Arg Ile Val Leu Leu His Ser Arg Met Thr Thr Glu His Arg Arg
580 585 590
Arg Ser Ala Glu Leu Leu Glu Ser Leu Leu Gly Pro Asn Gly Asn Arg
595 600 605
Lys Thr Ile Thr Val Val Gly Thr Gln Ala Ile Glu Ala Ser Leu Asp
610 615 620
Ile Asp Leu Asp Ile Leu Arg Thr Glu Leu Cys Pro Ala Pro Ser Leu
625 630 635 640
Val Gln Arg Ala Gly Arg Val Trp Arg Arg Asn Asp Pro Tyr Arg Ser
645 650 655
Ser Arg Ile Thr Ala Asp His Lys Pro Ile Ser Val Val Phe Ile Ala
660 665 670
Glu Ala Lys Asp Trp Gln Val Leu Pro Tyr Leu Arg Ala Glu Thr Ser
675 680 685
Arg Thr Gln Arg Trp Leu Glu Lys His Asn Gln Met Phe Leu Pro Gln
690 695 700
Met Ala Gln Glu Phe Ile Asp Ala Ala Thr Val Asp Leu Asp Thr Ala
705 710 715 720
Thr Ser Glu Met Asp Leu Asp Ala Leu Ala Leu Met Gly Ile His Leu
725 730 735
Met Lys Ala Asp Gly Ala Lys Ala Arg Ile Gln Asp Val Leu Asn Ser
740 745 750
Asp Ser Lys Val Ser Asp Phe Ala Leu Leu Thr Ser Lys Asn Glu Ile
755 760 765
Asp Glu Ala Gln Thr Arg Leu Ile Glu Glu Gly Thr His Leu Arg Ile
770 775 780
Ile Leu Gly Asp Glu Asn Glu Ser Ile Pro Gly Gly Trp Lys His Gly
785 790 795 800
Leu Ser Ser Leu Leu Lys Leu Lys Ala Ser Asp Arg Glu Ser Leu Arg
805 810 815
Thr Ala Leu Leu Ala Ser Ile Pro Leu Leu Val Ser Glu Lys Gln Lys
820 825 830
Gln Leu Leu Tyr Gln His Asn Leu Val Pro Leu Ser Ser Ser Lys Thr
835 840 845
Val Leu Ala Gly Phe Tyr Phe Leu Pro Lys Ala Gln Asn Phe Tyr Ser
850 855 860
Lys Asn Leu Gly Phe Ile Trp Pro Glu Glu Lys Asp
865 870 875
<210> 21
<211> 773
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自梅毒螺原体（Spiroplasma syrphidicola）
<400> 21
Met Asn Tyr Lys Lys Leu Ile Leu Gly Leu Asp Leu Gly Ile Ala Ser
1 5 10 15
Cys Gly Trp Ala Val Thr Gly Gln Met Glu Asp Gly Asn Trp Val Leu
20 25 30
Asp Asp Phe Gly Val Arg Leu Phe Gln Thr Pro Glu Asn Ser Lys Asp
35 40 45
Gly Thr Thr Asn Ala Ala Ala Arg Arg Leu Lys Arg Gly Ala Arg Arg
50 55 60
Leu Ile Lys Arg Arg Lys Asn Arg Ile Lys Asp Leu Lys Asn Leu Phe
65 70 75 80
Glu Lys Ile Asn Phe Ile Asn Lys Ala Ser Leu Asp Lys Tyr Ile Asn
85 90 95
Glu His Ser Ala Thr Asn Leu Val Glu Asp Phe Asn Arg His Glu Leu
100 105 110
Tyr Asn Pro Tyr Phe Leu Arg Ser Ile Gly Ile Thr Glu Lys Leu Thr
115 120 125
Arg Glu Glu Leu Val Trp Ser Leu Ile His Ile Ala Asn Arg Arg Gly
130 135 140
Tyr Lys Asn Lys Phe Ala Phe Asp Ile Glu Gly Asp Gly Lys Lys Arg
145 150 155 160
Glu Thr Lys Leu Asp Glu Ala Ile Ser Asn Ala Leu Ile Ser Ser Asn
165 170 175
Leu Thr Ile Ser Gln Glu Ile Val Arg Asn Lys Lys Phe Arg Asp Ala
180 185 190
Lys Asn Lys Lys Ala Leu Leu Val Arg Asn Lys Gly Gly Lys Glu Gly
195 200 205
Glu Asn Asn Phe Gln Phe Leu Phe Ala Arg Asp Asp Tyr Lys Lys Glu
210 215 220
Val Asp Leu Leu Leu Ala Lys Gln Ala Lys Phe Tyr Pro Glu Leu Thr
225 230 235 240
Glu Glu Ile Arg Ala Lys Ala Ala Asp Ile Ile Phe Arg Gln Arg Asp
245 250 255
Phe Glu Asp Gly Pro Gly Pro Lys Lys Gln Glu Leu Arg Glu Ile Tyr
260 265 270
Lys Lys Glu Asn Lys Gln Phe Ser Lys Asn Phe Thr Gln Leu Glu Gly
275 280 285
Arg Cys Thr Phe Leu Arg Glu Leu Ser Val Gly Tyr Lys Ser Ser Ile
290 295 300
Leu Phe Asp Leu Phe His Ile Ile Ser Glu Val Ser Lys Ile Ser Lys
305 310 315 320
Tyr Ile Glu Glu Asn Asp Gln Leu Ala Gln Asp Ile Ile Ser Ser Phe
325 330 335
Leu Tyr Asn Glu Ala Gly Lys Lys Gly Lys Thr Leu Leu Lys Glu Ile
340 345 350
Leu Lys Lys His His Ile Asn Asp Asp Ile Phe Asp Thr Asn Ala Tyr
355 360 365
Lys Asn Ile Asp Phe Lys Thr Asn Tyr Leu Asn Leu Leu Lys Glu Val
370 375 380
Phe Gly Asn Asp Val Leu Lys Asn Leu Ser Leu Asn Arg Leu Glu Asp
385 390 395 400
Asn Ile Tyr His Gln Leu Gly Phe Ile Ile His Thr Asn Ile Thr Pro
405 410 415
Glu Arg Lys Glu Lys Ala Ile Asn Gln Trp Leu Leu Glu Asn Asn Ile
420 425 430
Ile Leu Ala Lys Glu Lys Leu Asn Ile Leu Leu Lys Pro Asn Ser Ser
435 440 445
Ile Ser Thr Thr Val Lys Thr Ser Phe Lys Trp Met Ser Ile Ala Ile
450 455 460
Ser Asn Phe Leu Lys Gly Ile Pro Tyr Gly Lys Phe Gln Ala Gln Phe
465 470 475 480
Ile Lys Glu Asp Asn Phe Lys Leu Pro Glu Ser Tyr Ala Lys Gln Tyr
485 490 495
Gln Lys Tyr Leu Thr Gly Glu Lys Thr Phe Glu Met Phe Ala Pro Ile
500 505 510
Ile Asp Pro Asp Leu Trp Arg Asn Pro Ile Val Phe Arg Ala Ile Asn
515 520 525
Gln Ala Arg Lys Val Ile Lys Lys Leu Phe Glu Lys Tyr Thr Phe Ile
530 535 540
Asp Gln Ile Asn Ile Glu Leu Thr Arg Glu Met Gly Leu Ser Phe Ser
545 550 555 560
Asp Arg Lys Lys Val Lys Glu Arg Gln Asp Asp Ser Leu Lys Glu Asn
565 570 575
Ala Lys Ala Lys Glu Phe Leu Met Ala Asn Gly Ile Ile Val Asn Asp
580 585 590
Thr Asn Val Leu Lys Tyr Lys Leu Trp Ile Gln Gln Asn Lys Lys Ser
595 600 605
Leu Tyr Ser Gly Lys Glu Ile Thr Ile Ala Asp Leu Gly Ala Ser Asn
610 615 620
Val Leu Gln Ile Asp His Ile Ile Pro Tyr Ser Lys Leu Ala Asp Asp
625 630 635 640
Ser Phe Asn Asn Lys Val Leu Val Phe Ser Lys Glu Asn Gln Glu Lys
645 650 655
Gly Asn Gln Phe Ala Asp Gln Tyr Val Lys Ser Leu Gly Thr Glu Asn
660 665 670
Tyr Asn Asn Tyr Lys Lys Arg Val Asn Tyr Leu Leu Phe Gln Asn Gln
675 680 685
Ile Asn Gln Lys Lys Ala Glu Tyr Leu Leu Cys Ser Asn Gln Asn Glu
690 695 700
Glu Ile Leu Asn Asp Phe Val Ser Arg Asn Leu Asn Asp Thr Arg Tyr
705 710 715 720
Ile Thr Arg Tyr Val Thr Asn Trp Leu Lys Ala Glu Phe Glu Leu Gln
725 730 735
Ser Arg Phe Gly Leu Ala Lys Pro Lys Ile Met Thr Leu Asn Gly Ala
740 745 750
Ile Thr Ser Arg Phe Arg Arg Thr Trp Leu Arg Asn Ser Pro Trp Gly
755 760 765
Leu Glu Lys Lys Ser
770
<210> 22
<211> 1380
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自中间普氏菌（Prevotella intermedia）
<400> 22
Met Lys Arg Ile Leu Gly Leu Asp Leu Gly Thr Thr Ser Ile Gly Trp
1 5 10 15
Ala Leu Val Asn Glu Ala Glu Asn Asn Asn Glu Ala Ser Ser Ile Val
20 25 30
Arg Leu Gly Val Arg Val Asn Pro Leu Thr Val Asp Glu Lys Ser Asn
35 40 45
Phe Glu Lys Gly Lys Ala Ile Thr Thr Asn Ala Asp Arg Gln Leu Arg
50 55 60
His Gly Ala Arg Ile Asn Leu Gln Arg Tyr Lys Leu Arg Arg Gln Asn
65 70 75 80
Leu His Asp Cys Leu Gln Lys Gln Gly Trp Leu Gly Thr Glu Ala Met
85 90 95
Tyr Glu Glu Gly Lys Ala Ser Thr Phe Glu Thr Tyr Lys Leu Arg Ala
100 105 110
Lys Ala Ala Glu Glu Glu Ile Ser Leu His Glu Phe Ala Arg Val Leu
115 120 125
Phe Met Leu Asn Lys Lys Arg Gly Tyr Lys Ser Asn Arg Lys Ala Asn
130 135 140
Asn Lys Glu Asp Gly Gln Leu Phe Asp Gly Met Thr Ile Ala Lys Lys
145 150 155 160
Leu Tyr Glu Glu His Leu Thr Pro Ala Glu Tyr Ser Leu Gln Leu Leu
165 170 175
Asn Lys Gly Lys Lys Phe Thr Gln Gly Tyr Tyr Arg Ser Asp Leu Asn
180 185 190
Ala Glu Leu Glu Arg Ile Trp Asp Glu Gln Lys Lys Tyr Tyr Pro Glu
195 200 205
Ile Leu Thr Asp Glu Phe Lys Gln Gln Leu Glu Gly Lys Thr Lys Thr
210 215 220
Asn Thr Ser Lys Ile Phe Leu Ala Lys Tyr Gly Ile Tyr Ser Ala Asp
225 230 235 240
Leu Lys Gly Leu Asp Arg Lys Phe Gln Pro Leu Lys Trp Arg Val Glu
245 250 255
Ala Leu Gln Gln Gln Val Asp Lys Glu Val Leu Ala Phe Val Ile Ser
260 265 270
Asp Leu Lys Gly Gln Ile Ala Asn Thr Ser Gly Leu Leu Gly Ala Ile
275 280 285
Ser Asp Arg Ser Lys Glu Leu Tyr Phe Asn Lys Gln Thr Val Gly Gln
290 295 300
Tyr Leu Trp Ala Ser Leu Glu Glu Asn Pro His Ile Ser Ile Lys Asn
305 310 315 320
Lys Pro Phe Tyr Arg Gln Asp Tyr Leu Asp Glu Phe Glu Lys Ile Trp
325 330 335
Glu Thr Gln Ala Ala Phe His Lys Gln Leu Thr Pro Glu Leu Lys Gln
340 345 350
Glu Ile Arg Asp Ile Ile Ile Phe Tyr Gln Arg Pro Leu Lys Ser Lys
355 360 365
Lys Ser Leu Ile Ser Val Cys Glu Leu Glu Gln Arg Lys Val Lys Ala
370 375 380
Thr Ile Asp Gly Lys Glu Lys Glu Ile Thr Ile Gly Pro Lys Val Ala
385 390 395 400
Pro Lys Ser Ser Pro Val Phe Gln Glu Phe Arg Ile Trp Gln Asn Leu
405 410 415
Asn Asn Val Leu Leu Ile Asp Asn Asp Thr Asn Glu Lys Arg Pro Leu
420 425 430
Asp Glu Val Glu Arg Asn Leu Leu Tyr Lys Glu Leu Ser Ile Lys Ala
435 440 445
Lys Leu Ser Lys Thr Glu Ala Leu Lys Ile Leu Asn Lys Lys Gly Lys
450 455 460
Gln Trp Asp Leu Asn Tyr Arg Glu Leu Glu Gly Asn Arg Thr Gln Ala
465 470 475 480
Ile Leu Phe Asp Cys Tyr Asn Arg Ile Ile Thr Leu Thr Gly His Glu
485 490 495
Glu Cys Asp Phe Lys Lys Ile Lys Ala Ser Glu Ile Arg His Tyr Val
500 505 510
Ser Thr Ile Phe Lys Asn Leu Gly Phe Ser Thr Glu Ile Leu Asp Phe
515 520 525
Asp Pro Ser Leu Lys Lys His Glu Leu Glu Lys Gln Pro Met Tyr Gln
530 535 540
Leu Trp His Leu Leu Tyr Ser Tyr Glu Ser Asp Asn Ser Arg Thr Gly
545 550 555 560
Asn Glu Ser Leu Leu Arg Lys Leu Glu Thr Thr Phe Gly Phe Pro Glu
565 570 575
Glu Tyr Ala Thr Val Leu Cys Asp Val Val Phe Glu Glu Asp Tyr Gly
580 585 590
Asn Leu Ser Val Lys Ala Met Arg Glu Ile Leu Pro Tyr Leu Gln Ala
595 600 605
Gly Asn Asp Tyr Ser Gln Ala Cys Ala Tyr Ala Gly Tyr Asn His Ser
610 615 620
Arg His Ser Leu Thr Lys Glu Glu Leu Asp Gln Lys Val Tyr Lys Glu
625 630 635 640
Arg Leu Glu Leu Leu Pro Lys Asn Ser Leu Arg Asn Pro Val Val Glu
645 650 655
Lys Ile Leu Asn Gln Met Ile Asn Val Ile Asn Ala Ile Ile Asp Glu
660 665 670
Tyr Gly Lys Pro Asp Glu Ile Arg Ile Glu Met Ala Arg Glu Leu Lys
675 680 685
Ser Ser Ala Ala Asp Arg Lys Lys Thr Thr His Ala Ile Ser Gln Gly
690 695 700
Asn Ala Glu Asn Gln Arg Ile Arg Glu Ile Leu Glu Lys Glu Phe Ser
705 710 715 720
Leu Ser Tyr Ile Ser Arg Asn Asp Ile Ile Lys Tyr Lys Leu Tyr Glu
725 730 735
Glu Leu Glu Pro Asn Tyr Tyr Lys Thr Leu Tyr Ser Asp Thr Tyr Ile
740 745 750
Thr Lys Asp Lys Leu Phe Ser Lys Asp Phe Asp Ile Glu His Ile Ile
755 760 765
Pro Lys Ala Arg Leu Phe Asp Asp Ser Phe Ser Asn Lys Thr Leu Glu
770 775 780
Ala Arg Asn Ile Asn Leu Glu Lys Ser Asn Lys Thr Ala Phe Asp Phe
785 790 795 800
Ile Lys Glu Lys Tyr Gly Glu Asp Gly Ala Glu Ala Tyr Lys Lys Lys
805 810 815
Leu Asp Met Leu Leu Glu Asn Asp Ala Ile Ser Arg Pro Lys Tyr Asn
820 825 830
Asn Leu Leu Arg Ala Glu Ala Asp Ile Pro Ser Asp Phe Ile Asn Arg
835 840 845
Asp Leu Arg Asn Thr Gln Tyr Ile Ala Lys Lys Ala Cys Glu Ile Leu
850 855 860
Gly Glu Leu Val Lys Thr Val Thr Pro Thr Thr Gly Lys Ile Thr Asn
865 870 875 880
Arg Leu Arg Glu Asp Trp Gln Leu Val Asp Val Met Lys Glu Leu Asn
885 890 895
Phe Glu Lys Tyr Glu Lys Leu Gly Leu Thr Glu Ile Val Glu Asp Arg
900 905 910
Asp Gly Arg Lys Ile Lys Arg Ile Lys Asp Trp Thr Lys Arg Asn Asp
915 920 925
His Arg His His Ala Met Asp Ala Leu Ala Ile Ala Phe Thr Lys Pro
930 935 940
Ser Phe Ile Gln Tyr Leu Asn Asn Leu Asn Ala Arg Ser Asn Lys Gly
945 950 955 960
Asp Ser Ile Tyr Ala Ile Glu Asn Lys Glu Leu His Tyr Glu Glu Gly
965 970 975
Lys Leu Arg Phe Asn Ala Pro Ile Pro Val Asn Glu Phe Arg Ala Glu
980 985 990
Ala Lys Arg His Leu Ser Ala Ile Leu Val Ser Ile Lys Ala Lys Asn
995 1000 1005
Lys Val Met Thr Gln Asn Val Asn Lys Ile Lys Thr Lys His Gly Ile
1010 1015 1020
Ile Lys Lys Ile Gln Leu Thr Pro Arg Gly Pro Leu His Asn Glu Thr
1025 1030 1035 1040
Ile Tyr Gly Thr Lys Met Arg Pro Ile Ile Lys Met Val Lys Val Gly
1045 1050 1055
Ala Ala Leu Asp Glu Ala Thr Ile Asn Lys Val Ser Ser Pro Ala Ile
1060 1065 1070
Arg Glu Ala Leu Leu Lys Arg Leu Asn Glu Tyr Ser Gly Asn Ala Lys
1075 1080 1085
Lys Ala Phe Thr Gly Lys Asn Thr Leu Glu Lys Asn Pro Ile Tyr Leu
1090 1095 1100
Asn Ala Gly Arg Thr Lys Thr Val Pro Ser Leu Val Lys Thr Val Glu
1105 1110 1115 1120
Trp Glu Ser Phe His Pro Thr Arg Lys Leu Ile Asp Lys Asp Leu Asn
1125 1130 1135
Val Asp Lys Val Val Asp Lys Gly Ile Arg Glu Ile Leu Lys Ala Arg
1140 1145 1150
Leu Glu Glu Phe Asn Gly Asp Ala Lys Lys Ala Phe Ser Asn Leu Glu
1155 1160 1165
Glu Asn Pro Ile Tyr Leu Asp Glu Ala Lys Lys Ile Ala Leu Lys Arg
1170 1175 1180
Val Ser Ile Glu Gly Val Leu Ser Ala Ile Pro Leu His Thr Leu Lys
1185 1190 1195 1200
Asn Gln Ala Gly Lys Pro Ile Thr Gly Lys Asp Gly Lys Pro Val Leu
1205 1210 1215
Gly Asn Tyr Val Gln Thr Ser Asn Asn His His Ile Ala Phe Tyr Tyr
1220 1225 1230
Asp Glu Asp Gly Asn Leu Gln Asp Asn Ala Val Ser Phe Phe Glu Ala
1235 1240 1245
Ala Glu Arg Lys Ser Gln Gly Ile Pro Val Ile Asp Lys Asp Tyr Asn
1250 1255 1260
Arg Asp Lys Gly Trp Arg Phe Leu Phe Thr Met Lys Gln Asn Glu Tyr
1265 1270 1275 1280
Phe Val Phe Pro Asn Glu Ala Thr Gly Phe Ile Pro Ser Glu Val Asp
1285 1290 1295
Leu Thr Asp Glu Ala Asn Tyr Gly Ile Ile Ser Pro Asn Leu Tyr Arg
1300 1305 1310
Val Gln Lys Val Ser Arg Ile Asp Lys Gly Thr Ser Ala Ser Arg Asp
1315 1320 1325
Tyr Trp Phe Arg His His Leu Glu Thr Ile Leu Asn Asp Asp Ala Lys
1330 1335 1340
Leu Lys Asn Leu Ala Phe Lys Arg Ile Arg Gly Leu Leu Glu Leu Lys
1345 1350 1355 1360
Asp Ile Ile Lys Val Arg Ile Asn Ser Thr Gly Lys Ile Val Ala Val
1365 1370 1375
Gly Glu Tyr Asp
1380
<210> 23
<211> 535
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自台湾螺原体（Spiroplasma taiwanense）
<400> 23
Met Trp Ser Arg Lys Ile Leu Lys Ala Gly Ser Arg Leu Phe Asp Glu
1 5 10 15
Ala Asn Leu Ser Asp Lys Ile Ala Ser Lys Arg Arg Glu Gln Arg Gly
20 25 30
Arg Arg Arg Asn Leu Arg Arg Lys Ile Thr Trp Lys Gln Asp Leu Ile
35 40 45
Asn Leu Phe Val Lys Tyr Asn Phe Leu Gln Lys Glu Asn Asp Phe Tyr
50 55 60
Glu Leu Asp Phe Asn Phe Asp Leu Leu Glu Leu Arg Lys Lys Ala Ile
65 70 75 80
Asn Ser Lys Ile Glu Leu Glu Gln Leu Leu Ile Ile Leu Phe Asn Tyr
85 90 95
Ile Lys His Arg Gly Ser Phe Asn Tyr Arg Glu Asp Leu Ser Glu Leu
100 105 110
Lys Asn Ile Ser Gln Glu Glu Leu Glu Thr Ser Ser Glu Phe Lys Leu
115 120 125
Pro Val Asp Ile Gln Phe Glu Leu Lys Glu Glu Asn Asn Lys Phe Arg
130 135 140
Glu Ile Asn Asn Glu Lys Ser Leu Ile Asn His Glu Trp Tyr Val Lys
145 150 155 160
Glu Ile Asn Leu Ile Leu Asp Ala Gln Ile Glu Asn Lys Leu Ile Asn
165 170 175
Leu Asp Phe Lys Lys Asp Tyr Leu Lys Leu Phe Asn Arg Lys Arg Glu
180 185 190
Tyr Tyr Asp Gly Pro Gly Pro Lys Asp Lys Asn Leu Leu Asn Pro Ser
195 200 205
Lys Tyr Gly Trp Lys Asn Gln Glu Glu Phe Phe Asp Arg Phe Ala Gly
210 215 220
Lys Asp Thr Tyr Asp Ser Lys Glu Gln Arg Ala Pro Lys His Ser Leu
225 230 235 240
Thr Ser Tyr Leu Phe Asn Ile Leu Asn Asp Leu Asn Asn Leu Ser Ile
245 250 255
Asn Gly Asp Arg Asn Gln Leu Thr Tyr Glu Asn Lys Lys Asp Leu Ile
260 265 270
Asn Leu Thr Leu Ile Asn Gln Lys Glu Lys Ala Glu Asn Ile Thr Leu
275 280 285
Lys Lys Ile Ala Lys Tyr Leu Lys Ile Asn Glu Lys Asn Ile Thr Gly
290 295 300
Tyr Arg Leu Lys Pro Asn Ser Asn Glu Ser Ile Phe Thr Val Phe Glu
305 310 315 320
Ser Ala Asn Lys Met Arg Ser Ile Leu Val Lys Asn Asn Lys Ser Ile
325 330 335
Asp Phe Ile Cys Leu Glu Asn Ile Asp Lys Ile Asp Lys Ile Val Asp
340 345 350
Ile Leu Thr Lys Tyr Gln Ser Ile Glu Asp Lys Ser Leu Lys Leu Glu
355 360 365
Glu Leu Asn Phe Asp Phe Phe Asp Lys Glu Thr Cys Glu Lys Leu Ala
370 375 380
Val Ile Ser Leu Thr Gly Thr His Ala Leu Ser Lys Lys Thr Met Ser
385 390 395 400
Lys Leu Ile Glu Glu Met Phe His Asp Asn Leu Asn His Met Glu Ala
405 410 415
Leu Ala Lys Leu Lys Ile Lys Pro Asp Tyr Lys Leu Lys Val Asp Leu
420 425 430
Thr Asn Phe Lys Thr Ile Pro Ile Leu Arg Glu Lys Ile Asn Glu Met
435 440 445
Tyr Ile Ser Pro Val Val Lys Arg Ala Leu Ile Glu Ser Leu Lys Ile
450 455 460
Ile Lys Glu Leu Glu Arg His Phe Lys Asp Phe Glu Ile Lys Asp Ile
465 470 475 480
Val Ile Glu Met Ala Lys Lys Asn Ser Ala Glu Lys Lys Gln Phe Ile
485 490 495
Ser Lys Ile Gln Arg Gln Asn Val Asp Leu Val Lys Lys Leu Ser Asn
500 505 510
Asp Tyr Ser Leu Asp Glu Asn Lys Leu Asn Phe Lys Met Lys Glu Lys
515 520 525
Phe Leu Leu Leu Ser Glu Gln
530 535
<210> 24
<211> 1281
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自海豚链球菌（Streptococcus iniae）
<400> 24
Met Arg Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Met
20 25 30
Arg Ile Gln Gly Thr Thr Asp Arg Thr Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Asn Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Thr Arg Arg Arg Tyr Thr Arg Arg Lys Tyr Arg Ile Lys
65 70 75 80
Glu Leu Gln Lys Ile Phe Ser Ser Glu Met Asn Glu Leu Asp Ile Ala
85 90 95
Phe Phe Pro Arg Leu Ser Glu Ser Phe Leu Val Ser Asp Asp Lys Glu
100 105 110
Phe Glu Asn His Pro Ile Phe Gly Asn Leu Lys Asp Glu Ile Thr Tyr
115 120 125
His Asn Asp Tyr Pro Thr Ile Tyr His Leu Arg Gln Thr Leu Ala Asp
130 135 140
Ser Asp Gln Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Ile Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asn Leu Asp Ser
165 170 175
Glu Asn Thr Asp Val His Val Leu Phe Leu Asn Leu Val Asn Ile Tyr
180 185 190
Asn Asn Leu Phe Glu Glu Asp Ile Val Glu Thr Ala Ser Ile Asp Ala
195 200 205
Glu Lys Ile Leu Thr Ser Lys Thr Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Glu Ile Pro Asn Gln Lys Arg Asn Met Leu Phe Gly Asn
225 230 235 240
Leu Val Ser Leu Ala Leu Gly Leu Thr Pro Asn Phe Lys Thr Asn Phe
245 250 255
Glu Leu Leu Glu Asp Ala Lys Leu Gln Ile Ser Lys Asp Ser Tyr Glu
260 265 270
Glu Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Ile Ala Ala Lys Lys Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Ile Thr Val Lys Gly Ala Ser Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Val Gln Arg Tyr Glu Glu His Gln Gln Asp Leu Ala Leu Leu Lys
325 330 335
Asn Leu Val Lys Lys Gln Ile Pro Glu Lys Tyr Lys Glu Ile Phe Asp
340 345 350
Asn Lys Glu Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Lys Thr Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Tyr Ile Lys Pro Ile Leu Leu Lys Leu Asp
370 375 380
Gly Thr Glu Lys Leu Ile Ser Lys Leu Glu Arg Glu Asp Phe Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Asn Glu Leu Lys Ala Ile Ile Arg Arg Gln Glu Lys Phe Tyr Pro Phe
420 425 430
Leu Lys Glu Asn Gln Lys Lys Ile Glu Lys Leu Phe Thr Phe Lys Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Asn Gly Gln Ser Ser Phe Ala Trp
450 455 460
Leu Lys Arg Gln Ser Asn Glu Ser Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Gln Glu Ala Ser Ala Arg Ala Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Thr Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys His Ser
500 505 510
Pro Leu Tyr Glu Met Phe Met Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Gln Thr Glu Gly Met Lys Arg Pro Val Phe Leu Ser Ser Glu Asp
530 535 540
Lys Glu Glu Ile Val Asn Leu Leu Phe Lys Lys Glu Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Glu Tyr Phe Ser Lys Met Lys Cys Phe His
565 570 575
Thr Val Thr Ile Leu Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Phe Lys Asp Lys Ala Phe Leu Asp
595 600 605
Asp Glu Ala Asn Gln Asp Ile Leu Glu Glu Ile Val Trp Thr Leu Thr
610 615 620
Leu Phe Glu Asp Gln Ala Met Ile Glu Arg Arg Leu Val Lys Tyr Ala
625 630 635 640
Asp Val Phe Glu Lys Ser Val Leu Lys Lys Leu Lys Lys Arg His Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Gln Lys Leu Ile Asn Gly Ile Lys Asp
660 665 670
Lys Gln Thr Gly Lys Thr Ile Leu Gly Phe Leu Lys Asp Asp Gly Val
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile Asn Asp Ser Ser Leu Asp Phe
690 695 700
Ala Lys Ile Ile Lys Asn Glu Gln Glu Lys Thr Ile Lys Asn Glu Ser
705 710 715 720
Leu Glu Glu Thr Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys
725 730 735
Gly Ile Leu Gln Ser Ile Lys Ile Val Asp Glu Ile Val Lys Ile Met
740 745 750
Gly Gln Asn Pro Asp Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Ser Thr Met Gln Gly Ile Lys Asn Ser Arg Gln Arg Leu Arg Lys Leu
770 775 780
Glu Glu Val His Lys Asn Thr Gly Ser Lys Ile Leu Lys Glu Tyr Asn
785 790 795 800
Val Ser Asn Thr Gln Leu Gln Ser Asp Arg Leu Tyr Leu Tyr Leu Leu
805 810 815
Gln Asp Gly Lys Asp Met Tyr Thr Gly Lys Glu Leu Asp Tyr Asp Asn
820 825 830
Leu Ser Gln Tyr Asp Ile Asp His Ile Ile Pro Gln Ser Phe Ile Lys
835 840 845
Asp Asn Ser Ile Asp Asn Thr Val Leu Thr Thr Gln Ala Ser Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Asn Ile Glu Thr Val Asn Lys Met Lys
865 870 875 880
Ser Phe Trp Tyr Lys Gln Leu Lys Ser Gly Ala Ile Ser Gln Arg Lys
885 890 895
Phe Asp His Leu Thr Lys Ala Glu Arg Gly Ala Leu Ser Asp Phe Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Phe Asn Ser Asn Leu Thr
930 935 940
Glu Asp Ser Lys Ser Asn Arg Asn Val Lys Ile Ile Thr Leu Lys Ser
945 950 955 960
Lys Met Val Ser Asp Phe Arg Lys Asp Phe Gly Phe Tyr Lys Leu Arg
965 970 975
Glu Val Asn Asp Tyr His His Ala Gln Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Leu Lys Lys Tyr Pro Lys Leu Glu Ala Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys His Tyr Asp Leu Ala Lys Leu Met Ile Gln
1010 1015 1020
Pro Asp Ser Ser Leu Gly Lys Ala Thr Thr Arg Met Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Leu Met Asn Phe Phe Lys Lys Glu Ile Lys Leu Ala Asp Asp Thr
1045 1050 1055
Ile Phe Thr Arg Pro Gln Ile Glu Val Asn Thr Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Val Lys Asp Met Gln Thr Ile Arg Lys Val Met Ser
1075 1080 1085
Tyr Pro Gln Val Asn Ile Val Met Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Trp Pro Lys Gly Asp Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Ser Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Ile Ile Ala Tyr Ser Val Leu Val Val Ala Lys Ile Ala Lys Gly
1140 1145 1150
Lys Thr Gln Lys Leu Lys Thr Ile Lys Glu Leu Val Gly Ile Lys Ile
1155 1160 1165
Met Glu Gln Asp Glu Phe Glu Lys Asp Pro Ile Ala Phe Leu Glu Lys
1170 1175 1180
Lys Gly Tyr Gln Asp Ile Gln Thr Ser Ser Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Leu Leu Ala Ser
1205 1210 1215
Ala Lys Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Asn Lys Tyr
1220 1225 1230
Val Lys Phe Leu Tyr Leu Ala Ser His Tyr Thr Lys Phe Thr Gly Lys
1235 1240 1245
Glu Glu Asp Arg Glu Lys Lys Arg Ser Tyr Val Glu Ser His Leu Tyr
1250 1255 1260
Tyr Phe Asp Val Arg Leu Ser Gln Val Phe Arg Val Thr Asn Val Glu
1265 1270 1275 1280
Phe
<210> 25
<211> 1352
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自Belliella baltica
<400> 25
Met Lys Lys Ile Leu Gly Leu Asp Leu Gly Thr Thr Ser Ile Gly Trp
1 5 10 15
Ala Phe Ile Lys Glu Pro Glu Lys Asp Val Val Gly Ser Glu Ile Val
20 25 30
Asp Met Gly Val Arg Ile Val Pro Leu Ser Ser Asp Glu Glu Asn Asp
35 40 45
Phe Ala Lys Gly Asn Thr Ile Ser Ile Asn Ala Asp Arg Thr Leu Lys
50 55 60
Arg Gly Ala Arg Arg Asn Leu Gln Arg Phe Lys Gln Arg Arg Asn Ala
65 70 75 80
Leu Leu Glu Ile Phe Lys Glu Lys Lys Leu Ile Ser Thr Asn Phe Lys
85 90 95
Tyr Ala Glu Asp Gly Pro Ser Ser Thr Phe Ser Thr Leu Asn Leu Arg
100 105 110
Ala Lys Ala Ala Lys Glu Lys Ile Glu Leu Gln Asp Leu Val Lys Val
115 120 125
Leu Leu Gln Ile Asn Lys Lys Arg Gly Tyr Lys Ser Ser Arg Lys Ala
130 135 140
Lys Ser Glu Glu Asp Asp Gly Ser Ala Ile Asp Ser Met Gly Ile Ala
145 150 155 160
Lys Glu Leu Tyr Glu Asn Asp Leu Thr Pro Gly Gln Trp Val Tyr Glu
165 170 175
Ala Leu Gln Lys Gly Arg Lys Asn Val Pro Asp Phe Tyr Arg Ser Asp
180 185 190
Leu Gln Glu Glu Phe Lys Lys Ile Val Asn Tyr Gln Ser Glu Phe Phe
195 200 205
Pro Asp Ile Phe Asn Ala Ser Phe Val Glu Asp Trp Met Gly Lys Ala
210 215 220
Ser Thr Pro Thr Lys Gln Tyr Phe Asn Lys Lys Gly Val Gln Leu Ala
225 230 235 240
Glu Asn Lys Gly Lys Arg Glu Glu Arg Arg Leu Gln Glu Tyr Lys Trp
245 250 255
Arg Ala Glu Ala Val Asn Phe Lys Ile Asp Leu Ser Glu Ile Ala Leu
260 265 270
Ile Leu Ser Gln Ile Asn Ser Gln Ile Ser Asn Ser Ser Gly Tyr Leu
275 280 285
Gly Ala Ile Ser Asp Arg Ser Lys Glu Leu Tyr Phe Lys Asn Leu Thr
290 295 300
Val Gly Gln Tyr Leu Tyr Gln Gln Ile Lys Lys Asn Pro His Thr Arg
305 310 315 320
Leu Lys Gly Gln Val Phe Tyr Arg Gln Asp Tyr Leu Asp Glu Phe Glu
325 330 335
Arg Ile Trp Ser Val Gln Ser Ser Phe Tyr Pro Gln Leu Asn Asp Ala
340 345 350
Leu Lys Arg Glu Val Arg Asp Ile Thr Ile Phe Phe Gln Arg Arg Leu
355 360 365
Lys Ser Gln Lys His Leu Ile Ser Asn Cys Glu Phe Glu Asp His His
370 375 380
Lys Val Val Pro Lys Ser His Pro Val Phe Gln Glu Phe Arg Ile Trp
385 390 395 400
Gln Asn Leu Asn Asn Leu Leu Leu Ile Lys Lys Asp Asn Leu Asn Glu
405 410 415
Lys Phe Asp Leu Glu Leu Glu Ser Lys Ile Ala Leu Ala Asn Glu Leu
420 425 430
Ala Phe Lys Arg Glu Leu Asn Val Lys Asp Ala Leu Lys Ile Leu Gly
435 440 445
Leu Lys Pro Asn Glu Trp Glu Phe Asn Phe Thr Lys Ile Glu Gly Asn
450 455 460
Arg Thr Asn Gln Ala Phe Phe Asp Ala Phe Ala Lys Ile Ile Glu Leu
465 470 475 480
Glu Asp Gly Glu Pro Ile Asp Leu Gly Asp Leu Lys Ala Asp Asp Ile
485 490 495
Leu Asp Gln Phe Ser Glu Ala Phe Leu Arg Ile Gly Ile Asp Thr Glu
500 505 510
Leu Leu Gln Val Asn Ser Asp Ile Glu Gly Ala Glu Tyr Glu Lys Gln
515 520 525
Ser Tyr Ile Gln Phe Trp His Leu Leu Tyr Ser Ser Glu Asp Asp Gln
530 535 540
Lys Leu Lys Leu Asn Leu Ile Arg Lys Phe Gly Phe Lys Pro Glu His
545 550 555 560
Ala Lys Ile Leu Ala Ser Ile Ser Leu Gln Asp Asp His Ala Ser Leu
565 570 575
Ser Ser Arg Ala Ile Lys Lys Ile Leu Pro His Leu Gln Ser Gly Leu
580 585 590
Ile Tyr Asp Lys Ala Cys Thr Tyr Ala Gly Tyr Asn His Ser Ser Ser
595 600 605
Phe Thr Lys Asp Glu Asn Glu Lys Arg Glu Leu Arg Ala Glu Leu Glu
610 615 620
Leu Leu Lys Lys Asn Ser Leu Arg Asn Pro Val Val Glu Lys Ile Leu
625 630 635 640
Asn Gln Met Ile Asn Val Val Asn Ala Ile Leu Lys Asp Pro Glu Leu
645 650 655
Gly Arg Pro Asp Glu Ile Arg Val Glu Met Ala Arg Glu Leu Lys Ala
660 665 670
Asn Ala Glu Gln Arg Lys Asn Met Thr Ser Asn Ile Ala Ser Ala Thr
675 680 685
Arg Asp His Asp Lys Tyr Arg Glu Ile Leu Lys Ser Glu Phe Gly Leu
690 695 700
Lys Arg Val Thr Lys Asn Asp Leu Leu Arg Tyr Lys Leu Trp Leu Glu
705 710 715 720
Thr Asp Gly Ile Ser Leu Tyr Thr Gly Lys Pro Ile Glu Ala Ser Lys
725 730 735
Leu Phe Ser Lys Glu Tyr Asp Ile Glu His Ile Ile Pro Lys Ala Arg
740 745 750
Leu Phe Asp Asp Ser Phe Ser Asn Lys Thr Ile Cys Glu Arg Gln Leu
755 760 765
Asn Ile Asp Lys Ala Asn Val Thr Ala Phe Ser Phe Leu Gln Asn Lys
770 775 780
Leu Ser Ala Asp Glu Phe Glu Gln Tyr Gln Ser Arg Val Lys Ser Leu
785 790 795 800
Tyr Gly Lys Leu Ser Lys Ala Lys Ile Gln Lys Leu Leu Met Ala Asn
805 810 815
Asp Lys Ile Pro Glu Asp Phe Ile Ala Arg Gln Leu Gln Glu Thr Arg
820 825 830
Tyr Ile Ser Lys Lys Ala Lys Glu Ile Leu Phe Glu Ile Ser Arg Arg
835 840 845
Val Ser Val Thr Thr Gly Thr Ile Thr Asp Lys Leu Arg Glu Asp Trp
850 855 860
Gly Leu Val Glu Ile Met Lys Glu Leu Asn Trp Glu Lys Tyr Asp Lys
865 870 875 880
Leu Gly Leu Thr Tyr Thr Ile Glu Gly Lys His Gly Glu Arg Leu Asn
885 890 895
Lys Ile Lys Asp Trp Ser Lys Arg Asn Asp His Arg His His Ala Met
900 905 910
Asp Ala Leu Thr Val Ala Leu Thr Lys Pro Ala Tyr Ile Gln Tyr Leu
915 920 925
Asn Asn Leu Asn Ala Lys Gly Leu Asn Asn Lys Lys Gly Thr Glu Val
930 935 940
Phe Ala Ile Glu Gln Lys Tyr Leu Lys Arg Glu Asn Gly Lys Leu Cys
945 950 955 960
Phe Ile Pro Pro Ile Glu Asn Ile Arg Ser Glu Ala Lys Lys His Leu
965 970 975
Ser Arg Ile Leu Val Ser Tyr Lys Ala Lys Asn Lys Val Val Thr Ile
980 985 990
Asn Lys Asn Lys Thr Lys Ser Lys Ala Gly Leu Asn Glu Gln Ile Ala
995 1000 1005
Leu Thr Pro Arg Gly Gln Leu His Lys Glu Thr Val Tyr Gly Lys Ser
1010 1015 1020
Phe His Tyr Ser Thr Lys Phe Glu Lys Ile Gly Ala Ser Phe Asn Val
1025 1030 1035 1040
Gln Lys Ile Asn Thr Val Ala Lys Lys Glu Glu Arg Glu Ala Leu Leu
1045 1050 1055
Lys Arg Leu Ala Glu Asn Gly Asn Asp Pro Lys Lys Ala Phe Thr Gly
1060 1065 1070
Lys Asn Thr Leu Asn Lys Met Pro Ile Tyr Leu Asp Leu Gly Lys Asn
1075 1080 1085
Ile Lys Leu Ser Glu Lys Val Lys Thr Val Val Leu Glu Gln Asn Tyr
1090 1095 1100
Thr Ile Arg Lys Asn Ile Asp Pro Asp Leu Lys Val Asp Lys Val Ile
1105 1110 1115 1120
Asp Val Gly Ile Lys Arg Ile Leu Glu Ser Arg Leu Glu Glu Phe Gly
1125 1130 1135
Gly Asn Ala Lys Leu Ala Phe Ser Asn Leu Glu Glu Asn Pro Ile Trp
1140 1145 1150
Leu Asn Lys Glu Lys Gly Ile Ser Ile Lys Arg Val Lys Ile Ser Gly
1155 1160 1165
Val Ser Asn Val Glu Ser Leu His Val Lys Lys Asp His Phe Gly Glu
1170 1175 1180
Pro Ile Leu Asp Gln Glu Gly Asn Glu Ile Pro Val Asp Phe Val Ser
1185 1190 1195 1200
Thr Gly Asn Asn His His Val Ala Ile Tyr Glu Asp Glu Asn Gly Asn
1205 1210 1215
Leu Gln Glu Glu Val Val Ser Phe Phe Glu Ala Val Val Arg Gln Asn
1220 1225 1230
Gln Gly Leu Pro Ile Ile Lys Lys Asn His Thr Leu Gly Trp Lys Phe
1235 1240 1245
Leu Phe Thr Leu Lys Gln Asn Glu Tyr Phe Val Phe Pro Ser Asp Asp
1250 1255 1260
Phe Val Pro Ala Asp Val Asp Leu Met Asp Glu Gln Asn Tyr His Leu
1265 1270 1275 1280
Ile Ser Pro Asn Leu Phe Arg Val Gln Lys Ile Ala Arg Lys Asn Tyr
1285 1290 1295
Val Phe Asn Asn His Leu Glu Thr Lys Ala Val Asp Asn Asp Leu Leu
1300 1305 1310
Lys Ser Lys Lys Glu Leu Ser Lys Ile Thr Tyr His Phe Tyr Gln Thr
1315 1320 1325
Pro Glu His Leu Arg Gly Ile Ile Lys Ile Arg Ile Asn His Leu Gly
1330 1335 1340
Lys Ile Ile Gln Ile Gly Glu Tyr
1345 1350
<210> 26
<211> 1509
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自热带冷弯菌（Psychroflexus torquisI）
<400> 26
Met Lys Arg Ile Leu Gly Leu Asp Leu Gly Thr Asn Ser Ile Gly Trp
1 5 10 15
Ser Leu Ile Glu His Asp Phe Lys Asn Lys Gln Gly Gln Ile Glu Gly
20 25 30
Leu Gly Val Arg Ile Ile Pro Met Ser Gln Glu Ile Leu Gly Lys Phe
35 40 45
Asp Ala Gly Gln Ser Ile Ser Gln Thr Ala Asp Arg Thr Lys Tyr Arg
50 55 60
Gly Val Arg Arg Leu Tyr Gln Arg Asp Asn Leu Arg Arg Glu Arg Leu
65 70 75 80
His Arg Val Leu Lys Ile Leu Asp Phe Leu Pro Lys His Tyr Ser Glu
85 90 95
Ser Ile Asp Phe Gln Asp Lys Val Gly Gln Phe Lys Pro Lys Gln Glu
100 105 110
Val Lys Leu Asn Tyr Arg Lys Asn Glu Lys Asn Lys His Glu Phe Val
115 120 125
Phe Met Asn Ser Phe Ile Glu Met Val Ser Glu Phe Lys Asn Ala Gln
130 135 140
Pro Glu Leu Phe Tyr Asn Lys Gly Asn Gly Glu Glu Thr Lys Ile Pro
145 150 155 160
Tyr Asp Trp Thr Leu Tyr Tyr Leu Arg Lys Lys Ala Leu Thr Gln Gln
165 170 175
Ile Thr Lys Glu Glu Leu Ala Trp Leu Ile Leu Asn Phe Asn Gln Lys
180 185 190
Arg Gly Tyr Tyr Gln Leu Arg Gly Glu Asp Ile Asp Glu Asp Lys Asn
195 200 205
Lys Lys Tyr Met Gln Leu Lys Val Asn Asn Leu Ile Asp Ser Gly Ala
210 215 220
Lys Val Lys Gly Lys Val Leu Tyr Asn Val Ile Phe Asp Asn Gly Trp
225 230 235 240
Lys Tyr Glu Lys Gln Ile Val Asn Lys Asp Glu Trp Glu Gly Arg Thr
245 250 255
Lys Glu Phe Ile Ile Thr Thr Lys Thr Leu Lys Asn Gly Asn Ile Lys
260 265 270
Arg Thr Tyr Lys Ala Val Asp Ser Glu Ile Asp Trp Ala Ala Ile Lys
275 280 285
Ala Lys Thr Glu Gln Asp Ile Asn Lys Ala Asn Lys Thr Val Gly Glu
290 295 300
Tyr Ile Tyr Glu Ser Leu Leu Asp Asn Pro Ser Gln Lys Ile Arg Gly
305 310 315 320
Lys Leu Val Lys Thr Ile Glu Arg Lys Phe Tyr Lys Glu Glu Phe Glu
325 330 335
Lys Leu Leu Ser Lys Gln Ile Glu Leu Gln Pro Glu Leu Phe Asn Glu
340 345 350
Ser Leu Tyr Lys Ala Cys Ile Lys Glu Leu Tyr Pro Arg Asn Glu Asn
355 360 365
His Gln Ser Asn Asn Lys Lys Gln Gly Phe Glu Tyr Leu Phe Thr Glu
370 375 380
Asp Ile Ile Phe Tyr Gln Arg Pro Leu Lys Ser Gln Lys Ser Asn Ile
385 390 395 400
Ser Gly Cys Gln Phe Glu His Lys Ile Tyr Lys Gln Lys Asn Lys Lys
405 410 415
Thr Gly Lys Leu Glu Leu Ile Lys Glu Pro Ile Lys Thr Ile Ser Arg
420 425 430
Ser His Pro Leu Phe Gln Glu Phe Arg Ile Trp Gln Trp Leu Gln Asn
435 440 445
Leu Lys Ile Tyr Asn Lys Glu Lys Ile Glu Asn Gly Lys Leu Glu Asp
450 455 460
Val Thr Thr Gln Leu Leu Pro Asn Asn Glu Ala Tyr Val Thr Leu Phe
465 470 475 480
Asp Phe Leu Asn Thr Lys Lys Glu Leu Glu Gln Lys Gln Phe Ile Glu
485 490 495
Tyr Phe Val Lys Lys Lys Leu Ile Asp Lys Lys Glu Lys Glu His Phe
500 505 510
Arg Trp Asn Phe Val Glu Asp Lys Lys Tyr Pro Phe Ser Glu Thr Arg
515 520 525
Ala Gln Phe Leu Ser Arg Leu Ala Lys Val Lys Gly Ile Lys Asn Thr
530 535 540
Glu Asp Phe Leu Asn Lys Asn Thr Gln Val Gly Ser Lys Glu Asn Ser
545 550 555 560
Pro Phe Ile Lys Arg Ile Glu Gln Leu Trp His Ile Ile Tyr Ser Val
565 570 575
Ser Asp Leu Lys Glu Tyr Glu Lys Ala Leu Glu Lys Phe Ala Glu Lys
580 585 590
His Asn Leu Glu Lys Asp Ser Phe Leu Lys Asn Phe Lys Lys Phe Pro
595 600 605
Pro Phe Val Ser Asp Tyr Ala Ser Tyr Ser Lys Lys Ala Ile Ser Lys
610 615 620
Leu Leu Pro Ile Met Arg Met Gly Lys Tyr Trp Ser Glu Ser Ala Val
625 630 635 640
Pro Thr Gln Val Lys Glu Arg Ser Leu Ser Ile Met Glu Arg Val Lys
645 650 655
Val Leu Pro Leu Lys Glu Gly Tyr Ser Asp Lys Asp Leu Ala Asp Leu
660 665 670
Leu Ser Arg Val Ser Asp Asp Asp Ile Pro Lys Gln Leu Ile Lys Ser
675 680 685
Phe Ile Ser Phe Lys Asp Lys Asn Pro Leu Lys Gly Leu Asn Thr Tyr
690 695 700
Gln Ala Asn Tyr Leu Val Tyr Gly Arg His Ser Glu Thr Gly Asp Ile
705 710 715 720
Gln His Trp Lys Thr Pro Glu Asp Ile Asp Arg Tyr Leu Asn Asn Phe
725 730 735
Lys Gln His Ser Leu Arg Asn Pro Ile Val Glu Gln Val Val Met Glu
740 745 750
Thr Leu Arg Val Val Arg Asp Ile Trp Glu His Tyr Gly Asn Asn Glu
755 760 765
Lys Asp Phe Phe Lys Glu Ile His Val Glu Leu Gly Arg Glu Met Lys
770 775 780
Ser Pro Ala Gly Lys Arg Glu Lys Leu Ser Gln Arg Asn Thr Glu Asn
785 790 795 800
Glu Asn Thr Asn His Arg Ile Arg Glu Val Leu Lys Glu Leu Met Asn
805 810 815
Asp Ala Ser Val Glu Gly Gly Val Arg Asp Tyr Ser Pro Ser Gln Gln
820 825 830
Glu Ile Leu Lys Leu Tyr Glu Glu Gly Ile Tyr Gln Asn Pro Asn Thr
835 840 845
Asn Tyr Leu Lys Val Asp Glu Asp Glu Ile Leu Lys Ile Arg Lys Lys
850 855 860
Asn Asn Pro Thr Gln Lys Glu Ile Gln Arg Tyr Lys Leu Trp Leu Glu
865 870 875 880
Gln Gly Tyr Ile Ser Pro Tyr Thr Gly Lys Ile Ile Pro Leu Thr Lys
885 890 895
Leu Phe Thr His Glu Tyr Gln Ile Glu His Ile Ile Pro Gln Ser Arg
900 905 910
Tyr Tyr Asp Asn Ser Leu Gly Asn Lys Ile Ile Cys Glu Ser Glu Val
915 920 925
Asn Glu Asp Lys Asp Asn Lys Thr Ala Tyr Glu Tyr Leu Lys Val Glu
930 935 940
Lys Gly Ser Ile Val Phe Gly His Lys Leu Leu Asn Leu Asp Glu Tyr
945 950 955 960
Glu Ala His Val Asn Lys Tyr Phe Lys Lys Asn Lys Thr Lys Leu Lys
965 970 975
Asn Leu Leu Ser Glu Asp Ile Pro Glu Gly Phe Ile Asn Arg Gln Leu
980 985 990
Asn Asp Ser Arg Tyr Ile Ser Lys Leu Val Lys Gly Leu Leu Ser Asn
995 1000 1005
Ile Val Arg Glu Asn Gly Glu Gln Glu Ala Thr Ser Lys Asn Leu Ile
1010 1015 1020
Pro Val Thr Gly Val Val Thr Ser Lys Leu Lys Gln Asp Trp Gly Leu
1025 1030 1035 1040
Asn Asp Lys Trp Asn Glu Ile Ile Ala Pro Arg Phe Lys Arg Leu Asn
1045 1050 1055
Lys Leu Thr Asn Ser Asn Asp Phe Gly Phe Trp Asp Asn Asp Ile Asn
1060 1065 1070
Ala Phe Arg Ile Gln Val Pro Asp Ser Leu Ile Lys Gly Phe Ser Lys
1075 1080 1085
Lys Arg Ile Asp His Arg His His Ala Leu Asp Ala Leu Val Val Ala
1090 1095 1100
Cys Thr Ser Arg Asn His Thr His Tyr Leu Ser Ala Leu Asn Ala Glu
1105 1110 1115 1120
Asn Lys Asn Tyr Ser Leu Arg Asp Lys Leu Val Ile Lys Asn Glu Asn
1125 1130 1135
Gly Asp Tyr Thr Lys Thr Phe Gln Ile Pro Trp Gln Gly Phe Thr Ile
1140 1145 1150
Glu Ala Lys Asn Asn Leu Glu Lys Thr Val Val Ser Phe Lys Lys Asn
1155 1160 1165
Leu Arg Val Ile Asn Lys Thr Asn Asn Lys Phe Trp Ser Tyr Lys Asp
1170 1175 1180
Glu Asn Gly Asn Leu Asn Leu Gly Lys Asp Gly Lys Pro Lys Lys Lys
1185 1190 1195 1200
Leu Arg Lys Gln Thr Lys Gly Tyr Asn Trp Ala Ile Arg Lys Pro Leu
1205 1210 1215
His Lys Glu Thr Val Ser Gly Ile Tyr Asn Ile Asn Ala Pro Lys Asn
1220 1225 1230
Lys Ile Ala Thr Ser Val Arg Thr Leu Leu Thr Glu Ile Lys Asn Glu
1235 1240 1245
Lys His Leu Ala Lys Ile Thr Asp Leu Arg Ile Arg Glu Thr Ile Leu
1250 1255 1260
Pro Asn His Leu Lys His Tyr Leu Asn Asn Lys Gly Glu Ala Asn Phe
1265 1270 1275 1280
Ser Glu Ala Phe Ser Gln Gly Gly Ile Glu Asp Leu Asn Lys Lys Ile
1285 1290 1295
Thr Thr Leu Asn Glu Gly Lys Lys His Gln Pro Ile Tyr Arg Val Lys
1300 1305 1310
Ile Phe Glu Val Gly Ser Lys Phe Ser Ile Ser Glu Asp Glu Asn Ser
1315 1320 1325
Ala Lys Ser Lys Lys Tyr Val Glu Ala Ala Lys Gly Thr Asn Leu Phe
1330 1335 1340
Phe Ala Ile Tyr Leu Asp Glu Glu Asn Lys Lys Arg Asn Tyr Glu Thr
1345 1350 1355 1360
Ile Pro Leu Asn Glu Val Ile Thr His Gln Lys Gln Val Ala Gly Phe
1365 1370 1375
Pro Lys Ser Glu Arg Leu Ser Val Gln Pro Asp Ser Gln Lys Gly Thr
1380 1385 1390
Phe Leu Phe Thr Leu Ser Pro Asn Asp Leu Val Tyr Val Pro Asn Asn
1395 1400 1405
Glu Glu Leu Glu Asn Arg Asp Leu Phe Asn Leu Gly Asn Leu Asn Val
1410 1415 1420
Glu Gln Ile Ser Arg Ile Tyr Lys Phe Thr Asp Ser Ser Asp Lys Thr
1425 1430 1435 1440
Cys Asn Phe Ile Pro Phe Gln Val Ser Lys Leu Ile Phe Asn Leu Lys
1445 1450 1455
Lys Lys Glu Gln Lys Lys Leu Asp Val Asp Phe Ile Ile Gln Asn Glu
1460 1465 1470
Phe Gly Leu Gly Ser Pro Gln Ser Lys Asn Gln Lys Ser Ile Asp Asp
1475 1480 1485
Val Met Ile Lys Glu Lys Cys Ile Lys Leu Lys Ile Asp Arg Leu Gly
1490 1495 1500
Asn Ile Ser Lys Ala
1505
<210> 27
<211> 1388
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自嗜热链球菌（Streptococcus thermophilus）
<400> 27
Met Thr Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Thr Thr Asp Asn Tyr Lys Val Pro Ser Lys Lys Met
20 25 30
Lys Val Leu Gly Asn Thr Ser Lys Lys Tyr Ile Lys Lys Asn Leu Leu
35 40 45
Gly Val Leu Leu Phe Asp Ser Gly Ile Thr Ala Glu Gly Arg Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Thr Glu Met Ala Thr Leu Asp Asp Ala
85 90 95
Phe Phe Gln Arg Leu Asp Asp Ser Phe Leu Val Pro Asp Asp Lys Arg
100 105 110
Asp Ser Lys Tyr Pro Ile Phe Gly Asn Leu Val Glu Glu Lys Ala Tyr
115 120 125
His Asp Glu Phe Pro Thr Ile Tyr His Leu Arg Lys Tyr Leu Ala Asp
130 135 140
Ser Thr Lys Lys Ala Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Tyr Arg Gly His Phe Leu Ile Glu Gly Glu Phe Asn Ser
165 170 175
Lys Asn Asn Asp Ile Gln Lys Asn Phe Gln Asp Phe Leu Asp Thr Tyr
180 185 190
Asn Ala Ile Phe Glu Ser Asp Leu Ser Leu Glu Asn Ser Lys Gln Leu
195 200 205
Glu Glu Ile Val Lys Asp Lys Ile Ser Lys Leu Glu Lys Lys Asp Arg
210 215 220
Ile Leu Lys Leu Phe Pro Gly Glu Lys Asn Ser Gly Ile Phe Ser Glu
225 230 235 240
Phe Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Arg Lys Cys Phe
245 250 255
Asn Leu Asp Glu Lys Ala Ser Leu His Phe Ser Lys Glu Ser Tyr Asp
260 265 270
Glu Asp Leu Glu Thr Leu Leu Gly Tyr Ile Gly Asp Asp Tyr Ser Asp
275 280 285
Val Phe Leu Lys Ala Lys Lys Leu Tyr Asp Ala Ile Leu Leu Ser Gly
290 295 300
Phe Leu Thr Val Thr Asp Asn Glu Thr Glu Ala Pro Leu Ser Ser Ala
305 310 315 320
Met Ile Lys Arg Tyr Asn Glu His Lys Glu Asp Leu Ala Leu Leu Lys
325 330 335
Glu Tyr Ile Arg Asn Ile Ser Leu Lys Thr Tyr Asn Glu Val Phe Lys
340 345 350
Asp Asp Thr Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Lys Thr Asn
355 360 365
Gln Glu Asp Phe Tyr Val Tyr Leu Lys Lys Leu Leu Ala Glu Phe Glu
370 375 380
Gly Ala Asp Tyr Phe Leu Glu Lys Ile Asp Arg Glu Asp Phe Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro Tyr Gln Ile His Leu
405 410 415
Gln Glu Met Arg Ala Ile Leu Asp Lys Gln Ala Lys Phe Tyr Pro Phe
420 425 430
Leu Ala Lys Asn Lys Glu Arg Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Asp Phe Ala Trp
450 455 460
Ser Ile Arg Lys Arg Asn Glu Lys Ile Thr Pro Trp Asn Phe Glu Asp
465 470 475 480
Val Ile Asp Lys Glu Ser Ser Ala Glu Ala Phe Ile Asn Arg Met Thr
485 490 495
Ser Phe Asp Leu Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Thr Phe Asn Val Tyr Asn Glu Leu Thr Lys Val Arg
515 520 525
Phe Ile Ala Glu Ser Met Arg Asp Tyr Gln Phe Leu Asp Ser Lys Gln
530 535 540
Lys Lys Asp Ile Val Arg Leu Tyr Phe Lys Asp Lys Arg Lys Val Thr
545 550 555 560
Asp Lys Asp Ile Ile Glu Tyr Leu His Ala Ile Tyr Gly Tyr Asp Gly
565 570 575
Ile Glu Leu Lys Gly Ile Glu Lys Gln Phe Asn Ser Ser Leu Ser Thr
580 585 590
Tyr His Asp Leu Leu Asn Ile Ile Asn Asp Lys Glu Phe Leu Asp Asp
595 600 605
Ser Ser Asn Glu Ala Ile Ile Glu Glu Ile Ile His Thr Leu Thr Ile
610 615 620
Phe Glu Asp Arg Glu Met Ile Lys Gln Arg Leu Ser Lys Phe Glu Asn
625 630 635 640
Ile Phe Asp Lys Ser Val Leu Lys Lys Leu Ser Arg Arg His Tyr Thr
645 650 655
Gly Trp Gly Lys Leu Ser Ala Lys Leu Ile Asn Gly Ile Arg Asp Glu
660 665 670
Lys Ser Gly Asn Thr Ile Leu Asp Tyr Leu Ile Asp Asp Gly Ile Ser
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ala Leu Ser Phe Lys
690 695 700
Lys Lys Ile Gln Lys Ala Gln Ile Ile Gly Asp Glu Asp Lys Gly Asn
705 710 715 720
Ile Lys Glu Val Val Lys Ser Leu Pro Gly Ser Pro Ala Ile Lys Lys
725 730 735
Gly Ile Leu Gln Ser Ile Lys Ile Val Asp Glu Leu Val Lys Val Met
740 745 750
Gly Gly Arg Lys Pro Glu Ser Ile Val Val Glu Met Ala Arg Glu Asn
755 760 765
Gln Tyr Thr Asn Gln Gly Lys Ser Asn Ser Gln Gln Arg Leu Lys Arg
770 775 780
Leu Glu Lys Ser Leu Lys Glu Leu Gly Ser Lys Ile Leu Lys Glu Asn
785 790 795 800
Ile Pro Ala Lys Leu Ser Lys Ile Asp Asn Asn Ala Leu Gln Asn Asp
805 810 815
Arg Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly
820 825 830
Asp Asp Leu Asp Ile Asp Arg Leu Ser Asn Tyr Asp Ile Asp His Ile
835 840 845
Ile Pro Gln Ala Phe Leu Lys Asp Asn Ser Ile Asp Asn Lys Val Leu
850 855 860
Val Ser Ser Ala Ser Asn Arg Gly Lys Ser Asp Asp Val Pro Ser Leu
865 870 875 880
Glu Val Val Lys Lys Arg Lys Thr Phe Trp Tyr Gln Leu Leu Lys Ser
885 890 895
Lys Leu Ile Ser Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg
900 905 910
Gly Gly Leu Ser Pro Glu Asp Lys Ala Gly Phe Ile Gln Arg Gln Leu
915 920 925
Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Arg Leu Leu Asp Glu
930 935 940
Lys Phe Asn Asn Lys Lys Asp Glu Asn Asn Arg Ala Val Arg Thr Val
945 950 955 960
Lys Ile Ile Thr Leu Lys Ser Thr Leu Val Ser Gln Phe Arg Lys Asp
965 970 975
Phe Glu Leu Tyr Lys Val Arg Glu Ile Asn Asp Phe His His Ala His
980 985 990
Asp Ala Tyr Leu Asn Ala Val Val Ala Ser Ala Leu Leu Lys Lys Tyr
995 1000 1005
Pro Lys Leu Glu Pro Glu Phe Val Tyr Gly Asp Tyr Pro Lys Tyr Asn
1010 1015 1020
Ser Phe Arg Glu Arg Lys Ser Ala Thr Glu Lys Val Tyr Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Ile Phe Lys Lys Ser Ile Ser Leu Ala Asp Gly Arg
1045 1050 1055
Val Ile Glu Arg Pro Leu Ile Glu Val Asn Glu Glu Thr Gly Glu Ser
1060 1065 1070
Val Trp Asn Lys Glu Ser Asp Leu Ala Thr Val Arg Arg Val Leu Ser
1075 1080 1085
Tyr Pro Gln Val Asn Val Val Lys Lys Val Glu Glu Gln Asn His Gly
1090 1095 1100
Leu Asp Arg Gly Lys Pro Lys Gly Leu Phe Asn Ala Asn Leu Ser Ser
1105 1110 1115 1120
Lys Pro Lys Pro Asn Ser Asn Glu Asn Leu Val Gly Ala Lys Glu Tyr
1125 1130 1135
Leu Asp Pro Lys Lys Tyr Gly Gly Tyr Ala Gly Ile Ser Asn Ser Phe
1140 1145 1150
Thr Val Leu Val Lys Gly Thr Ile Glu Lys Gly Ala Lys Lys Lys Ile
1155 1160 1165
Thr Asn Val Leu Glu Phe Gln Gly Ile Ser Ile Leu Asp Arg Ile Asn
1170 1175 1180
Tyr Arg Lys Asp Lys Leu Asn Phe Leu Leu Glu Lys Gly Tyr Lys Asp
1185 1190 1195 1200
Ile Glu Leu Ile Ile Glu Leu Pro Lys Tyr Ser Leu Phe Glu Leu Ser
1205 1210 1215
Asp Gly Ser Arg Arg Met Leu Ala Ser Ile Leu Ser Thr Asn Asn Lys
1220 1225 1230
Arg Gly Glu Ile His Lys Gly Asn Gln Ile Phe Leu Ser Gln Lys Phe
1235 1240 1245
Val Lys Leu Leu Tyr His Ala Lys Arg Ile Ser Asn Thr Ile Asn Glu
1250 1255 1260
Asn His Arg Lys Tyr Val Glu Asn His Lys Lys Glu Phe Glu Glu Leu
1265 1270 1275 1280
Phe Tyr Tyr Ile Leu Glu Phe Asn Glu Asn Tyr Val Gly Ala Lys Lys
1285 1290 1295
Asn Gly Lys Leu Leu Asn Ser Ala Phe Gln Ser Trp Gln Asn His Ser
1300 1305 1310
Ile Asp Glu Leu Cys Ser Ser Phe Ile Gly Pro Thr Gly Ser Glu Arg
1315 1320 1325
Lys Gly Leu Phe Glu Leu Thr Ser Arg Gly Ser Ala Ala Asp Phe Glu
1330 1335 1340
Phe Leu Gly Val Lys Ile Pro Arg Tyr Arg Asp Tyr Thr Pro Ser Ser
1345 1350 1355 1360
Leu Leu Lys Asp Ala Thr Leu Ile His Gln Ser Val Thr Gly Leu Tyr
1365 1370 1375
Glu Thr Arg Ile Asp Leu Ala Lys Leu Gly Glu Gly
1380 1385
<210> 28
<211> 1334
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自英诺克李斯特菌（ Listeria innocua）
<400> 28
Met Lys Lys Pro Tyr Thr Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Leu Thr Asp Gln Tyr Asp Leu Val Lys Arg Lys Met
20 25 30
Lys Ile Ala Gly Asp Ser Glu Lys Lys Gln Ile Lys Lys Asn Phe Trp
35 40 45
Gly Val Arg Leu Phe Asp Glu Gly Gln Thr Ala Ala Asp Arg Arg Met
50 55 60
Ala Arg Thr Ala Arg Arg Arg Ile Glu Arg Arg Arg Asn Arg Ile Ser
65 70 75 80
Tyr Leu Gln Gly Ile Phe Ala Glu Glu Met Ser Lys Thr Asp Ala Asn
85 90 95
Phe Phe Cys Arg Leu Ser Asp Ser Phe Tyr Val Asp Asn Glu Lys Arg
100 105 110
Asn Ser Arg His Pro Phe Phe Ala Thr Ile Glu Glu Glu Val Glu Tyr
115 120 125
His Lys Asn Tyr Pro Thr Ile Tyr His Leu Arg Glu Glu Leu Val Asn
130 135 140
Ser Ser Glu Lys Ala Asp Leu Arg Leu Val Tyr Leu Ala Leu Ala His
145 150 155 160
Ile Ile Lys Tyr Arg Gly Asn Phe Leu Ile Glu Gly Ala Leu Asp Thr
165 170 175
Gln Asn Thr Ser Val Asp Gly Ile Tyr Lys Gln Phe Ile Gln Thr Tyr
180 185 190
Asn Gln Val Phe Ala Ser Gly Ile Glu Asp Gly Ser Leu Lys Lys Leu
195 200 205
Glu Asp Asn Lys Asp Val Ala Lys Ile Leu Val Glu Lys Val Thr Arg
210 215 220
Lys Glu Lys Leu Glu Arg Ile Leu Lys Leu Tyr Pro Gly Glu Lys Ser
225 230 235 240
Ala Gly Met Phe Ala Gln Phe Ile Ser Leu Ile Val Gly Ser Lys Gly
245 250 255
Asn Phe Gln Lys Pro Phe Asp Leu Ile Glu Lys Ser Asp Ile Glu Cys
260 265 270
Ala Lys Asp Ser Tyr Glu Glu Asp Leu Glu Ser Leu Leu Ala Leu Ile
275 280 285
Gly Asp Glu Tyr Ala Glu Leu Phe Val Ala Ala Lys Asn Ala Tyr Ser
290 295 300
Ala Val Val Leu Ser Ser Ile Ile Thr Val Ala Glu Thr Glu Thr Asn
305 310 315 320
Ala Lys Leu Ser Ala Ser Met Ile Glu Arg Phe Asp Thr His Glu Glu
325 330 335
Asp Leu Gly Glu Leu Lys Ala Phe Ile Lys Leu His Leu Pro Lys His
340 345 350
Tyr Glu Glu Ile Phe Ser Asn Thr Glu Lys His Gly Tyr Ala Gly Tyr
355 360 365
Ile Asp Gly Lys Thr Lys Gln Ala Asp Phe Tyr Lys Tyr Met Lys Met
370 375 380
Thr Leu Glu Asn Ile Glu Gly Ala Asp Tyr Phe Ile Ala Lys Ile Glu
385 390 395 400
Lys Glu Asn Phe Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ala Ile
405 410 415
Pro His Gln Leu His Leu Glu Glu Leu Glu Ala Ile Leu His Gln Gln
420 425 430
Ala Lys Tyr Tyr Pro Phe Leu Lys Glu Asn Tyr Asp Lys Ile Lys Ser
435 440 445
Leu Val Thr Phe Arg Ile Pro Tyr Phe Val Gly Pro Leu Ala Asn Gly
450 455 460
Gln Ser Glu Phe Ala Trp Leu Thr Arg Lys Ala Asp Gly Glu Ile Arg
465 470 475 480
Pro Trp Asn Ile Glu Glu Lys Val Asp Phe Gly Lys Ser Ala Val Asp
485 490 495
Phe Ile Glu Lys Met Thr Asn Lys Asp Thr Tyr Leu Pro Lys Glu Asn
500 505 510
Val Leu Pro Lys His Ser Leu Cys Tyr Gln Lys Tyr Leu Val Tyr Asn
515 520 525
Glu Leu Thr Lys Val Arg Tyr Ile Asn Asp Gln Gly Lys Thr Ser Tyr
530 535 540
Phe Ser Gly Gln Glu Lys Glu Gln Ile Phe Asn Asp Leu Phe Lys Gln
545 550 555 560
Lys Arg Lys Val Lys Lys Lys Asp Leu Glu Leu Phe Leu Arg Asn Met
565 570 575
Ser His Val Glu Ser Pro Thr Ile Glu Gly Leu Glu Asp Ser Phe Asn
580 585 590
Ser Ser Tyr Ser Thr Tyr His Asp Leu Leu Lys Val Gly Ile Lys Gln
595 600 605
Glu Ile Leu Asp Asn Pro Val Asn Thr Glu Met Leu Glu Asn Ile Val
610 615 620
Lys Ile Leu Thr Val Phe Glu Asp Lys Arg Met Ile Lys Glu Gln Leu
625 630 635 640
Gln Gln Phe Ser Asp Val Leu Asp Gly Val Val Leu Lys Lys Leu Glu
645 650 655
Arg Arg His Tyr Thr Gly Trp Gly Arg Leu Ser Ala Lys Leu Leu Met
660 665 670
Gly Ile Arg Asp Lys Gln Ser His Leu Thr Ile Leu Asp Tyr Leu Met
675 680 685
Asn Asp Asp Gly Leu Asn Arg Asn Leu Met Gln Leu Ile Asn Asp Ser
690 695 700
Asn Leu Ser Phe Lys Ser Ile Ile Glu Lys Glu Gln Val Thr Thr Ala
705 710 715 720
Asp Lys Asp Ile Gln Ser Ile Val Ala Asp Leu Ala Gly Ser Pro Ala
725 730 735
Ile Lys Lys Gly Ile Leu Gln Ser Leu Lys Ile Val Asp Glu Leu Val
740 745 750
Ser Val Met Gly Tyr Pro Pro Gln Thr Ile Val Val Glu Met Ala Arg
755 760 765
Glu Asn Gln Thr Thr Gly Lys Gly Lys Asn Asn Ser Arg Pro Arg Tyr
770 775 780
Lys Ser Leu Glu Lys Ala Ile Lys Glu Phe Gly Ser Gln Ile Leu Lys
785 790 795 800
Glu His Pro Thr Asp Asn Gln Glu Leu Arg Asn Asn Arg Leu Tyr Leu
805 810 815
Tyr Tyr Leu Gln Asn Gly Lys Asp Met Tyr Thr Gly Gln Asp Leu Asp
820 825 830
Ile His Asn Leu Ser Asn Tyr Asp Ile Asp His Ile Val Pro Gln Ser
835 840 845
Phe Ile Thr Asp Asn Ser Ile Asp Asn Leu Val Leu Thr Ser Ser Ala
850 855 860
Gly Asn Arg Glu Lys Gly Asp Asp Val Pro Pro Leu Glu Ile Val Arg
865 870 875 880
Lys Arg Lys Val Phe Trp Glu Lys Leu Tyr Gln Gly Asn Leu Met Ser
885 890 895
Lys Arg Lys Phe Asp Tyr Leu Thr Lys Ala Glu Arg Gly Gly Leu Thr
900 905 910
Glu Ala Asp Lys Ala Arg Phe Ile His Arg Gln Leu Val Glu Thr Arg
915 920 925
Gln Ile Thr Lys Asn Val Ala Asn Ile Leu His Gln Arg Phe Asn Tyr
930 935 940
Glu Lys Asp Asp His Gly Asn Thr Met Lys Gln Val Arg Ile Val Thr
945 950 955 960
Leu Lys Ser Ala Leu Val Ser Gln Phe Arg Lys Gln Phe Gln Leu Tyr
965 970 975
Lys Val Arg Asp Val Asn Asp Tyr His His Ala His Asp Ala Tyr Leu
980 985 990
Asn Gly Val Val Ala Asn Thr Leu Leu Lys Val Tyr Pro Gln Leu Glu
995 1000 1005
Pro Glu Phe Val Tyr Gly Asp Tyr His Gln Phe Asp Trp Phe Lys Ala
1010 1015 1020
Asn Lys Ala Thr Ala Lys Lys Gln Phe Tyr Thr Asn Ile Met Leu Phe
1025 1030 1035 1040
Phe Ala Gln Lys Asp Arg Ile Ile Asp Glu Asn Gly Glu Ile Leu Trp
1045 1050 1055
Asp Lys Lys Tyr Leu Asp Thr Val Lys Lys Val Met Ser Tyr Arg Gln
1060 1065 1070
Met Asn Ile Val Lys Lys Thr Glu Ile Gln Lys Gly Glu Phe Ser Lys
1075 1080 1085
Ala Thr Ile Lys Pro Lys Gly Asn Ser Ser Lys Leu Ile Pro Arg Lys
1090 1095 1100
Thr Asn Trp Asp Pro Met Lys Tyr Gly Gly Leu Asp Ser Pro Asn Met
1105 1110 1115 1120
Ala Tyr Ala Val Val Ile Glu Tyr Ala Lys Gly Lys Asn Lys Leu Val
1125 1130 1135
Phe Glu Lys Lys Ile Ile Arg Val Thr Ile Met Glu Arg Lys Ala Phe
1140 1145 1150
Glu Lys Asp Glu Lys Ala Phe Leu Glu Glu Gln Gly Tyr Arg Gln Pro
1155 1160 1165
Lys Val Leu Ala Lys Leu Pro Lys Tyr Thr Leu Tyr Glu Cys Glu Glu
1170 1175 1180
Gly Arg Arg Arg Met Leu Ala Ser Ala Asn Glu Ala Gln Lys Gly Asn
1185 1190 1195 1200
Gln Gln Val Leu Pro Asn His Leu Val Thr Leu Leu His His Ala Ala
1205 1210 1215
Asn Cys Glu Val Ser Asp Gly Lys Ser Leu Asp Tyr Ile Glu Ser Asn
1220 1225 1230
Arg Glu Met Phe Ala Glu Leu Leu Ala His Val Ser Glu Phe Ala Lys
1235 1240 1245
Arg Tyr Thr Leu Ala Glu Ala Asn Leu Asn Lys Ile Asn Gln Leu Phe
1250 1255 1260
Glu Gln Asn Lys Glu Gly Asp Ile Lys Ala Ile Ala Gln Ser Phe Val
1265 1270 1275 1280
Asp Leu Met Ala Phe Asn Ala Met Gly Ala Pro Ala Ser Phe Lys Phe
1285 1290 1295
Phe Glu Thr Thr Ile Glu Arg Lys Arg Tyr Asn Asn Leu Lys Glu Leu
1300 1305 1310
Leu Asn Ser Thr Ile Ile Tyr Gln Ser Ile Thr Gly Leu Tyr Glu Ser
1315 1320 1325
Arg Lys Arg Leu Asp Asp
1330
<210> 29
<211> 984
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自空肠弯曲杆菌（空肠弯曲杆菌（Campylobacter jejuni））
<400> 29
Met Ala Arg Ile Leu Ala Phe Asp Ile Gly Ile Ser Ser Ile Gly Trp
1 5 10 15
Ala Phe Ser Glu Asn Asp Glu Leu Lys Asp Cys Gly Val Arg Ile Phe
20 25 30
Thr Lys Val Glu Asn Pro Lys Thr Gly Glu Ser Leu Ala Leu Pro Arg
35 40 45
Arg Leu Ala Arg Ser Ala Arg Lys Arg Leu Ala Arg Arg Lys Ala Arg
50 55 60
Leu Asn His Leu Lys His Leu Ile Ala Asn Glu Phe Lys Leu Asn Tyr
65 70 75 80
Glu Asp Tyr Gln Ser Phe Asp Glu Ser Leu Ala Lys Ala Tyr Lys Gly
85 90 95
Ser Leu Ile Ser Pro Tyr Glu Leu Arg Phe Arg Ala Leu Asn Glu Leu
100 105 110
Leu Ser Lys Gln Asp Phe Ala Arg Val Ile Leu His Ile Ala Lys Arg
115 120 125
Arg Gly Tyr Asp Asp Ile Lys Asn Ser Asp Asp Lys Glu Lys Gly Ala
130 135 140
Ile Leu Lys Ala Ile Lys Gln Asn Glu Glu Lys Leu Ala Asn Tyr Gln
145 150 155 160
Ser Val Gly Glu Tyr Leu Tyr Lys Glu Tyr Phe Gln Lys Phe Lys Glu
165 170 175
Asn Ser Lys Glu Phe Thr Asn Val Arg Asn Lys Lys Glu Ser Tyr Glu
180 185 190
Arg Cys Ile Ala Gln Ser Phe Leu Lys Asp Glu Leu Lys Leu Ile Phe
195 200 205
Lys Lys Gln Arg Glu Phe Gly Phe Ser Phe Ser Lys Lys Phe Glu Glu
210 215 220
Glu Val Leu Ser Val Ala Phe Tyr Lys Arg Ala Leu Lys Asp Phe Ser
225 230 235 240
His Leu Val Gly Asn Cys Ser Phe Phe Thr Asp Glu Lys Arg Ala Pro
245 250 255
Lys Asn Ser Pro Leu Ala Phe Met Phe Val Ala Leu Thr Arg Ile Ile
260 265 270
Asn Leu Leu Asn Asn Leu Lys Asn Thr Glu Gly Ile Leu Tyr Thr Lys
275 280 285
Asp Asp Leu Asn Ala Leu Leu Asn Glu Val Leu Lys Asn Gly Thr Leu
290 295 300
Thr Tyr Lys Gln Thr Lys Lys Leu Leu Gly Leu Ser Asp Asp Tyr Glu
305 310 315 320
Phe Lys Gly Glu Lys Gly Thr Tyr Phe Ile Glu Phe Lys Lys Tyr Lys
325 330 335
Glu Phe Ile Lys Ala Leu Gly Glu His Asn Leu Ser Gln Asp Asp Leu
340 345 350
Asn Glu Ile Ala Lys Asp Ile Thr Leu Ile Lys Asp Glu Ile Lys Leu
355 360 365
Lys Lys Ala Leu Ala Lys Tyr Asp Leu Asn Gln Asn Gln Ile Asp Ser
370 375 380
Leu Ser Lys Leu Glu Phe Lys Asp His Leu Asn Ile Ser Phe Lys Ala
385 390 395 400
Leu Lys Leu Val Thr Pro Leu Met Leu Glu Gly Lys Lys Tyr Asp Glu
405 410 415
Ala Cys Asn Glu Leu Asn Leu Lys Val Ala Ile Asn Glu Asp Lys Lys
420 425 430
Asp Phe Leu Pro Ala Phe Asn Glu Thr Tyr Tyr Lys Asp Glu Val Thr
435 440 445
Asn Pro Val Val Leu Arg Ala Ile Lys Glu Tyr Arg Lys Val Leu Asn
450 455 460
Ala Leu Leu Lys Lys Tyr Gly Lys Val His Lys Ile Asn Ile Glu Leu
465 470 475 480
Ala Arg Glu Val Gly Lys Asn His Ser Gln Arg Ala Lys Ile Glu Lys
485 490 495
Glu Gln Asn Glu Asn Tyr Lys Ala Lys Lys Asp Ala Glu Leu Glu Cys
500 505 510
Glu Lys Leu Gly Leu Lys Ile Asn Ser Lys Asn Ile Leu Lys Leu Arg
515 520 525
Leu Phe Lys Glu Gln Lys Glu Phe Cys Ala Tyr Ser Gly Glu Lys Ile
530 535 540
Lys Ile Ser Asp Leu Gln Asp Glu Lys Met Leu Glu Ile Asp His Ile
545 550 555 560
Tyr Pro Tyr Ser Arg Ser Phe Asp Asp Ser Tyr Met Asn Lys Val Leu
565 570 575
Val Phe Thr Lys Gln Asn Gln Glu Lys Leu Asn Gln Thr Pro Phe Glu
580 585 590
Ala Phe Gly Asn Asp Ser Ala Lys Trp Gln Lys Ile Glu Val Leu Ala
595 600 605
Lys Asn Leu Pro Thr Lys Lys Gln Lys Arg Ile Leu Asp Lys Asn Tyr
610 615 620
Lys Asp Lys Glu Gln Lys Asn Phe Lys Asp Arg Asn Leu Asn Asp Thr
625 630 635 640
Arg Tyr Ile Ala Arg Leu Val Leu Asn Tyr Thr Lys Asp Tyr Leu Asp
645 650 655
Phe Leu Pro Leu Ser Asp Asp Glu Asn Thr Lys Leu Asn Asp Thr Gln
660 665 670
Lys Gly Ser Lys Val His Val Glu Ala Lys Ser Gly Met Leu Thr Ser
675 680 685
Ala Leu Arg His Thr Trp Gly Phe Ser Ala Lys Asp Arg Asn Asn His
690 695 700
Leu His His Ala Ile Asp Ala Val Ile Ile Ala Tyr Ala Asn Asn Ser
705 710 715 720
Ile Val Lys Ala Phe Ser Asp Phe Lys Lys Glu Gln Glu Ser Asn Ser
725 730 735
Ala Glu Leu Tyr Ala Lys Lys Ile Ser Glu Leu Asp Tyr Lys Asn Lys
740 745 750
Arg Lys Phe Phe Glu Pro Phe Ser Gly Phe Arg Gln Lys Val Leu Asp
755 760 765
Lys Ile Asp Glu Ile Phe Val Ser Lys Pro Glu Arg Lys Lys Pro Ser
770 775 780
Gly Ala Leu His Glu Glu Thr Phe Arg Lys Glu Glu Glu Phe Tyr Gln
785 790 795 800
Ser Tyr Gly Gly Lys Glu Gly Val Leu Lys Ala Leu Glu Leu Gly Lys
805 810 815
Ile Arg Lys Val Asn Gly Lys Ile Val Lys Asn Gly Asp Met Phe Arg
820 825 830
Val Asp Ile Phe Lys His Lys Lys Thr Asn Lys Phe Tyr Ala Val Pro
835 840 845
Ile Tyr Thr Met Asp Phe Ala Leu Lys Val Leu Pro Asn Lys Ala Val
850 855 860
Ala Arg Ser Lys Lys Gly Glu Ile Lys Asp Trp Ile Leu Met Asp Glu
865 870 875 880
Asn Tyr Glu Phe Cys Phe Ser Leu Tyr Lys Asp Ser Leu Ile Leu Ile
885 890 895
Gln Thr Lys Asp Met Gln Glu Pro Glu Phe Val Tyr Tyr Asn Ala Phe
900 905 910
Thr Ser Ser Thr Val Ser Leu Ile Val Ser Lys His Asp Asn Lys Phe
915 920 925
Glu Thr Leu Ser Lys Asn Gln Lys Ile Leu Phe Lys Asn Ala Asn Glu
930 935 940
Lys Glu Val Ile Ala Lys Ser Ile Gly Ile Gln Asn Leu Lys Val Phe
945 950 955 960
Glu Lys Tyr Ile Val Ser Ala Leu Gly Glu Val Thr Lys Ala Glu Phe
965 970 975
Arg Gln Arg Glu Asp Phe Lys Lys
980
<210> 30
<211> 1082
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自脑膜炎奈瑟菌（Neisseria. meningitidis）
<400> 30
Met Ala Ala Phe Lys Pro Asn Pro Ile Asn Tyr Ile Leu Gly Leu Asp
1 5 10 15
Ile Gly Ile Ala Ser Val Gly Trp Ala Met Val Glu Ile Asp Glu Asp
20 25 30
Glu Asn Pro Ile Cys Leu Ile Asp Leu Gly Val Arg Val Phe Glu Arg
35 40 45
Ala Glu Val Pro Lys Thr Gly Asp Ser Leu Ala Met Ala Arg Arg Leu
50 55 60
Ala Arg Ser Val Arg Arg Leu Thr Arg Arg Arg Ala His Arg Leu Leu
65 70 75 80
Arg Ala Arg Arg Leu Leu Lys Arg Glu Gly Val Leu Gln Ala Ala Asp
85 90 95
Phe Asp Glu Asn Gly Leu Ile Lys Ser Leu Pro Asn Thr Pro Trp Gln
100 105 110
Leu Arg Ala Ala Ala Leu Asp Arg Lys Leu Thr Pro Leu Glu Trp Ser
115 120 125
Ala Val Leu Leu His Leu Ile Lys His Arg Gly Tyr Leu Ser Gln Arg
130 135 140
Lys Asn Glu Gly Glu Thr Ala Asp Lys Glu Leu Gly Ala Leu Leu Lys
145 150 155 160
Gly Val Ala Asp Asn Ala His Ala Leu Gln Thr Gly Asp Phe Arg Thr
165 170 175
Pro Ala Glu Leu Ala Leu Asn Lys Phe Glu Lys Glu Ser Gly His Ile
180 185 190
Arg Asn Gln Arg Gly Asp Tyr Ser His Thr Phe Ser Arg Lys Asp Leu
195 200 205
Gln Ala Glu Leu Ile Leu Leu Phe Glu Lys Gln Lys Glu Phe Gly Asn
210 215 220
Pro His Val Ser Gly Gly Leu Lys Glu Gly Ile Glu Thr Leu Leu Met
225 230 235 240
Thr Gln Arg Pro Ala Leu Ser Gly Asp Ala Val Gln Lys Met Leu Gly
245 250 255
His Cys Thr Phe Glu Pro Ala Glu Pro Lys Ala Ala Lys Asn Thr Tyr
260 265 270
Thr Ala Glu Arg Phe Ile Trp Leu Thr Lys Leu Asn Asn Leu Arg Ile
275 280 285
Leu Glu Gln Gly Ser Glu Arg Pro Leu Thr Asp Thr Glu Arg Ala Thr
290 295 300
Leu Met Asp Glu Pro Tyr Arg Lys Ser Lys Leu Thr Tyr Ala Gln Ala
305 310 315 320
Arg Lys Leu Leu Gly Leu Glu Asp Thr Ala Phe Phe Lys Gly Leu Arg
325 330 335
Tyr Gly Lys Asp Asn Ala Glu Ala Ser Thr Leu Met Glu Met Lys Ala
340 345 350
Tyr His Ala Ile Ser Arg Ala Leu Glu Lys Glu Gly Leu Lys Asp Lys
355 360 365
Lys Ser Pro Leu Asn Leu Ser Pro Glu Leu Gln Asp Glu Ile Gly Thr
370 375 380
Ala Phe Ser Leu Phe Lys Thr Asp Glu Asp Ile Thr Gly Arg Leu Lys
385 390 395 400
Asp Arg Ile Gln Pro Glu Ile Leu Glu Ala Leu Leu Lys His Ile Ser
405 410 415
Phe Asp Lys Phe Val Gln Ile Ser Leu Lys Ala Leu Arg Arg Ile Val
420 425 430
Pro Leu Met Glu Gln Gly Lys Arg Tyr Asp Glu Ala Cys Ala Glu Ile
435 440 445
Tyr Gly Asp His Tyr Gly Lys Lys Asn Thr Glu Glu Lys Ile Tyr Leu
450 455 460
Pro Pro Ile Pro Ala Asp Glu Ile Arg Asn Pro Val Val Leu Arg Ala
465 470 475 480
Leu Ser Gln Ala Arg Lys Val Ile Asn Gly Val Val Arg Arg Tyr Gly
485 490 495
Ser Pro Ala Arg Ile His Ile Glu Thr Ala Arg Glu Val Gly Lys Ser
500 505 510
Phe Lys Asp Arg Lys Glu Ile Glu Lys Arg Gln Glu Glu Asn Arg Lys
515 520 525
Asp Arg Glu Lys Ala Ala Ala Lys Phe Arg Glu Tyr Phe Pro Asn Phe
530 535 540
Val Gly Glu Pro Lys Ser Lys Asp Ile Leu Lys Leu Arg Leu Tyr Glu
545 550 555 560
Gln Gln His Gly Lys Cys Leu Tyr Ser Gly Lys Glu Ile Asn Leu Gly
565 570 575
Arg Leu Asn Glu Lys Gly Tyr Val Glu Ile Asp His Ala Leu Pro Phe
580 585 590
Ser Arg Thr Trp Asp Asp Ser Phe Asn Asn Lys Val Leu Val Leu Gly
595 600 605
Ser Glu Asn Gln Asn Lys Gly Asn Gln Thr Pro Tyr Glu Tyr Phe Asn
610 615 620
Gly Lys Asp Asn Ser Arg Glu Trp Gln Glu Phe Lys Ala Arg Val Glu
625 630 635 640
Thr Ser Arg Phe Pro Arg Ser Lys Lys Gln Arg Ile Leu Leu Gln Lys
645 650 655
Phe Asp Glu Asp Gly Phe Lys Glu Arg Asn Leu Asn Asp Thr Arg Tyr
660 665 670
Val Asn Arg Phe Leu Cys Gln Phe Val Ala Asp Arg Met Arg Leu Thr
675 680 685
Gly Lys Gly Lys Lys Arg Val Phe Ala Ser Asn Gly Gln Ile Thr Asn
690 695 700
Leu Leu Arg Gly Phe Trp Gly Leu Arg Lys Val Arg Ala Glu Asn Asp
705 710 715 720
Arg His His Ala Leu Asp Ala Val Val Val Ala Cys Ser Thr Val Ala
725 730 735
Met Gln Gln Lys Ile Thr Arg Phe Val Arg Tyr Lys Glu Met Asn Ala
740 745 750
Phe Asp Gly Lys Thr Ile Asp Lys Glu Thr Gly Glu Val Leu His Gln
755 760 765
Lys Thr His Phe Pro Gln Pro Trp Glu Phe Phe Ala Gln Glu Val Met
770 775 780
Ile Arg Val Phe Gly Lys Pro Asp Gly Lys Pro Glu Phe Glu Glu Ala
785 790 795 800
Asp Thr Pro Glu Lys Leu Arg Thr Leu Leu Ala Glu Lys Leu Ser Ser
805 810 815
Arg Pro Glu Ala Val His Glu Tyr Val Thr Pro Leu Phe Val Ser Arg
820 825 830
Ala Pro Asn Arg Lys Met Ser Gly Gln Gly His Met Glu Thr Val Lys
835 840 845
Ser Ala Lys Arg Leu Asp Glu Gly Val Ser Val Leu Arg Val Pro Leu
850 855 860
Thr Gln Leu Lys Leu Lys Asp Leu Glu Lys Met Val Asn Arg Glu Arg
865 870 875 880
Glu Pro Lys Leu Tyr Glu Ala Leu Lys Ala Arg Leu Glu Ala His Lys
885 890 895
Asp Asp Pro Ala Lys Ala Phe Ala Glu Pro Phe Tyr Lys Tyr Asp Lys
900 905 910
Ala Gly Asn Arg Thr Gln Gln Val Lys Ala Val Arg Val Glu Gln Val
915 920 925
Gln Lys Thr Gly Val Trp Val Arg Asn His Asn Gly Ile Ala Asp Asn
930 935 940
Ala Thr Met Val Arg Val Asp Val Phe Glu Lys Gly Asp Lys Tyr Tyr
945 950 955 960
Leu Val Pro Ile Tyr Ser Trp Gln Val Ala Lys Gly Ile Leu Pro Asp
965 970 975
Arg Ala Val Val Gln Gly Lys Asp Glu Glu Asp Trp Gln Leu Ile Asp
980 985 990
Asp Ser Phe Asn Phe Lys Phe Ser Leu His Pro Asn Asp Leu Val Glu
995 1000 1005
Val Ile Thr Lys Lys Ala Arg Met Phe Gly Tyr Phe Ala Ser Cys His
1010 1015 1020
Arg Gly Thr Gly Asn Ile Asn Ile Arg Ile His Asp Leu Asp His Lys
1025 1030 1035 1040
Ile Gly Lys Asn Gly Ile Leu Glu Gly Ile Gly Val Lys Thr Ala Leu
1045 1050 1055
Ser Phe Gln Lys Tyr Gln Ile Asp Glu Leu Gly Lys Glu Ile Arg Pro
1060 1065 1070
Cys Arg Leu Lys Lys Arg Pro Pro Val Arg
1075 1080
<210> 31
<211> 1367
<212> PRT
<213> 人工序列
<220>
<223> Cas9，来自酿脓链球菌（Streptococcus pyogenes）
<400> 31
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Asp Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Gly Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Ala Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Ile Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Arg Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Arg Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Ser Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Ala Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Gly Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly His Ser Leu
705 710 715 720
His Glu Gln Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Ile Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Ile Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser
1010 1015 1020
Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn
1025 1030 1035 1040
Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile
1045 1050 1055
Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val
1060 1065 1070
Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
1075 1080 1085
Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe
1090 1095 1100
Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala
1105 1110 1115 1120
Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro
1125 1130 1135
Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys
1140 1145 1150
Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met
1155 1160 1165
Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys
1170 1175 1180
Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1185 1190 1195 1200
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala
1205 1210 1215
Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr
1250 1255 1260
Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile
1265 1270 1275 1280
Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His
1285 1290 1295
Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe
1300 1305 1310
Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
1315 1320 1325
Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala
1330 1335 1340
Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
1345 1350 1355 1360
Leu Ser Gln Leu Gly Gly Asp
1365
<210> 32
<211> 528
<212> DNA
<213> 人工序列
<220>
<223> zfp编码序列
<400> 32
atggcccagg ctgctcttga gcccggagag aaaccctaca agtgcccgga gtgcggaaag 60
tccttctctg agcggagtca cctccgagag caccagcgga ctcatacggg cgaaaaacca 120
tacaagtgcc cagaatgtgg taaatctttt tctcgggctg acaacctgac tgaacatcag 180
cgcacgcaca ccggtgaaaa accttacaag tgtccagagt gtggcaagag cttttctagt 240
agaaggacct gtcgagcgca tcagcggact cacaccggcg aaaaacccta taagtgtccg 300
gaatgtggaa agagctttag ccgcaacgac acccttactg aacaccagcg aacacacacg 360
ggagaaaaac catataaatg tccggaatgt ggcaaaagtt ttagtcggag tgataaactt 420
acggagcacc aacggacaca caccggagag aagccatata agtgtcctga atgtggaaag 480
tccttctcac agcttgctca tctgcgagca catcagcgca cacacacc 528
<210> 33
<211> 176
<212> PRT
<213> 人工序列
<220>
<223> ZFP氨基酸序列
<400> 33
Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro
1 5 10 15
Glu Cys Gly Lys Ser Phe Ser Glu Arg Ser His Leu Arg Glu His Gln
20 25 30
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
35 40 45
Ser Phe Ser Arg Ala Asp Asn Leu Thr Glu His Gln Arg Thr His Thr
50 55 60
Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Ser
65 70 75 80
Arg Arg Thr Cys Arg Ala His Gln Arg Thr His Thr Gly Glu Lys Pro
85 90 95
Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Arg Asn Asp Thr Leu
100 105 110
Thr Glu His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro
115 120 125
Glu Cys Gly Lys Ser Phe Ser Arg Ser Asp Lys Leu Thr Glu His Gln
130 135 140
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
145 150 155 160
Ser Phe Ser Gln Leu Ala His Leu Arg Ala His Gln Arg Thr His Thr
165 170 175
<210> 34
<211> 555
<212> DNA
<213> 人工序列
<220>
<223> ZNF-E2C核苷酸
<400> 34
atggcgcagg cggcgctgga accgggcgaa aaaccgtata aatgcccgga atgcggcaaa 60
agctttagcc gcaaagatag cctggtgcgc catcagcgca cccataccgg cgaaaaaccg 120
tataaatgcc cggaatgcgg caaaagcttt agccagagcg gcgatctgcg ccgccatcag 180
cgcacccata ccggcgaaaa accgtataaa tgcccggaat gcggcaaaag ctttagcgat 240
tgccgcgatc tggcgcgcca tcagcgcacc cataccggcg aaaaaccgta taaatgcccg 300
gaatgcggca aaagctttag ccagagcagc catctggtgc gccatcagcg cacccatacc 360
ggcgaaaaac cgtataaatg cccggaatgc ggcaaaagct ttagcgattg ccgcgatctg 420
gcgcgccatc agcgcaccca taccggcgaa aaaccgtata aatgcccgga atgcggcaaa 480
agctttagcc gcagcgataa actggtgcgc catcagcgca cccataccgg caaaaaaacc 540
agcggccagg cgggc 555
<210> 35
<211> 185
<212> PRT
<213> 人工序列
<220>
<223> ZNF-E2C氨基酸
<400> 35
Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro
1 5 10 15
Glu Cys Gly Lys Ser Phe Ser Arg Lys Asp Ser Leu Val Arg His Gln
20 25 30
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
35 40 45
Ser Phe Ser Gln Ser Gly Asp Leu Arg Arg His Gln Arg Thr His Thr
50 55 60
Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Asp
65 70 75 80
Cys Arg Asp Leu Ala Arg His Gln Arg Thr His Thr Gly Glu Lys Pro
85 90 95
Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu
100 105 110
Val Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro
115 120 125
Glu Cys Gly Lys Ser Phe Ser Asp Cys Arg Asp Leu Ala Arg His Gln
130 135 140
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
145 150 155 160
Ser Phe Ser Arg Ser Asp Lys Leu Val Arg His Gln Arg Thr His Thr
165 170 175
Gly Lys Lys Thr Ser Gly Gln Ala Gly
180 185
<210> 36
<211> 555
<212> DNA
<213> 人工序列
<220>
<223> ZNF-E3核苷酸
<400> 36
atggcgcagg cggcgctgga accgggcgaa aaaccgtata aatgcccgga atgcggcaaa 60
agctttagcg atccgggcgc gctggtgcgc catcagcgca cccataccgg cgaaaaaccg 120
tataaatgcc cggaatgcgg caaaagcttt agccagagca gccatctggt gcgccatcag 180
cgcacccata ccggcgaaaa accgtataaa tgcccggaat gcggcaaaag ctttagcgat 240
tgccgcgatc tggcgcgcca tcagcgcacc cataccggcg aaaaaccgta taaatgcccg 300
gaatgcggca aaagctttag ccagagcagc catctggtgc gccatcagcg cacccatacc 360
ggcgaaaaac cgtataaatg cccggaatgc ggcaaaagct ttagcgattg ccgcgatctg 420
gcgcgccatc agcgcaccca taccggcgaa aaaccgtata aatgcccgga atgcggcaaa 480
agctttagcc agagcagcca tctggtgcgc catcagcgca cccataccgg caaaaaaacc 540
agcggccagg cgggc 555
<210> 37
<211> 185
<212> PRT
<213> 人工序列
<220>
<223> ZNF-E3氨基酸
<400> 37
Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro
1 5 10 15
Glu Cys Gly Lys Ser Phe Ser Asp Pro Gly Ala Leu Val Arg His Gln
20 25 30
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
35 40 45
Ser Phe Ser Gln Ser Ser His Leu Val Arg His Gln Arg Thr His Thr
50 55 60
Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Asp
65 70 75 80
Cys Arg Asp Leu Ala Arg His Gln Arg Thr His Thr Gly Glu Lys Pro
85 90 95
Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu
100 105 110
Val Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro
115 120 125
Glu Cys Gly Lys Ser Phe Ser Asp Cys Arg Asp Leu Ala Arg His Gln
130 135 140
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
145 150 155 160
Ser Phe Ser Gln Ser Ser His Leu Val Arg His Gln Arg Thr His Thr
165 170 175
Gly Lys Lys Thr Ser Gly Gln Ala Gly
180 185
<210> 38
<211> 528
<212> DNA
<213> 人工序列
<220>
<223> ZNF-TRCa核苷酸
<400> 38
atggcgcagg cggctcttga acccggggag aaaccctata aatgccctga gtgtggcaag 60
agtttttcaa ccacaggaaa cttgacagtc caccaacgga cccacaccgg cgagaaacca 120
tacaagtgtc cggagtgtgg taagtctttc tcaagtcctg ccgaccttac cagacatcaa 180
cgcacacata caggtgaaaa accttacaag tgcccagagt gcggaaaaag tttttcacaa 240
tctggcgacc tccgcaggca ccagcgcact cacaccggtg aaaaaccata caagtgtcct 300
gagtgcggga agagttttag tcaacgagct catctggagc gacaccaaag gactcatact 360
ggggagaaac cgtacaaatg tcccgaatgt gggaagagct tctctaccaa gaattccctt 420
acagagcacc agcgcacgca tacgggagag aagccgtata agtgtccgga atgtggcaag 480
agcttttcca gaagtgacca ccttacaacc caccagagga cgcacacc 528
<210> 39
<211> 176
<212> PRT
<213> 人工序列
<220>
<223> ZNF-TRCa氨基酸
<400> 39
Met Ala Gln Ala Ala Leu Glu Pro Gly Glu Lys Pro Tyr Lys Cys Pro
1 5 10 15
Glu Cys Gly Lys Ser Phe Ser Thr Thr Gly Asn Leu Thr Val His Gln
20 25 30
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
35 40 45
Ser Phe Ser Ser Pro Ala Asp Leu Thr Arg His Gln Arg Thr His Thr
50 55 60
Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln
65 70 75 80
Ser Gly Asp Leu Arg Arg His Gln Arg Thr His Thr Gly Glu Lys Pro
85 90 95
Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Arg Ala His Leu
100 105 110
Glu Arg His Gln Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro
115 120 125
Glu Cys Gly Lys Ser Phe Ser Thr Lys Asn Ser Leu Thr Glu His Gln
130 135 140
Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly Lys
145 150 155 160
Ser Phe Ser Arg Ser Asp His Leu Thr Thr His Gln Arg Thr His Thr
165 170 175
<210> 40
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> AAVS1位点
<400> 40
agacggccgc gtcagagc 18
<210> 41
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 锌指1
<400> 41
Glu Arg Ser His Leu Arg Glu
1 5
<210> 42
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 锌指2
<400> 42
Arg Ala Asp Asn Leu Thr Glu
1 5
<210> 43
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 锌指3
<400> 43
Ser Arg Arg Thr Cys Arg Ala
1 5
<210> 44
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 锌指4
<400> 44
Arg Asn Asp Thr Leu Thr Glu
1 5
<210> 45
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 锌指5
<400> 45
Arg Ser Asp Lys Leu Thr Glu
1 5
<210> 46
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 锌指6
<400> 46
Gln Leu Ala His Leu Arg Ala
1 5
<210> 47
<211> 51
<212> DNA
<213> 人工序列
<220>
<223> 核定位信号
<400> 47
atggctccaa agaaaaagag gaaagtggga atccacggag tccccgccgc t 51
<210> 48
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> GGS接头核酸
<400> 48
ggtggatctg gcggtggatc tggtggcggt 30
<210> 49
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> GGS接头氨基酸
<400> 49
Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
1 5 10
<210> 50
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> GGS4x接头核酸
<400> 50
ggagggagtg gtgggtccgg tggtagtggc ggatcc 36
<210> 51
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> GGS4x接头氨基酸
<400> 51
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
1 5 10
<210> 52
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> GGS5x接头核酸
<400> 52
ggaggctccg gtgggtctgg tgggagcggt ggtagtggcg gatcc 45
<210> 53
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> GGS5x接头氨基酸
<400> 53
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
1 5 10 15
<210> 54
<211> 54
<212> DNA
<213> 人工序列
<220>
<223> GGS6x接头核酸
<400> 54
ggaggcagtg gtgggagcgg tggttccggg ggtagtggtg gttccggggg atcc 54
<210> 55
<211> 18
<212> PRT
<213> 人工序列
<220>
<223> GGS6x接头氨基酸
<400> 55
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15
Gly Ser
<210> 56
<211> 63
<212> DNA
<213> 人工序列
<220>
<223> GGS7x接头核酸
<400> 56
ggaggttctg gaggctccgg tgggtccggg ggaagtgggg ggtcaggcgg atcaggagga 60
tcc 63
<210> 57
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> GGS7x接头氨基酸
<400> 57
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15
Gly Ser Gly Gly Ser
20
<210> 58
<211> 75
<212> DNA
<213> 人工序列
<220>
<223> GGS8x接头核酸
<400> 58
ggaggtagcg gaggttccgg agggagcggc gggagtgggg gaagcggggg aagtggagga 60
tccgggggag gatcc 75
<210> 59
<211> 21
<212> PRT
<213> 人工序列
<220>
<223> GGS8x接头氨基酸
<400> 59
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15
Gly Ser Gly Gly Ser
20
<210> 60
<211> 48
<212> DNA
<213> 人工序列
<220>
<223> 接头XTEN 核酸
<400> 60
tccggtagcg aaacaccggg gacttcagaa tcggccaccc cggagtct 48
<210> 61
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 接头XTEN氨基酸
<400> 61
Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser
1 5 10 15
<210> 62
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 接头B 核酸
<400> 62
ggaagcgccg gtagtgcggc tgggtctggc gagttc 36
<210> 63
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 接头B氨基酸
<400> 63
Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe
1 5 10
<210> 64
<211> 4104
<212> DNA
<213> 人工序列
<220>
<223> 人Cas9 (hCas9) 核酸
<400> 64
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agac 4104
<210> 65
<211> 4104
<212> DNA
<213> 人工序列
<220>
<223> 切口酶Cas9 (nCas9) 核酸
<400> 65
atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac aggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agac 4104
<210> 66
<211> 4104
<212> DNA
<213> 人工序列
<220>
<223> 死Cas9 (dCas9) 核酸
<400> 66
atggacaaga agtactccat tgggctcgct atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggctgct 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaagcta gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agac 4104
<210> 67
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 超活性PiggyBac (PB)转座酶核酸
<400> 67
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 68
<211> 1020
<212> DNA
<213> 人工序列
<220>
<223> 超活性睡美人(SB100)转座酶核酸
<400> 68
atgggaaaat caaaagaaat cagccaagac ctcagaaaaa gaattgtaga cctccacaag 60
tctggttcat ccttgggagc aatttccaaa cgcctggcgg taccacgttc atctgtacaa 120
acaatagtac gcaagtataa acaccatggg accacgcagc cgtcataccg ctcaggaagg 180
agacgcgttc tgtctcctag agatgaacgt actttggtgc gaaaagtgca aatcaatccc 240
agaacaacag caaaggacct tgtgaagatg ctggaggaaa caggtacaaa agtatctata 300
tccacagtaa aacgagtcct atatcgacat aacctgaaag gccactcagc aaggaagaag 360
ccactgctcc aaaaccgaca taagaaagcc agactacggt ttgcaactgc acatggggac 420
aaagatcgta ctttttggag aaatgtcctc tggtctgatg aaacaaaaat agaactgttt 480
ggccataatg accatcgtta tgtttggagg aagaaggggg aggcttgcaa gccgaagaac 540
accatcccaa ccgtgaagca cgggggtggc agcatcatgt tgtgggggtg ctttgctgca 600
ggagggactg gtgcacttca caaaatagat ggcatcatgg acgccgtgca gtatgtggat 660
atattgaagc aacatctcaa gacatcagtc aggaagttaa agcttggtcg caaatgggtc 720
ttccaacacg acaatgaccc caagcatact tccaaagttg tggcaaaatg gcttaaggac 780
aacaaagtca aggtattgga gtggccatca caaagccctg acctcaatcc tatagaaaat 840
ttgtgggcag aactgaaaaa gcgtgtgcga gcaaggaggc ctacaaacct gactcagtta 900
caccagctct gtcaggagga atgggccaaa attcacccaa attattgtgg gaagcttgtg 960
gaaggctacc cgaaacgttt gacccaagtt aaacaattta aaggcaatgc taccaaatac 1020
<210> 69
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 人Cas9 (hCas9)氨基酸
<400> 69
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 70
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 切口酶Cas9 (nCas9)氨基酸
<400> 70
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 71
<211> 1368
<212> PRT
<213> 人工序列
<220>
<223> 死Cas9 (dCas9)氨基酸
<400> 71
Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Ala Ala Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Ala Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365
<210> 72
<211> 1069
<212> PRT
<213> 金黄色葡萄球菌（Staphylococcus aureus）
<400> 72
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Lys Arg Asn Tyr Ile Leu Gly Leu Asp Ile Gly Ile Thr Ser Val
20 25 30
Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly
35 40 45
Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg
50 55 60
Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile
65 70 75 80
Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His
85 90 95
Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu
100 105 110
Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu
115 120 125
Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr
130 135 140
Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala
145 150 155 160
Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys
165 170 175
Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr
180 185 190
Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln
195 200 205
Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg
210 215 220
Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys
225 230 235 240
Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe
245 250 255
Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr
260 265 270
Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn
275 280 285
Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe
290 295 300
Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu
305 310 315 320
Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys
325 330 335
Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr
340 345 350
Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala
355 360 365
Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu
370 375 380
Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser
385 390 395 400
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile
405 410 415
Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala
420 425 430
Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln
435 440 445
Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro
450 455 460
Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile
465 470 475 480
Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg
485 490 495
Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys
500 505 510
Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr
515 520 525
Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp
530 535 540
Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu
545 550 555 560
Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro
565 570 575
Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys
580 585 590
Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu
595 600 605
Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile
610 615 620
Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu
625 630 635 640
Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp
645 650 655
Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu
660 665 670
Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys
675 680 685
Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp
690 695 700
Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp
705 710 715 720
Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys
725 730 735
Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys
740 745 750
Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu
755 760 765
Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp
770 775 780
Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile
785 790 795 800
Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu
805 810 815
Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu
820 825 830
Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His
835 840 845
Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly
850 855 860
Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr
865 870 875 880
Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile
885 890 895
Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp
900 905 910
Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr
915 920 925
Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val
930 935 940
Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser
945 950 955 960
Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala
965 970 975
Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly
980 985 990
Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile
995 1000 1005
Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met
1010 1015 1020
Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr
1025 1030 1035 1040
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu
1045 1050 1055
Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1060 1065
<210> 73
<211> 340
<212> PRT
<213> 人工序列
<220>
<223> 超活性睡美人(SB100)转座酶氨基酸
<400> 73
Met Gly Lys Ser Lys Glu Ile Ser Gln Asp Leu Arg Lys Arg Ile Val
1 5 10 15
Asp Leu His Lys Ser Gly Ser Ser Leu Gly Ala Ile Ser Lys Arg Leu
20 25 30
Ala Val Pro Arg Ser Ser Val Gln Thr Ile Val Arg Lys Tyr Lys His
35 40 45
His Gly Thr Thr Gln Pro Ser Tyr Arg Ser Gly Arg Arg Arg Val Leu
50 55 60
Ser Pro Arg Asp Glu Arg Thr Leu Val Arg Lys Val Gln Ile Asn Pro
65 70 75 80
Arg Thr Thr Ala Lys Asp Leu Val Lys Met Leu Glu Glu Thr Gly Thr
85 90 95
Lys Val Ser Ile Ser Thr Val Lys Arg Val Leu Tyr Arg His Asn Leu
100 105 110
Lys Gly His Ser Ala Arg Lys Lys Pro Leu Leu Gln Asn Arg His Lys
115 120 125
Lys Ala Arg Leu Arg Phe Ala Thr Ala His Gly Asp Lys Asp Arg Thr
130 135 140
Phe Trp Arg Asn Val Leu Trp Ser Asp Glu Thr Lys Ile Glu Leu Phe
145 150 155 160
Gly His Asn Asp His Arg Tyr Val Trp Arg Lys Lys Gly Glu Ala Cys
165 170 175
Lys Pro Lys Asn Thr Ile Pro Thr Val Lys His Gly Gly Gly Ser Ile
180 185 190
Met Leu Trp Gly Cys Phe Ala Ala Gly Gly Thr Gly Ala Leu His Lys
195 200 205
Ile Asp Gly Ile Met Asp Ala Val Gln Tyr Val Asp Ile Leu Lys Gln
210 215 220
His Leu Lys Thr Ser Val Arg Lys Leu Lys Leu Gly Arg Lys Trp Val
225 230 235 240
Phe Gln His Asp Asn Asp Pro Lys His Thr Ser Lys Val Val Ala Lys
245 250 255
Trp Leu Lys Asp Asn Lys Val Lys Val Leu Glu Trp Pro Ser Gln Ser
260 265 270
Pro Asp Leu Asn Pro Ile Glu Asn Leu Trp Ala Glu Leu Lys Lys Arg
275 280 285
Val Arg Ala Arg Arg Pro Thr Asn Leu Thr Gln Leu His Gln Leu Cys
290 295 300
Gln Glu Glu Trp Ala Lys Ile His Pro Asn Tyr Cys Gly Lys Leu Val
305 310 315 320
Glu Gly Tyr Pro Lys Arg Leu Thr Gln Val Lys Gln Phe Lys Gly Asn
325 330 335
Ala Thr Lys Tyr
340
<210> 74
<211> 1236
<212> PRT
<213> 人工序列
<220>
<223> cpf1
<400> 74
Met Ala Pro Lys Lys Lys Arg Lys Val Ser Lys Leu Glu Lys Phe Thr
1 5 10 15
Asn Cys Tyr Ser Leu Ser Lys Thr Leu Arg Phe Lys Ala Ile Pro Val
20 25 30
Gly Lys Thr Gln Glu Asn Ile Asp Asn Lys Arg Leu Leu Val Glu Asp
35 40 45
Glu Lys Arg Ala Glu Asp Tyr Lys Gly Val Lys Lys Leu Leu Asp Arg
50 55 60
Tyr Tyr Leu Ser Phe Ile Asn Asp Val Leu His Ser Ile Lys Leu Lys
65 70 75 80
Asn Leu Asn Asn Tyr Ile Ser Leu Phe Arg Lys Lys Thr Arg Thr Glu
85 90 95
Lys Glu Asn Lys Glu Leu Glu Asn Leu Glu Ile Asn Leu Arg Lys Glu
100 105 110
Ile Ala Lys Ala Phe Lys Gly Asn Glu Gly Tyr Lys Ser Leu Phe Lys
115 120 125
Lys Asp Ile Ile Glu Thr Ile Leu Pro Glu Phe Leu Asp Asp Lys Asp
130 135 140
Glu Ile Ala Leu Val Asn Ser Phe Asn Gly Phe Thr Thr Ala Phe Thr
145 150 155 160
Gly Phe Phe Asp Asn Arg Glu Asn Met Phe Ser Glu Glu Ala Lys Ser
165 170 175
Thr Ser Ile Ala Phe Arg Cys Ile Asn Glu Asn Leu Thr Arg Tyr Ile
180 185 190
Ser Asn Met Asp Ile Phe Glu Lys Val Asp Ala Ile Phe Asp Lys His
195 200 205
Glu Val Gln Glu Ile Lys Glu Lys Ile Leu Asn Ser Asp Tyr Asp Val
210 215 220
Glu Asp Phe Phe Glu Gly Glu Phe Phe Asn Phe Val Leu Thr Gln Glu
225 230 235 240
Gly Ile Asp Val Tyr Asn Ala Ile Ile Gly Gly Phe Val Thr Glu Ser
245 250 255
Gly Glu Lys Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln
260 265 270
Lys Thr Lys Gln Lys Leu Pro Lys Phe Lys Pro Leu Tyr Lys Gln Val
275 280 285
Leu Ser Asp Arg Glu Ser Leu Ser Phe Tyr Gly Glu Gly Tyr Thr Ser
290 295 300
Asp Glu Glu Val Leu Glu Val Phe Arg Asn Thr Leu Asn Lys Asn Ser
305 310 315 320
Glu Ile Phe Ser Ser Ile Lys Lys Leu Glu Lys Leu Phe Lys Asn Phe
325 330 335
Asp Glu Tyr Ser Ser Ala Gly Ile Phe Val Lys Asn Gly Pro Ala Ile
340 345 350
Ser Thr Ile Ser Lys Asp Ile Phe Gly Glu Trp Asn Val Ile Arg Asp
355 360 365
Lys Trp Asn Ala Glu Tyr Asp Asp Ile His Leu Lys Lys Lys Ala Val
370 375 380
Val Thr Glu Lys Tyr Glu Asp Asp Arg Arg Lys Ser Phe Lys Lys Ile
385 390 395 400
Gly Ser Phe Ser Leu Glu Gln Leu Gln Glu Tyr Ala Asp Ala Asp Leu
405 410 415
Ser Val Val Glu Lys Leu Lys Glu Ile Ile Ile Gln Lys Val Asp Glu
420 425 430
Ile Tyr Lys Val Tyr Gly Ser Ser Glu Lys Leu Phe Asp Ala Asp Phe
435 440 445
Val Leu Glu Lys Ser Leu Lys Lys Asn Asp Ala Val Val Ala Ile Met
450 455 460
Lys Asp Leu Leu Asp Ser Val Lys Ser Phe Glu Asn Tyr Ile Lys Ala
465 470 475 480
Phe Phe Gly Glu Gly Lys Glu Thr Asn Arg Asp Glu Ser Phe Tyr Gly
485 490 495
Asp Phe Val Leu Ala Tyr Asp Ile Leu Leu Lys Val Asp His Ile Tyr
500 505 510
Asp Ala Ile Arg Asn Tyr Val Thr Gln Lys Pro Tyr Ser Lys Asp Lys
515 520 525
Phe Lys Leu Tyr Phe Gln Asn Pro Gln Phe Met Gly Gly Trp Asp Lys
530 535 540
Asp Lys Glu Thr Asp Tyr Arg Ala Thr Ile Leu Arg Tyr Gly Ser Lys
545 550 555 560
Tyr Tyr Leu Ala Ile Met Asp Lys Lys Tyr Ala Lys Cys Leu Gln Lys
565 570 575
Ile Asp Lys Asp Asp Val Asn Gly Asn Tyr Glu Lys Ile Asn Tyr Lys
580 585 590
Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Lys
595 600 605
Lys Trp Met Ala Tyr Tyr Asn Pro Ser Glu Asp Ile Gln Lys Ile Tyr
610 615 620
Lys Asn Gly Thr Phe Lys Lys Gly Asp Met Phe Asn Leu Asn Asp Cys
625 630 635 640
His Lys Leu Ile Asp Phe Phe Lys Asp Ser Ile Ser Arg Tyr Pro Lys
645 650 655
Trp Ser Asn Ala Tyr Asp Phe Asn Phe Ser Glu Thr Glu Lys Tyr Lys
660 665 670
Asp Ile Ala Gly Phe Tyr Arg Glu Val Glu Glu Gln Gly Tyr Lys Val
675 680 685
Ser Phe Glu Ser Ala Ser Lys Lys Glu Val Asp Lys Leu Val Glu Glu
690 695 700
Gly Lys Leu Tyr Met Phe Gln Ile Tyr Asn Lys Asp Phe Ser Asp Lys
705 710 715 720
Ser His Gly Thr Pro Asn Leu His Thr Met Tyr Phe Lys Leu Leu Phe
725 730 735
Asp Glu Asn Asn His Gly Gln Ile Arg Leu Ser Gly Gly Ala Glu Leu
740 745 750
Phe Met Arg Arg Ala Ser Leu Lys Lys Glu Glu Leu Val Val His Pro
755 760 765
Ala Asn Ser Pro Ile Ala Asn Lys Asn Pro Asp Asn Pro Lys Lys Thr
770 775 780
Thr Thr Leu Ser Tyr Asp Val Tyr Lys Asp Lys Arg Phe Ser Glu Asp
785 790 795 800
Gln Tyr Glu Leu His Ile Pro Ile Ala Ile Asn Lys Cys Pro Lys Asn
805 810 815
Ile Phe Lys Ile Asn Thr Glu Val Arg Val Leu Leu Lys His Asp Asp
820 825 830
Asn Pro Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr
835 840 845
Ile Val Val Val Asp Gly Lys Gly Asn Ile Val Glu Gln Tyr Ser Leu
850 855 860
Asn Glu Ile Ile Asn Asn Phe Asn Gly Ile Arg Ile Lys Thr Asp Tyr
865 870 875 880
His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln
885 890 895
Asn Trp Thr Ser Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Ile
900 905 910
Ser Gln Val Val His Lys Ile Cys Glu Leu Val Glu Lys Tyr Asp Ala
915 920 925
Val Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe Lys Asn Ser Arg Val
930 935 940
Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Met Leu Ile Asp
945 950 955 960
Lys Leu Asn Tyr Met Val Asp Lys Lys Ser Asn Pro Cys Ala Thr Gly
965 970 975
Gly Ala Leu Lys Gly Tyr Gln Ile Thr Asn Lys Phe Glu Ser Phe Lys
980 985 990
Ser Met Ser Thr Gln Asn Gly Phe Ile Phe Tyr Ile Pro Ala Trp Leu
995 1000 1005
Thr Ser Lys Ile Asp Pro Ser Thr Gly Phe Val Asn Leu Leu Lys Thr
1010 1015 1020
Lys Tyr Thr Ser Ile Ala Asp Ser Lys Lys Phe Ile Ser Ser Phe Asp
1025 1030 1035 1040
Arg Ile Met Tyr Val Pro Glu Glu Asp Leu Phe Glu Phe Ala Leu Asp
1045 1050 1055
Tyr Lys Asn Phe Ser Arg Thr Asp Ala Asp Tyr Ile Lys Lys Trp Lys
1060 1065 1070
Leu Tyr Ser Tyr Gly Asn Arg Ile Arg Ile Phe Arg Asn Pro Lys Lys
1075 1080 1085
Asn Asn Val Phe Asp Trp Glu Glu Val Cys Leu Thr Ser Ala Tyr Lys
1090 1095 1100
Glu Leu Phe Asn Lys Tyr Gly Ile Asn Tyr Gln Gln Gly Asp Ile Arg
1105 1110 1115 1120
Ala Leu Leu Cys Glu Gln Ser Asp Lys Ala Phe Tyr Ser Ser Phe Met
1125 1130 1135
Ala Leu Met Ser Leu Met Leu Gln Met Arg Asn Ser Ile Thr Gly Arg
1140 1145 1150
Thr Asp Val Asp Phe Leu Ile Ser Pro Val Lys Asn Ser Asp Gly Ile
1155 1160 1165
Phe Tyr Asp Ser Arg Asn Tyr Glu Ala Gln Glu Asn Ala Ile Leu Pro
1170 1175 1180
Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Val Leu
1185 1190 1195 1200
Trp Ala Ile Gly Gln Phe Lys Lys Ala Glu Asp Glu Lys Leu Asp Lys
1205 1210 1215
Val Lys Ile Ala Ile Ser Asn Lys Glu Trp Leu Glu Tyr Ala Gln Thr
1220 1225 1230
Ser Val Lys His
1235
<210> 75
<211> 990
<212> PRT
<213> 人工序列
<220>
<223> CasX
<400> 75
Met Ala Pro Lys Lys Lys Arg Lys Val Ser Met Gln Glu Ile Lys Arg
1 5 10 15
Ile Asn Lys Ile Arg Arg Arg Leu Val Lys Asp Ser Asn Thr Lys Lys
20 25 30
Ala Gly Lys Thr Gly Pro Met Lys Thr Leu Leu Val Arg Val Met Thr
35 40 45
Pro Asp Leu Arg Glu Arg Leu Glu Asn Leu Arg Lys Lys Pro Glu Asn
50 55 60
Ile Pro Gln Pro Ile Ser Asn Thr Ser Arg Ala Asn Leu Asn Lys Leu
65 70 75 80
Leu Thr Asp Tyr Thr Glu Met Lys Lys Ala Ile Leu His Val Tyr Trp
85 90 95
Glu Glu Phe Gln Lys Asp Pro Val Gly Leu Met Ser Arg Val Ala Gln
100 105 110
Pro Ala Pro Lys Asn Ile Asp Gln Arg Lys Leu Ile Pro Val Lys Asp
115 120 125
Gly Asn Glu Arg Leu Thr Ser Ser Gly Phe Ala Cys Ser Gln Cys Cys
130 135 140
Gln Pro Leu Tyr Val Tyr Lys Leu Glu Gln Val Asn Asp Lys Gly Lys
145 150 155 160
Pro His Thr Asn Tyr Phe Gly Arg Cys Asn Val Ser Glu His Glu Arg
165 170 175
Leu Ile Leu Leu Ser Pro His Lys Pro Glu Ala Asn Asp Glu Leu Val
180 185 190
Thr Tyr Ser Leu Gly Lys Phe Gly Gln Arg Ala Leu Asp Phe Tyr Ser
195 200 205
Ile His Val Thr Arg Glu Ser Asn His Pro Val Lys Pro Leu Glu Gln
210 215 220
Ile Gly Gly Asn Ser Cys Ala Ser Gly Pro Val Gly Lys Ala Leu Ser
225 230 235 240
Asp Ala Cys Met Gly Ala Val Ala Ser Phe Leu Thr Lys Tyr Gln Asp
245 250 255
Ile Ile Leu Glu His Gln Lys Val Ile Lys Lys Asn Glu Lys Arg Leu
260 265 270
Ala Asn Leu Lys Asp Ile Ala Ser Ala Asn Gly Leu Ala Phe Pro Lys
275 280 285
Ile Thr Leu Pro Pro Gln Pro His Thr Lys Glu Gly Ile Glu Ala Tyr
290 295 300
Asn Asn Val Val Ala Gln Ile Val Ile Trp Val Asn Leu Asn Leu Trp
305 310 315 320
Gln Lys Leu Lys Ile Gly Arg Asp Glu Ala Lys Pro Leu Gln Arg Leu
325 330 335
Lys Gly Phe Pro Ser Phe Pro Leu Val Glu Arg Gln Ala Asn Glu Val
340 345 350
Asp Trp Trp Asp Met Val Cys Asn Val Lys Lys Leu Ile Asn Glu Lys
355 360 365
Lys Glu Asp Gly Lys Val Phe Trp Gln Asn Leu Ala Gly Tyr Lys Arg
370 375 380
Gln Glu Ala Leu Leu Pro Tyr Leu Ser Ser Glu Glu Asp Arg Lys Lys
385 390 395 400
Gly Lys Lys Phe Ala Arg Tyr Gln Phe Gly Asp Leu Leu Leu His Leu
405 410 415
Glu Lys Lys His Gly Glu Asp Trp Gly Lys Val Tyr Asp Glu Ala Trp
420 425 430
Glu Arg Ile Asp Lys Lys Val Glu Gly Leu Ser Lys His Ile Lys Leu
435 440 445
Glu Glu Glu Arg Arg Ser Glu Asp Ala Gln Ser Lys Ala Ala Leu Thr
450 455 460
Asp Trp Leu Arg Ala Lys Ala Ser Phe Val Ile Glu Gly Leu Lys Glu
465 470 475 480
Ala Asp Lys Asp Glu Phe Cys Arg Cys Glu Leu Lys Leu Gln Lys Trp
485 490 495
Tyr Gly Asp Leu Arg Gly Lys Pro Phe Ala Ile Glu Ala Glu Asn Ser
500 505 510
Ile Leu Asp Ile Ser Gly Phe Ser Lys Gln Tyr Asn Cys Ala Phe Ile
515 520 525
Trp Gln Lys Asp Gly Val Lys Lys Leu Asn Leu Tyr Leu Ile Ile Asn
530 535 540
Tyr Phe Lys Gly Gly Lys Leu Arg Phe Lys Lys Ile Lys Pro Glu Ala
545 550 555 560
Phe Glu Ala Asn Arg Phe Tyr Thr Val Ile Asn Lys Lys Ser Gly Glu
565 570 575
Ile Val Pro Met Glu Val Asn Phe Asn Phe Asp Asp Pro Asn Leu Ile
580 585 590
Ile Leu Pro Leu Ala Phe Gly Lys Arg Gln Gly Arg Glu Phe Ile Trp
595 600 605
Asn Asp Leu Leu Ser Leu Glu Thr Gly Ser Leu Lys Leu Ala Asn Gly
610 615 620
Arg Val Ile Glu Lys Thr Leu Tyr Asn Arg Arg Thr Arg Gln Asp Glu
625 630 635 640
Pro Ala Leu Phe Val Ala Leu Thr Phe Glu Arg Arg Glu Val Leu Asp
645 650 655
Ser Ser Asn Ile Lys Pro Met Asn Leu Ile Gly Ile Asp Arg Gly Glu
660 665 670
Asn Ile Pro Ala Val Ile Ala Leu Thr Asp Pro Glu Gly Cys Pro Leu
675 680 685
Ser Arg Phe Lys Asp Ser Leu Gly Asn Pro Thr His Ile Leu Arg Ile
690 695 700
Gly Glu Ser Tyr Lys Glu Lys Gln Arg Thr Ile Gln Ala Ala Lys Glu
705 710 715 720
Val Glu Gln Arg Arg Ala Gly Gly Tyr Ser Arg Lys Tyr Ala Ser Lys
725 730 735
Ala Lys Asn Leu Ala Asp Asp Met Val Arg Asn Thr Ala Arg Asp Leu
740 745 750
Leu Tyr Tyr Ala Val Thr Gln Asp Ala Met Leu Ile Phe Glu Asn Leu
755 760 765
Ser Arg Gly Phe Gly Arg Gln Gly Lys Arg Thr Phe Met Ala Glu Arg
770 775 780
Gln Tyr Thr Arg Met Glu Asp Trp Leu Thr Ala Lys Leu Ala Tyr Glu
785 790 795 800
Gly Leu Pro Ser Lys Thr Tyr Leu Ser Lys Thr Leu Ala Gln Tyr Thr
805 810 815
Ser Lys Thr Cys Ser Asn Cys Gly Phe Thr Ile Thr Ser Ala Asp Tyr
820 825 830
Asp Arg Val Leu Glu Lys Leu Lys Lys Thr Ala Thr Gly Trp Met Thr
835 840 845
Thr Ile Asn Gly Lys Glu Leu Lys Val Glu Gly Gln Ile Thr Tyr Tyr
850 855 860
Asn Arg Tyr Lys Arg Gln Asn Val Val Lys Asp Leu Ser Val Glu Leu
865 870 875 880
Asp Arg Leu Ser Glu Glu Ser Val Asn Asn Asp Ile Ser Ser Trp Thr
885 890 895
Lys Gly Arg Ser Gly Glu Ala Leu Ser Leu Leu Lys Lys Arg Phe Ser
900 905 910
His Arg Pro Val Gln Glu Lys Phe Val Cys Leu Asn Cys Gly Phe Glu
915 920 925
Thr His Ala Asp Glu Gln Ala Ala Leu Asn Ile Ala Arg Ser Trp Leu
930 935 940
Phe Leu Arg Ser Gln Glu Tyr Lys Lys Tyr Gln Thr Asn Lys Thr Thr
945 950 955 960
Gly Asn Thr Asp Lys Arg Ala Phe Val Glu Thr Trp Gln Ser Phe Tyr
965 970 975
Arg Lys Lys Leu Lys Glu Val Trp Lys Pro Ala Val Thr Ser
980 985 990
<210> 76
<211> 1069
<212> PRT
<213> 金黄色葡萄球菌（Staphylococcus aureus）
<400> 76
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Ile His Gly Val Pro Ala
1 5 10 15
Ala Lys Arg Asn Tyr Ile Leu Gly Leu Ala Ile Gly Ile Thr Ser Val
20 25 30
Gly Tyr Gly Ile Ile Asp Tyr Glu Thr Arg Asp Val Ile Asp Ala Gly
35 40 45
Val Arg Leu Phe Lys Glu Ala Asn Val Glu Asn Asn Glu Gly Arg Arg
50 55 60
Ser Lys Arg Gly Ala Arg Arg Leu Lys Arg Arg Arg Arg His Arg Ile
65 70 75 80
Gln Arg Val Lys Lys Leu Leu Phe Asp Tyr Asn Leu Leu Thr Asp His
85 90 95
Ser Glu Leu Ser Gly Ile Asn Pro Tyr Glu Ala Arg Val Lys Gly Leu
100 105 110
Ser Gln Lys Leu Ser Glu Glu Glu Phe Ser Ala Ala Leu Leu His Leu
115 120 125
Ala Lys Arg Arg Gly Val His Asn Val Asn Glu Val Glu Glu Asp Thr
130 135 140
Gly Asn Glu Leu Ser Thr Lys Glu Gln Ile Ser Arg Asn Ser Lys Ala
145 150 155 160
Leu Glu Glu Lys Tyr Val Ala Glu Leu Gln Leu Glu Arg Leu Lys Lys
165 170 175
Asp Gly Glu Val Arg Gly Ser Ile Asn Arg Phe Lys Thr Ser Asp Tyr
180 185 190
Val Lys Glu Ala Lys Gln Leu Leu Lys Val Gln Lys Ala Tyr His Gln
195 200 205
Leu Asp Gln Ser Phe Ile Asp Thr Tyr Ile Asp Leu Leu Glu Thr Arg
210 215 220
Arg Thr Tyr Tyr Glu Gly Pro Gly Glu Gly Ser Pro Phe Gly Trp Lys
225 230 235 240
Asp Ile Lys Glu Trp Tyr Glu Met Leu Met Gly His Cys Thr Tyr Phe
245 250 255
Pro Glu Glu Leu Arg Ser Val Lys Tyr Ala Tyr Asn Ala Asp Leu Tyr
260 265 270
Asn Ala Leu Asn Asp Leu Asn Asn Leu Val Ile Thr Arg Asp Glu Asn
275 280 285
Glu Lys Leu Glu Tyr Tyr Glu Lys Phe Gln Ile Ile Glu Asn Val Phe
290 295 300
Lys Gln Lys Lys Lys Pro Thr Leu Lys Gln Ile Ala Lys Glu Ile Leu
305 310 315 320
Val Asn Glu Glu Asp Ile Lys Gly Tyr Arg Val Thr Ser Thr Gly Lys
325 330 335
Pro Glu Phe Thr Asn Leu Lys Val Tyr His Asp Ile Lys Asp Ile Thr
340 345 350
Ala Arg Lys Glu Ile Ile Glu Asn Ala Glu Leu Leu Asp Gln Ile Ala
355 360 365
Lys Ile Leu Thr Ile Tyr Gln Ser Ser Glu Asp Ile Gln Glu Glu Leu
370 375 380
Thr Asn Leu Asn Ser Glu Leu Thr Gln Glu Glu Ile Glu Gln Ile Ser
385 390 395 400
Asn Leu Lys Gly Tyr Thr Gly Thr His Asn Leu Ser Leu Lys Ala Ile
405 410 415
Asn Leu Ile Leu Asp Glu Leu Trp His Thr Asn Asp Asn Gln Ile Ala
420 425 430
Ile Phe Asn Arg Leu Lys Leu Val Pro Lys Lys Val Asp Leu Ser Gln
435 440 445
Gln Lys Glu Ile Pro Thr Thr Leu Val Asp Asp Phe Ile Leu Ser Pro
450 455 460
Val Val Lys Arg Ser Phe Ile Gln Ser Ile Lys Val Ile Asn Ala Ile
465 470 475 480
Ile Lys Lys Tyr Gly Leu Pro Asn Asp Ile Ile Ile Glu Leu Ala Arg
485 490 495
Glu Lys Asn Ser Lys Asp Ala Gln Lys Met Ile Asn Glu Met Gln Lys
500 505 510
Arg Asn Arg Gln Thr Asn Glu Arg Ile Glu Glu Ile Ile Arg Thr Thr
515 520 525
Gly Lys Glu Asn Ala Lys Tyr Leu Ile Glu Lys Ile Lys Leu His Asp
530 535 540
Met Gln Glu Gly Lys Cys Leu Tyr Ser Leu Glu Ala Ile Pro Leu Glu
545 550 555 560
Asp Leu Leu Asn Asn Pro Phe Asn Tyr Glu Val Asp His Ile Ile Pro
565 570 575
Arg Ser Val Ser Phe Asp Asn Ser Phe Asn Asn Lys Val Leu Val Lys
580 585 590
Gln Glu Glu Asn Ser Lys Lys Gly Asn Arg Thr Pro Phe Gln Tyr Leu
595 600 605
Ser Ser Ser Asp Ser Lys Ile Ser Tyr Glu Thr Phe Lys Lys His Ile
610 615 620
Leu Asn Leu Ala Lys Gly Lys Gly Arg Ile Ser Lys Thr Lys Lys Glu
625 630 635 640
Tyr Leu Leu Glu Glu Arg Asp Ile Asn Arg Phe Ser Val Gln Lys Asp
645 650 655
Phe Ile Asn Arg Asn Leu Val Asp Thr Arg Tyr Ala Thr Arg Gly Leu
660 665 670
Met Asn Leu Leu Arg Ser Tyr Phe Arg Val Asn Asn Leu Asp Val Lys
675 680 685
Val Lys Ser Ile Asn Gly Gly Phe Thr Ser Phe Leu Arg Arg Lys Trp
690 695 700
Lys Phe Lys Lys Glu Arg Asn Lys Gly Tyr Lys His His Ala Glu Asp
705 710 715 720
Ala Leu Ile Ile Ala Asn Ala Asp Phe Ile Phe Lys Glu Trp Lys Lys
725 730 735
Leu Asp Lys Ala Lys Lys Val Met Glu Asn Gln Met Phe Glu Glu Lys
740 745 750
Gln Ala Glu Ser Met Pro Glu Ile Glu Thr Glu Gln Glu Tyr Lys Glu
755 760 765
Ile Phe Ile Thr Pro His Gln Ile Lys His Ile Lys Asp Phe Lys Asp
770 775 780
Tyr Lys Tyr Ser His Arg Val Asp Lys Lys Pro Asn Arg Glu Leu Ile
785 790 795 800
Asn Asp Thr Leu Tyr Ser Thr Arg Lys Asp Asp Lys Gly Asn Thr Leu
805 810 815
Ile Val Asn Asn Leu Asn Gly Leu Tyr Asp Lys Asp Asn Asp Lys Leu
820 825 830
Lys Lys Leu Ile Asn Lys Ser Pro Glu Lys Leu Leu Met Tyr His His
835 840 845
Asp Pro Gln Thr Tyr Gln Lys Leu Lys Leu Ile Met Glu Gln Tyr Gly
850 855 860
Asp Glu Lys Asn Pro Leu Tyr Lys Tyr Tyr Glu Glu Thr Gly Asn Tyr
865 870 875 880
Leu Thr Lys Tyr Ser Lys Lys Asp Asn Gly Pro Val Ile Lys Lys Ile
885 890 895
Lys Tyr Tyr Gly Asn Lys Leu Asn Ala His Leu Asp Ile Thr Asp Asp
900 905 910
Tyr Pro Asn Ser Arg Asn Lys Val Val Lys Leu Ser Leu Lys Pro Tyr
915 920 925
Arg Phe Asp Val Tyr Leu Asp Asn Gly Val Tyr Lys Phe Val Thr Val
930 935 940
Lys Asn Leu Asp Val Ile Lys Lys Glu Asn Tyr Tyr Glu Val Asn Ser
945 950 955 960
Lys Cys Tyr Glu Glu Ala Lys Lys Leu Lys Lys Ile Ser Asn Gln Ala
965 970 975
Glu Phe Ile Ala Ser Phe Tyr Asn Asn Asp Leu Ile Lys Ile Asn Gly
980 985 990
Glu Leu Tyr Arg Val Ile Gly Val Asn Asn Asp Leu Leu Asn Arg Ile
995 1000 1005
Glu Val Asn Met Ile Asp Ile Thr Tyr Arg Glu Tyr Leu Glu Asn Met
1010 1015 1020
Asn Asp Lys Arg Pro Pro Arg Ile Ile Lys Thr Ile Ala Ser Lys Thr
1025 1030 1035 1040
Gln Ser Ile Lys Lys Tyr Ser Thr Asp Ile Leu Gly Asn Leu Tyr Glu
1045 1050 1055
Val Lys Ser Lys Lys His Pro Gln Ile Ile Lys Lys Gly
1060 1065
<210> 77
<211> 3207
<212> DNA
<213> 金黄色葡萄球菌（Staphylococcus aureus）
<400> 77
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc caagcggaac 60
tacatcctgg gcctggacat cggcatcacc agcgtgggct acggcatcat cgactacgag 120
acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag aggccaacgt ggaaaacaac 180
gagggcaggc ggagcaagag aggcgccaga aggctgaagc ggcggaggcg gcatagaatc 240
cagagagtga agaagctgct gttcgactac aacctgctga ccgaccacag cgagctgagc 300
ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc agaagctgag cgaggaagag 360
ttctctgccg ccctgctgca cctggccaag agaagaggcg tgcacaacgt gaacgaggtg 420
gaagaggaca ccggcaacga gctgtccacc aaagagcaga tcagccggaa cagcaaggcc 480
ctggaagaga aatacgtggc cgaactgcag ctggaacggc tgaagaaaga cggcgaagtg 540
cggggcagca tcaacagatt caagaccagc gactacgtga aagaagccaa acagctgctg 600
aaggtgcaga aggcctacca ccagctggac cagagcttca tcgacaccta catcgacctg 660
ctggaaaccc ggcggaccta ctatgaggga cctggcgagg gcagcccctt cggctggaag 720
gacatcaaag aatggtacga gatgctgatg ggccactgca cctacttccc cgaggaactg 780
cggagcgtga agtacgccta caacgccgac ctgtacaacg ccctgaacga cctgaacaat 840
ctcgtgatca ccagggacga gaacgagaag ctggaatatt acgagaagtt ccagatcatc 900
gagaacgtgt tcaagcagaa gaagaagccc accctgaagc agatcgccaa agaaatcctc 960
gtgaacgaag aggatattaa gggctacaga gtgaccagca ccggcaagcc cgagttcacc 1020
aacctgaagg tgtaccacga catcaaggac attaccgccc ggaaagagat tattgagaac 1080
gccgagctgc tggatcagat tgccaagatc ctgaccatct accagagcag cgaggacatc 1140
caggaagaac tgaccaatct gaactccgag ctgacccagg aagagatcga gcagatctct 1200
aatctgaagg gctataccgg cacccacaac ctgagcctga aggccatcaa cctgatcctg 1260
gacgagctgt ggcacaccaa cgacaaccag atcgctatct tcaaccggct gaagctggtg 1320
cccaagaagg tggacctgtc ccagcagaaa gagatcccca ccaccctggt ggacgacttc 1380
atcctgagcc ccgtcgtgaa gagaagcttc atccagagca tcaaagtgat caacgccatc 1440
atcaagaagt acggcctgcc caacgacatc attatcgagc tggcccgcga gaagaactcc 1500
aaggacgccc agaaaatgat caacgagatg cagaagcgga accggcagac caacgagcgg 1560
atcgaggaaa tcatccggac caccggcaaa gagaacgcca agtacctgat cgagaagatc 1620
aagctgcacg acatgcagga aggcaagtgc ctgtacagcc tggaagccat ccctctggaa 1680
gatctgctga acaacccctt caactatgag gtggaccaca tcatccccag aagcgtgtcc 1740
ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg aagaaaacag caagaagggc 1800
aaccggaccc cattccagta cctgagcagc agcgacagca agatcagcta cgaaaccttc 1860
aagaagcaca tcctgaatct ggccaagggc aagggcagaa tcagcaagac caagaaagag 1920
tatctgctgg aagaacggga catcaacagg ttctccgtgc agaaagactt catcaaccgg 1980
aacctggtgg ataccagata cgccaccaga ggcctgatga acctgctgcg gagctacttc 2040
agagtgaaca acctggacgt gaaagtgaag tccatcaatg gcggcttcac cagctttctg 2100
cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt acaagcacca cgccgaggac 2160
gccctgatca ttgccaacgc cgatttcatc ttcaaagagt ggaagaaact ggacaaggcc 2220
aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg ccgagagcat gcccgagatc 2280
gaaaccgagc aggagtacaa agagatcttc atcacccccc accagatcaa gcacattaag 2340
gacttcaagg actacaagta cagccaccgg gtggacaaga agcctaatag agagctgatt 2400
aacgacaccc tgtactccac ccggaaggac gacaagggca acaccctgat cgtgaacaat 2460
ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa agctgatcaa caagagcccc 2520
gaaaagctgc tgatgtacca ccacgacccc cagacctacc agaaactgaa gctgattatg 2580
gaacagtacg gcgacgagaa gaatcccctg tacaagtact acgaggaaac cgggaactac 2640
ctgaccaagt actccaaaaa ggacaacggc cccgtgatca agaagattaa gtattacggc 2700
aacaaactga acgcccatct ggacatcacc gacgactacc ccaacagcag aaacaaggtc 2760
gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc tggacaatgg cgtgtacaag 2820
ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa actactacga agtgaatagc 2880
aagtgctatg aggaagctaa gaagctgaag aagatcagca accaggccga gtttatcgcc 2940
tccttctaca acaacgatct gatcaagatc aacggcgagc tgtatagagt gatcggcgtg 3000
aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg acatcaccta ccgcgagtac 3060
ctggaaaaca tgaacgacaa gaggcccccc aggatcatta agacaatcgc ctccaagacc 3120
cagagcatta agaagtacag cacagacatt ctgggcaacc tgtatgaagt gaaatctaag 3180
aagcaccctc agatcatcaa aaagggc 3207
<210> 78
<211> 3708
<212> DNA
<213> 人工序列
<220>
<223> cpf1编码序列
<400> 78
atggccccaa agaagaagcg gaaggtcagc aagctggaga agtttacaaa ctgctactcc 60
ctgtctaaga ccctgaggtt caaggccatc cctgtgggca agacccagga gaacatcgac 120
aataagcggc tgctggtgga ggacgagaag agagccgagg attataaggg cgtgaagaag 180
ctgctggatc gctactatct gtcttttatc aacgacgtgc tgcacagcat caagctgaag 240
aatctgaaca attacatcag cctgttccgg aagaaaacca gaaccgagaa ggagaataag 300
gagctggaga acctggagat caatctgcgg aaggagatcg ccaaggcctt caagggcaac 360
gagggctaca agtccctgtt taagaaggat atcatcgaga caatcctgcc agagttcctg 420
gacgataagg acgagatcgc cctggtgaac agcttcaatg gctttaccac agccttcacc 480
ggcttctttg ataacagaga gaatatgttt tccgaggagg ccaagagcac atccatcgcc 540
ttcaggtgta tcaacgagaa tctgacccgc tacatctcta atatggacat cttcgagaag 600
gtggacgcca tctttgataa gcacgaggtg caggagatca aggagaagat cctgaacagc 660
gactatgatg tggaggattt ctttgagggc gagttcttta actttgtgct gacacaggag 720
ggcatcgacg tgtataacgc catcatcggc ggcttcgtga ccgagagcgg cgagaagatc 780
aagggcctga acgagtacat caacctgtat aatcagaaaa ccaagcagaa gctgcctaag 840
tttaagccac tgtataagca ggtgctgagc gatcgggagt ctctgagctt ctacggcgag 900
ggctatacat ccgatgagga ggtgctggag gtgtttagaa acaccctgaa caagaacagc 960
gagatcttca gctccatcaa gaagctggag aagctgttca agaattttga cgagtactct 1020
agcgccggca tctttgtgaa gaacggcccc gccatcagca caatctccaa ggatatcttc 1080
ggcgagtgga acgtgatccg ggacaagtgg aatgccgagt atgacgatat ccacctgaag 1140
aagaaggccg tggtgaccga gaagtacgag gacgatcgga gaaagtcctt caagaagatc 1200
ggctcctttt ctctggagca gctgcaggag tacgccgacg ccgatctgtc tgtggtggag 1260
aagctgaagg agatcatcat ccagaaggtg gatgagatct acaaggtgta tggctcctct 1320
gagaagctgt tcgacgccga ttttgtgctg gagaagagcc tgaagaagaa cgacgccgtg 1380
gtggccatca tgaaggacct gctggattct gtgaagagct tcgagaatta catcaaggcc 1440
ttctttggcg agggcaagga gacaaacagg gacgagtcct tctatggcga ttttgtgctg 1500
gcctacgaca tcctgctgaa ggtggaccac atctacgatg ccatccgcaa ttatgtgacc 1560
cagaagccct actctaagga taagttcaag ctgtattttc agaaccctca gttcatgggc 1620
ggctgggaca aggataagga gacagactat cgggccacca tcctgagata cggctccaag 1680
tactatctgg ccatcatgga taagaagtac gccaagtgcc tgcagaagat cgacaaggac 1740
gatgtgaacg gcaattacga gaagatcaac tataagctgc tgcccggccc taataagatg 1800
ctgccaaagg tgttcttttc taagaagtgg atggcctact ataaccccag cgaggacatc 1860
cagaagatct acaagaatgg cacattcaag aagggcgata tgtttaacct gaatgactgt 1920
cacaagctga tcgacttctt taaggatagc atctcccggt atccaaagtg gtccaatgcc 1980
tacgatttca acttttctga gacagagaag tataaggaca tcgccggctt ttacagagag 2040
gtggaggagc agggctataa ggtgagcttc gagtctgcca gcaagaagga ggtggataag 2100
ctggtggagg agggcaagct gtatatgttc cagatctata acaaggactt ttccgataag 2160
tctcacggca cacccaatct gcacaccatg tacttcaagc tgctgtttga cgagaacaat 2220
cacggacaga tcaggctgag cggaggagca gagctgttca tgaggcgcgc ctccctgaag 2280
aaggaggagc tggtggtgca cccagccaac tcccctatcg ccaacaagaa tccagataat 2340
cccaagaaaa ccacaaccct gtcctacgac gtgtataagg ataagaggtt ttctgaggac 2400
cagtacgagc tgcacatccc aatcgccatc aataagtgcc ccaagaacat cttcaagatc 2460
aatacagagg tgcgcgtgct gctgaagcac gacgataacc cctatgtgat cggcatcgat 2520
aggggcgagc gcaatctgct gtatatcgtg gtggtggacg gcaagggcaa catcgtggag 2580
cagtattccc tgaacgagat catcaacaac ttcaacggca tcaggatcaa gacagattac 2640
cactctctgc tggacaagaa ggagaaggag aggttcgagg cccgccagaa ctggacctcc 2700
atcgagaata tcaaggagct gaaggccggc tatatctctc aggtggtgca caagatctgc 2760
gagctggtgg agaagtacga tgccgtgatc gccctggagg acctgaactc tggctttaag 2820
aatagccgcg tgaaggtgga gaagcaggtg tatcagaagt tcgagaagat gctgatcgat 2880
aagctgaact acatggtgga caagaagtct aatccttgtg caacaggcgg cgccctgaag 2940
ggctatcaga tcaccaataa gttcgagagc tttaagtcca tgtctaccca gaacggcttc 3000
atcttttaca tccctgcctg gctgacatcc aagatcgatc catctaccgg ctttgtgaac 3060
ctgctgaaaa ccaagtatac cagcatcgcc gattccaaga agttcatcag ctcctttgac 3120
aggatcatgt acgtgcccga ggaggatctg ttcgagtttg ccctggacta taagaacttc 3180
tctcgcacag acgccgatta catcaagaag tggaagctgt actcctacgg caaccggatc 3240
agaatcttcc ggaatcctaa gaagaacaac gtgttcgact gggaggaggt gtgcctgacc 3300
agcgcctata aggagctgtt caacaagtac ggcatcaatt atcagcaggg cgatatcaga 3360
gccctgctgt gcgagcagtc cgacaaggcc ttctactcta gctttatggc cctgatgagc 3420
ctgatgctgc agatgcggaa cagcatcaca ggccgcaccg acgtggattt tctgatcagc 3480
cctgtgaaga actccgacgg catcttctac gatagccgga actatgaggc ccaggagaat 3540
gccatcctgc caaagaacgc cgacgccaat ggcgcctata acatcgccag aaaggtgctg 3600
tgggccatcg gccagttcaa gaaggccgag gacgagaagc tggataaggt gaagatcgcc 3660
atctctaaca aggagtggct ggagtacgcc cagaccagcg tgaagcac 3708
<210> 79
<211> 2970
<212> DNA
<213> 人工序列
<220>
<223> CasX编码序列
<400> 79
atggccccaa agaagaagcg gaaggtcagc atgcaagaga tcaagagaat caacaagatc 60
agaaggagac tggtcaagga cagcaacaca aagaaggccg gcaagacagg ccccatgaaa 120
accctgctcg tcagagtgat gacccctgac ctgagagagc ggctggaaaa cctgagaaag 180
aagcccgaga acatccctca gcctatcagc aacaccagca gggccaacct gaacaagctg 240
ctgaccgact acaccgagat gaagaaagcc atcctgcacg tgtactggga agagttccag 300
aaagaccccg tgggcctgat gagcagagtt gctcagcccg ctcctaagaa catcgaccag 360
agaaagctga tccccgtgaa ggacggcaac gagagactga cctctagcgg ctttgcctgc 420
agccagtgtt gccagcctct gtacgtgtac aagctggaac aagtgaacga caagggcaag 480
ccccacacca actacttcgg cagatgcaac gtgtccgagc acgagaggct gatcctgctg 540
tctcctcaca agcccgaggc caacgatgag ctggtcacat acagcctggg caagttcgga 600
cagagagccc tggacttcta cagcatccac gtgaccaggg agagcaatca ccctgtgaag 660
cccctggaac agatcggcgg caatagctgt gcctctggac ctgtgggaaa agccctgagc 720
gacgcctgta tgggagccgt ggcatccttc ctgaccaagt accaggacat catcctggaa 780
caccagaaag tgatcaagaa gaacgagaaa agactggcca acctcaagga tatcgccagc 840
gctaacggcc tggcctttcc taagatcacc ctgcctccac agcctcacac caaagagggc 900
atcgaggcct acaacaacgt ggtggcccag atcgtgattt gggtcaacct gaatctgtgg 960
cagaagctga agatcggcag ggacgaagcc aagccactgc agagactgaa gggcttccct 1020
agcttccctc tggtggaaag acaggccaat gaagtggatt ggtgggacat ggtctgcaac 1080
gtgaagaagc tgatcaacga gaagaaagag gatggcaagg ttttctggca gaacctggcc 1140
ggctacaaga gacaagaagc cctgctgcct tacctgagca gcgaagagga ccggaagaag 1200
ggcaagaagt tcgccagata ccagttcggc gacctgctgc tgcacctgga aaagaagcac 1260
ggcgaggact ggggcaaagt gtacgatgag gcctgggaga gaatcgacaa gaaggtggaa 1320
ggcctgagca agcacattaa gctggaagag gaaagaagga gcgaggacgc ccaatctaaa 1380
gccgctctga ccgattggct gagagccaag gccagctttg tgatcgaggg cctgaaagag 1440
gccgacaagg acgagttctg cagatgcgag ctgaagctgc agaagtggta cggcgatctg 1500
agaggcaagc ccttcgccat tgaggccgag aacagcatcc tggacatcag cggcttcagc 1560
aagcagtaca actgcgcctt catttggcag aaagacggcg tcaagaaact gaacctgtac 1620
ctgatcatca attacttcaa aggcggcaag ctgcggttca agaagatcaa acccgaggcc 1680
ttcgaggcta acagattcta caccgtgatc aacaaaaagt ccggcgagat cgtgcccatg 1740
gaagtgaact tcaacttcga cgaccccaac ctgattatcc tgcctctggc cttcggcaag 1800
agacagggca gagagttcat ctggaacgat ctgctgagcc tggaaaccgg ctctctgaag 1860
ctggccaatg gcagagtgat cgagaaaacc ctgtacaaca ggagaaccag acaggacgag 1920
cctgctctgt ttgtggccct gaccttcgag agaagagagg tgctggacag cagcaacatc 1980
aagcccatga acctgatcgg catcgaccgg ggcgagaata tccctgctgt gatcgccctg 2040
acagaccctg aaggatgccc actgagcaga ttcaaggact ccctgggcaa ccctacacac 2100
atcctgagaa tcggcgagag ctacaaagag aagcagagga caatccaggc cgccaaagag 2160
gtggaacaga gaagagccgg cggatactct aggaagtacg ccagcaaggc caagaatctg 2220
gccgacgaca tggtccgaaa caccgccaga gatctgctgt actacgccgt gacacaggac 2280
gccatgctga tcttcgagaa tctgagcaga ggcttcggcc ggcagggcaa gagaaccttt 2340
atggccgaga ggcagtacac cagaatggaa gattggctca cagctaaact ggcctacgag 2400
ggactgccca gcaagaccta cctgtccaaa acactggccc agtatacctc caagacctgc 2460
agcaattgcg gcttcaccat caccagcgcc gactacgaca gagtgctgga aaagctcaag 2520
aaaaccgcca ccggctggat gaccaccatc aacggcaaag agctgaaggt tgagggccag 2580
atcacctact acaacaggta caagaggcag aacgtcgtga aggatctgag cgtggaactg 2640
gacagactga gcgaagagag cgtgaacaac gacatcagca gctggacaaa gggcagatca 2700
ggcgaggctc tgagcctgct gaagaagagg tttagccaca gacctgtgca agagaagttc 2760
gtgtgcctga actgcggctt cgagacacac gccgatgaac aggctgccct gaacattgcc 2820
agaagctggc tgttcctgag aagccaagag tacaagaagt accagaccaa caagaccacc 2880
ggcaacaccg acaagagggc ctttgtggaa acctggcaga gcttctacag aaaaaagctg 2940
aaagaagtct ggaagcccgc cgtgactagt 2970
<210> 80
<211> 3213
<212> DNA
<213> 金黄色葡萄球菌（Staphylococcus aureus）
<400> 80
gccaccatgg ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccaag 60
cggaactaca tcctgggcct ggccatcggc atcaccagcg tgggctacgg catcatcgac 120
tacgagacac gggacgtgat cgatgccggc gtgcggctgt tcaaagaggc caacgtggaa 180
aacaacgagg gcaggcggag caagagaggc gccagaaggc tgaagcggcg gaggcggcat 240
agaatccaga gagtgaagaa gctgctgttc gactacaacc tgctgaccga ccacagcgag 300
ctgagcggca tcaaccccta cgaggccaga gtgaagggcc tgagccagaa gctgagcgag 360
gaagagttct ctgccgccct gctgcacctg gccaagagaa gaggcgtgca caacgtgaac 420
gaggtggaag aggacaccgg caacgagctg tccaccaaag agcagatcag ccggaacagc 480
aaggccctgg aagagaaata cgtggccgaa ctgcagctgg aacggctgaa gaaagacggc 540
gaagtgcggg gcagcatcaa cagattcaag accagcgact acgtgaaaga agccaaacag 600
ctgctgaagg tgcagaaggc ctaccaccag ctggaccaga gcttcatcga cacctacatc 660
gacctgctgg aaacccggcg gacctactat gagggacctg gcgagggcag ccccttcggc 720
tggaaggaca tcaaagaatg gtacgagatg ctgatgggcc actgcaccta cttccccgag 780
gaactgcgga gcgtgaagta cgcctacaac gccgacctgt acaacgccct gaacgacctg 840
aacaatctcg tgatcaccag ggacgagaac gagaagctgg aatattacga gaagttccag 900
atcatcgaga acgtgttcaa gcagaagaag aagcccaccc tgaagcagat cgccaaagaa 960
atcctcgtga acgaagagga tattaagggc tacagagtga ccagcaccgg caagcccgag 1020
ttcaccaacc tgaaggtgta ccacgacatc aaggacatta ccgcccggaa agagattatt 1080
gagaacgccg agctgctgga tcagattgcc aagatcctga ccatctacca gagcagcgag 1140
gacatccagg aagaactgac caatctgaac tccgagctga cccaggaaga gatcgagcag 1200
atctctaatc tgaagggcta taccggcacc cacaacctga gcctgaaggc catcaacctg 1260
atcctggacg agctgtggca caccaacgac aaccagatcg ctatcttcaa ccggctgaag 1320
ctggtgccca agaaggtgga cctgtcccag cagaaagaga tccccaccac cctggtggac 1380
gacttcatcc tgagccccgt cgtgaagaga agcttcatcc agagcatcaa agtgatcaac 1440
gccatcatca agaagtacgg cctgcccaac gacatcatta tcgagctggc ccgcgagaag 1500
aactccaagg acgcccagaa aatgatcaac gagatgcaga agcggaaccg gcagaccaac 1560
gagcggatcg aggaaatcat ccggaccacc ggcaaagaga acgccaagta cctgatcgag 1620
aagatcaagc tgcacgacat gcaggaaggc aagtgcctgt acagcctgga agccatccct 1680
ctggaagatc tgctgaacaa ccccttcaac tatgaggtgg accacatcat ccccagaagc 1740
gtgtccttcg acaacagctt caacaacaag gtgctcgtga agcaggaaga aaacagcaag 1800
aagggcaacc ggaccccatt ccagtacctg agcagcagcg acagcaagat cagctacgaa 1860
accttcaaga agcacatcct gaatctggcc aagggcaagg gcagaatcag caagaccaag 1920
aaagagtatc tgctggaaga acgggacatc aacaggttct ccgtgcagaa agacttcatc 1980
aaccggaacc tggtggatac cagatacgcc accagaggcc tgatgaacct gctgcggagc 2040
tacttcagag tgaacaacct ggacgtgaaa gtgaagtcca tcaatggcgg cttcaccagc 2100
tttctgcggc ggaagtggaa gtttaagaaa gagcggaaca aggggtacaa gcaccacgcc 2160
gaggacgccc tgatcattgc caacgccgat ttcatcttca aagagtggaa gaaactggac 2220
aaggccaaaa aagtgatgga aaaccagatg ttcgaggaaa agcaggccga gagcatgccc 2280
gagatcgaaa ccgagcagga gtacaaagag atcttcatca ccccccacca gatcaagcac 2340
attaaggact tcaaggacta caagtacagc caccgggtgg acaagaagcc taatagagag 2400
ctgattaacg acaccctgta ctccacccgg aaggacgaca agggcaacac cctgatcgtg 2460
aacaatctga acggcctgta cgacaaggac aatgacaagc tgaaaaagct gatcaacaag 2520
agccccgaaa agctgctgat gtaccaccac gacccccaga cctaccagaa actgaagctg 2580
attatggaac agtacggcga cgagaagaat cccctgtaca agtactacga ggaaaccggg 2640
aactacctga ccaagtactc caaaaaggac aacggccccg tgatcaagaa gattaagtat 2700
tacggcaaca aactgaacgc ccatctggac atcaccgacg actaccccaa cagcagaaac 2760
aaggtcgtga agctgtccct gaagccctac agattcgacg tgtacctgga caatggcgtg 2820
tacaagttcg tgaccgtgaa gaatctggat gtgatcaaaa aagaaaacta ctacgaagtg 2880
aatagcaagt gctatgagga agctaagaag ctgaagaaga tcagcaacca ggccgagttt 2940
atcgcctcct tctacaacaa cgatctgatc aagatcaacg gcgagctgta tagagtgatc 3000
ggcgtgaaca acgacctgct gaaccggatc gaagtgaaca tgatcgacat cacctaccgc 3060
gagtacctgg aaaacatgaa cgacaagagg ccccccagga tcattaagac aatcgcctcc 3120
aagacccaga gcattaagaa gtacagcaca gacattctgg gcaacctgta tgaagtgaaa 3180
tctaagaagc accctcagat catcaaaaag ggc 3213
<210> 81
<211> 2976
<212> DNA
<213> 空肠弯曲杆菌（Campylobacter jejuni）
<400> 81
atggccccaa agaagaagcg gaaggtcgcc agaatcctgg ccttcgacat cggcatcagc 60
agcatcggct gggccttcag cgagaacgac gagctgaagg actgcggcgt gcggatcttc 120
accaaggtgg aaaaccccaa gaccggcgag agcctggccc tgcccagaag gctggccaga 180
agcgcccgga agagactggc cagacggaag gcccggctga accacctgaa gcacctgatc 240
gccaacgagt tcaagctgaa ctacgaggac taccagagct tcgacgagtc cctggccaag 300
gcctacaagg gcagcctgat cagcccctac gagctgcggt tccgggccct gaacgagctg 360
ctgagcaagc aggacttcgc cagagtgatc ctgcacattg ccaagcggag aggctacgac 420
gacatcaaga acagcgacga caaagagaag ggcgccatcc tgaaggccat caagcagaac 480
gaggaaaagc tggccaacta ccagtccgtg ggcgagtacc tgtacaaaga gtacttccag 540
aagttcaaag agaacagcaa agaattcacc aacgtgcgga acaagaaaga aagctacgag 600
cggtgtatcg cccagagctt cctgaaggat gagctgaagc tgatcttcaa gaagcagaga 660
gagttcggct tcagcttcag caagaaattc gaggaagagg tgctgagcgt cgccttctac 720
aagagagccc tgaaggactt cagccacctc gtgggcaact gcagcttctt caccgacgag 780
aagagagccc ccaagaacag ccccctggcc ttcatgttcg tggccctgac ccggatcatc 840
aacctgctga acaatctgaa gaacaccgag ggcatcctgt acaccaagga cgacctgaac 900
gccctgctga atgaggtgct gaagaacggc accctgacct acaagcagac caagaagctg 960
ctgggcctga gcgacgacta cgagtttaag ggcgagaagg gcacctactt catcgagttc 1020
aagaagtaca aagagttcat caaggccctg ggcgagcaca acctgagcca ggacgatctg 1080
aatgagatcg ccaaggacat caccctgatc aaggacgaga ttaagctgaa gaaggccctg 1140
gccaaatacg acctgaatca gaaccagatc gacagcctga gcaagctgga attcaaggat 1200
cacctgaaca tcagcttcaa ggctctgaag ctggtcaccc ccctgatgct ggaaggcaag 1260
aagtacgacg aggcctgcaa cgagctgaac ctgaaggtgg ccatcaacga ggacaagaag 1320
gacttcctgc ccgccttcaa cgaaacctac tacaaggacg aagtgaccaa ccccgtggtg 1380
ctgcgggcca tcaaagaata ccggaaggtg ctgaatgccc tgctcaagaa atacggcaag 1440
gtgcacaaga tcaacatcga gctggcccgg gaagtgggca agaaccacag ccagcgggcc 1500
aagatcgaga aagagcagaa cgaaaactac aaggccaaga aggacgctga gctggaatgc 1560
gagaagctgg gactgaagat caacagcaag aacatcctga agctgcggct gttcaaagaa 1620
cagaaagagt tctgcgccta cagcggcgag aagatcaaga tcagcgatct gcaggacgag 1680
aagatgctgg aaatcgacca catctacccc tacagccggt ccttcgacga cagctacatg 1740
aacaaggtgc tggtgttcac caaacagaac caggaaaaac tgaaccagac ccccttcgag 1800
gccttcggca acgacagcgc caagtggcag aaaatcgagg tgctggccaa gaacctgccc 1860
accaagaaac agaagagaat cctggacaag aattacaagg acaaagagca gaagaacttc 1920
aaggaccgga acctgaacga cacccggtat atcgcccggc tggtgctgaa ctacacaaag 1980
gactacctgg atttcctgcc cctgtccgac gacgagaaca ccaagctgaa cgatacccag 2040
aaaggctcca aggtgcacgt ggaagccaag agcggcatgc tgaccagcgc cctgagacac 2100
acctggggct tcagcgccaa ggatcggaac aaccatctgc accacgccat cgacgccgtg 2160
atcattgcct acgccaacaa cagcatcgtg aaggccttct ccgacttcaa gaaagaacag 2220
gaaagcaaca gcgccgagct gtacgccaag aagatctctg agctggacta caagaacaag 2280
cggaagttct tcgagccctt cagcggcttc cggcagaagg tgctggataa gatcgacgag 2340
atcttcgtgt ccaagcccga gcggaagaag ccctctggcg ccctgcacga ggaaaccttc 2400
agaaaagagg aagagttcta ccagtcctac ggcggcaaag aaggcgtgct gaaggccctc 2460
gagctgggca agatcagaaa agtgaacggc aagatcgtga agaacgggga catgttccgg 2520
gtggacatct tcaagcacaa aaagaccaac aagttctacg ccgtgcccat ctacaccatg 2580
gacttcgccc tgaaggtgct gcccaacaag gccgtggccc ggtccaagaa gggcgagatc 2640
aaggactgga ttctgatgga cgagaactac gagttctgct ttagcctgta caaggactcc 2700
ctgatcctga tccagaccaa ggacatgcag gaacccgagt tcgtctacta caacgccttc 2760
accagcagca ccgtgtccct gatcgtgtct aagcacgaca acaagttcga gacactgagc 2820
aagaaccaga agatcctgtt caagaacgcc aacgagaaag aagtgatcgc caagagcatc 2880
ggcatccaga atctgaaggt gttcgagaag tacatcgtgt ccgccctggg agaagtgaca 2940
aaggccgagt tccggcagag agaggacttc aaaaag 2976
<210> 82
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac D450N/R372A/K375A
<400> 82
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtggccagca acgccagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctgaac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 83
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac R275A/R277A
<400> 83
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcgccggcgc ctgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 84
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac K409A/K412A
<400> 84
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccgccccc gccgccatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 85
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac R460A/K461A
<400> 85
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcgcc 1380
gccaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 86
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac S351A/R372A/K375A/R388A/
D450N/W465A/S573A/M589V/S594L
<400> 86
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc gccatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtggccagca acgccagaga gatccccgag 1140
gtcctgaaga acagcaggtc cgcccccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctgaac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca gggcccccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccgcct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacgtgtgc cagaggtgtc tc 1782
<210> 87
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
R275A/R277A/N347S/K375A/T560A/S573A/M589V/S594L
<400> 87
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcgccggcgc ctgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc cgcccccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctgaac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca gggcccccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccgcct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacgtgtgc cagagctgtc tc 1782
<210> 88
<211> 1133
<212> DNA
<213> 人工序列
<220>
<223> 工程化的PiggyBac R245A/R275A/R277A/R372A/W465A
<400> 88
tcgacttcct gatcagatgc ctgaggatgg acgacaagag catcaggccc accctgcggg 60
agaacgacgt gttcaccccc gtggccaaga tctgggacct gttcatccac cagtgcatcc 120
agaactacac ccctggcgcc cacctgacca tcgacgagca gctgctgggc ttcgccggcg 180
cctgcccctt cagggtctat atccccaaca agcccagcaa gtacggcatc aagatcctga 240
tgatgtgcga cagcggcacc aagtacatga tcaacggcat gccctacctg ggcaggggca 300
cccagaccaa cggcgtgccc ctgggcgagt actacgtgaa ggagctgtcc aagcccgtcc 360
acggcagctg cagaaacatc acctgcgaca actggttcac cagcatcccc ctggccaaga 420
acctgctgca ggagccctac aagctgacca tcgtgggcac cgtggccagc aacaagagag 480
agatccccga ggtcctgaag aacagcaggt ccaggcccgt gggcaccagc atgttctgct 540
tcgacggccc cctgaccctg gtgtcctaca agcccaagcc cgccaagatg gtgtacctgc 600
tgtccagctg cgacgaggac gccagcatca acgagagcac cggcaagccc cagatggtga 660
tgtactacaa ccagaccaag ggcggcgtgg acaccctgga ccagatgtgc agcgtgatga 720
cctgcagcag aaagaccaac agggccccca tggccctgct gtacggcatg atcaacatcg 780
cctgcatcaa cagcttcatc atctacagcc acaacgtgag cagcaagggc gagaaggtgc 840
agagccggaa aaagttcatg cggaacctgt acatgggcct gacctccagc ttcatgagga 900
agaggctgga ggcccccacc ctgaagagat acctgaggga caacatcagc aacatcctgc 960
ccaaagaggt gcccggcacc agcgacgaca gcaccgagga gcccgtgatg aagaagagga 1020
cctactgcac ctactgtccc agcaagatca gaagaaaggc cagcgccagc tgcaagaagt 1080
gtaagaaggt catctgccgg gagcacaaca tcgacatgtg ccagagctgt ttc 1133
<210> 89
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
R372A/K375A/R388A/D450N/W465A/S573A/M589V/S594L
<400> 89
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtggccagca acgccagaga gatccccgag 1140
gtcctgaaga acagcaggtc cgcccccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctgaac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca gggcccccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccgcct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacgtgtgc cagaggtgtc tc 1782
<210> 90
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac M194A
<400> 90
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Ala Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 91
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac R245A
<400> 91
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Ala Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 92
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac R325A
<400> 92
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 93
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac R372A
<400> 93
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 94
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac R375A
<400> 94
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 95
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac R376A
<400> 95
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Ala Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 96
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac E377A
<400> 96
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Ala Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 97
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac E380A
<400> 97
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Ala Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 98
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac D450N
<400> 98
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 99
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 工程化的Piggybac S564P
<400> 99
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 100
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
<400> 100
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccacg ccagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 101
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
<400> 101
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tggccaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 102
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
<400> 102
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tggccgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 103
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
<400> 103
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtggccagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 104
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac R375A
<400> 104
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acgccagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 105
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac R376A
<400> 105
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaaggccga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 106
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac E377A
<400> 106
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagagc catccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 107
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac E380A
<400> 107
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgcc 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 108
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac D450N
<400> 108
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctgaac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtccca gcaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 109
<211> 1782
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac S564P
<400> 109
atgggcagca gcctggacga cgagcacatc ctgagcgccc tgctgcagag cgacgacgag 60
ctggtcggcg aggacagcga cagcgaggtg agcgaccacg tgagcgagga cgacgtgcag 120
tccgacaccg aggaggcctt catcgacgag gtgcacgagg tgcagcctac cagcagcggc 180
tccgagatcc tggacgagca gaacgtgatc gagcagcccg gcagctccct ggccagcaac 240
aggatcctga ccctgcccca gaggaccatc aggggcaaga acaagcactg ctggtccacc 300
tccaagccca ccaggcggag cagggtgtcc gccctgaaca tcgtgagaag ccagaggggc 360
cccaccagga tgtgcaggaa catctacgac cccctgctgt gcttcaagct gttcttcacc 420
gacgagatca tcagcgagat cgtgaagtgg accaacgccg agatcagcct gaagaggcgg 480
gagagcatga cctccgccac cttcagggac accaacgagg acgagatcta cgccttcttc 540
ggcatcctgg tgatgaccgc cgtgaggaag gacaaccaca tgagcaccga cgacctgttc 600
gacagatccc tgagcatggt gtacgtgagc gtgatgagca gggacagatt cgacttcctg 660
atcagatgcc tgaggatgga cgacaagagc atcaggccca ccctgcggga gaacgacgtg 720
ttcacccccg tgagaaagat ctgggacctg ttcatccacc agtgcatcca gaactacacc 780
cctggcgccc acctgaccat cgacgagcag ctgctgggct tcaggggcag gtgccccttc 840
agggtctata tccccaacaa gcccagcaag tacggcatca agatcctgat gatgtgcgac 900
agcggcacca agtacatgat caacggcatg ccctacctgg gcaggggcac ccagaccaac 960
ggcgtgcccc tgggcgagta ctacgtgaag gagctgtcca agcccgtcca cggcagctgc 1020
agaaacatca cctgcgacaa ctggttcacc agcatccccc tggccaagaa cctgctgcag 1080
gagccctaca agctgaccat cgtgggcacc gtgagaagca acaagagaga gatccccgag 1140
gtcctgaaga acagcaggtc caggcccgtg ggcaccagca tgttctgctt cgacggcccc 1200
ctgaccctgg tgtcctacaa gcccaagccc gccaagatgg tgtacctgct gtccagctgc 1260
gacgaggacg ccagcatcaa cgagagcacc ggcaagcccc agatggtgat gtactacaac 1320
cagaccaagg gcggcgtgga caccctggac cagatgtgca gcgtgatgac ctgcagcaga 1380
aagaccaaca ggtggcccat ggccctgctg tacggcatga tcaacatcgc ctgcatcaac 1440
agcttcatca tctacagcca caacgtgagc agcaagggcg agaaggtgca gagccggaaa 1500
aagttcatgc ggaacctgta catgggcctg acctccagct tcatgaggaa gaggctggag 1560
gcccccaccc tgaagagata cctgagggac aacatcagca acatcctgcc caaagaggtg 1620
cccggcacca gcgacgacag caccgaggag cccgtgatga agaagaggac ctactgcacc 1680
tactgtcccc ccaagatcag aagaaaggcc agcgccagct gcaagaagtg taagaaggtc 1740
atctgccggg agcacaacat cgacatgtgc cagagctgtt tc 1782
<210> 110
<211> 5931
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac
S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/592G/S594L
<400> 110
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga aacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agattccggt agcgaaacac cggggacttc agaatcggcc 4140
accccggagt ctggcagcag cctggacgac gagcacatcc tgagcgccct gctgcagagc 4200
gacgacgagc tggtcggcga ggacagcgac agcgaggtga gcgaccacgt gagcgaggac 4260
gacgtgcagt ccgacaccga ggaggccttc atcgacgagg tgcacgaggt gcagcctacc 4320
agcagcggct ccgagatcct ggacgagcag aacgtgatcg agcagcccgg cagctccctg 4380
gccagcaaca ggatcctgac cctgccccag aggaccatca ggggcaagaa caagcactgc 4440
tggtccacct ccaagcccac caggcggagc agggtgtccg ccctgaacat cgtgagaagc 4500
cagaggggcc ccaccaggat gtgcaggaac atctacgacc ccctgctgtg cttcaagctg 4560
ttcttcaccg acgagatcat cagcgagatc gtgaagtgga ccaacgccga gatcagcctg 4620
aagaggcggg agagcatgac ctccgccacc ttcagggaca ccaacgagga cgagatctac 4680
gccttcttcg gcatcctggt gatgaccgcc gtgaggaagg acaaccacat gagcaccgac 4740
gacctgttcg acagatccct gagcatggtg tacgtgagcg tgatgagcag ggacagattc 4800
gacttcctga tcagatgcct gaggatggac gacaagagca tcaggcccac cctgcgggag 4860
aacgacgtgt tcacccccgt gagaaagatc tgggacctgt tcatccacca gtgcatccag 4920
aactacaccc ctggcgccca cctgaccatc gacgagcagc tgctgggctt caggggcagg 4980
tgccccttca gggtctatat ccccaacaag cccagcaagt acggcatcaa gatcctgatg 5040
atgtgcgaca gcggcaccaa gtacatgatc aacggcatgc cctacctggg caggggcacc 5100
cagaccaacg gcgtgcccct gggcgagtac tacgtgaagg agctgtccaa gcccgtccac 5160
ggcagctgca gaaacatcac ctgcgacaac tggttcaccg ccatccccct ggccaagaac 5220
ctgctgcagg agccctacaa gctgaccatc gtgggcaccg tggccagcaa cgccagagag 5280
atccccgagg tcctgaagaa cagcaggtcc gcccccgtgg gcaccagcat gttctgcttc 5340
gacggccccc tgaccctggt gtcctacaag cccaagcccg ccaagatggt gtacctgctg 5400
tccagctgcg acgaggacgc cagcatcaac gagagcaccg gcaagcccca gatggtgatg 5460
tactacaacc agaccaaggg cggcgtggac accctgaacc agatgtgcag cgtgatgacc 5520
tgcagcagaa agaccaacag ggcccccatg gccctgctgt acggcatgat caacatcgcc 5580
tgcatcaaca gcttcatcat ctacagccac aacgtgagca gcaagggcga gaaggtgcag 5640
agccggaaaa agttcatgcg gaacctgtac atgggcctga cctccagctt catgaggaag 5700
aggctggagg cccccaccct gaagagatac ctgagggaca acatcagcaa catcctgccc 5760
aaagaggtgc ccggcaccag cgacgacagc accgaggagc ccgtgatgaa gaagaggacc 5820
tactgcacct actgtcccag caagatcaga agaaaggcca gcgccgcctg caagaagtgt 5880
aagaaggtca tctgccggga gcacaacatc gacgtgtgcc agggctgttt g 5931
<210> 111
<211> 5931
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac R245A/R275A/R277A/R372A/W465A/M589V
<400> 111
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga aacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agattccggt agcgaaacac cggggacttc agaatcggcc 4140
accccggagt ctggcagcag cctggacgac gagcacatcc tgagcgccct gctgcagagc 4200
gacgacgagc tggtcggcga ggacagcgac agcgaggtga gcgaccacgt gagcgaggac 4260
gacgtgcagt ccgacaccga ggaggccttc atcgacgagg tgcacgaggt gcagcctacc 4320
agcagcggct ccgagatcct ggacgagcag aacgtgatcg agcagcccgg cagctccctg 4380
gccagcaaca ggatcctgac cctgccccag aggaccatca ggggcaagaa caagcactgc 4440
tggtccacct ccaagcccac caggcggagc agggtgtccg ccctgaacat cgtgagaagc 4500
cagaggggcc ccaccaggat gtgcaggaac atctacgacc ccctgctgtg cttcaagctg 4560
ttcttcaccg acgagatcat cagcgagatc gtgaagtgga ccaacgccga gatcagcctg 4620
aagaggcggg agagcatgac ctccgccacc ttcagggaca ccaacgagga cgagatctac 4680
gccttcttcg gcatcctggt gatgaccgcc gtgaggaagg acaaccacat gagcaccgac 4740
gacctgttcg acagatccct gagcatggtg tacgtgagcg tgatgagcag ggacagattc 4800
gacttcctga tcagatgcct gaggatggac gacaagagca tcaggcccac cctgcgggag 4860
aacgacgtgt tcacccccgt gagaaagatc tgggacctgt tcatccacca gtgcatccag 4920
aactacaccc ctggcgccca cctgaccatc gacgagcagc tgctgggctt caggggcagg 4980
tgccccttca gggtctatat ccccaacaag cccagcaagt acggcatcaa gatcctgatg 5040
atgtgcgaca gcggcaccaa gtacatgatc aacggcatgc cctacctggg caggggcacc 5100
cagaccaacg gcgtgcccct gggcgagtac tacgtgaagg agctgtccaa gcccgtccac 5160
ggcagctgca gaaacatcac ctgcgacaac tggttcaccg ccatccccct ggccaagaac 5220
ctgctgcagg agccctacaa gctgaccatc gtgggcaccg tggccagcaa cgccagagag 5280
atccccgagg tcctgaagaa cagcaggtcc gcccccgtgg gcaccagcat gttctgcttc 5340
gacggccccc tgaccctggt gtcctacaag cccaagcccg ccaagatggt gtacctgctg 5400
tccagctgcg acgaggacgc cagcatcaac gagagcaccg gcaagcccca gatggtgatg 5460
tactacaacc agaccaaggg cggcgtggac accctgaacc agatgtgcag cgtgatgacc 5520
tgcagcagaa agaccaacag ggcccccatg gccctgctgt acggcatgat caacatcgcc 5580
tgcatcaaca gcttcatcat ctacagccac aacgtgagca gcaagggcga gaaggtgcag 5640
agccggaaaa agttcatgcg gaacctgtac atgggcctga cctccagctt catgaggaag 5700
aggctggagg cccccaccct gaagagatac ctgagggaca acatcagcaa catcctgccc 5760
aaagaggtgc ccggcaccag cgacgacagc accgaggagc ccgtgatgaa gaagaggacc 5820
tactgcacct actgtccccc caagatcaga agaaaggcca gcgccgcctg caagaagtgt 5880
aagaaggtca tctgccggga gcacaacatc gacatgtgcc agaggtgtct c 5931
<210> 112
<211> 5931
<212> DNA
<213> 人工序列
<220>
<223> 工程化的Piggybac R275A/325A/R372A/T560A
<400> 112
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga aacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agattccggt agcgaaacac cggggacttc agaatcggcc 4140
accccggagt ctggcagcag cctggacgac gagcacatcc tgagcgccct gctgcagagc 4200
gacgacgagc tggtcggcga ggacagcgac agcgaggtga gcgaccacgt gagcgaggac 4260
gacgtgcagt ccgacaccga ggaggccttc atcgacgagg tgcacgaggt gcagcctacc 4320
agcagcggct ccgagatcct ggacgagcag aacgtgatcg agcagcccgg cagctccctg 4380
gccagcaaca ggatcctgac cctgccccag aggaccatca ggggcaagaa caagcactgc 4440
tggtccacct ccaagcccac caggcggagc agggtgtccg ccctgaacat cgtgagaagc 4500
cagaggggcc ccaccaggat gtgcaggaac atctacgacc ccctgctgtg cttcaagctg 4560
ttcttcaccg acgagatcat cagcgagatc gtgaagtgga ccaacgccga gatcagcctg 4620
aagaggcggg agagcatgac ctccgccacc ttcagggaca ccaacgagga cgagatctac 4680
gccttcttcg gcatcctggt gatgaccgcc gtgaggaagg acaaccacat gagcaccgac 4740
gacctgttcg acagatccct gagcatggtg tacgtgagcg tgatgagcag ggacagattc 4800
gacttcctga tcagatgcct gaggatggac gacaagagca tcaggcccac cctgcgggag 4860
aacgacgtgt tcacccccgt gagaaagatc tgggacctgt tcatccacca gtgcatccag 4920
aactacaccc ctggcgccca cctgaccatc gacgagcagc tgctgggctt cgccggcagg 4980
tgccccttca gggtctatat ccccaacaag cccagcaagt acggcatcaa gatcctgatg 5040
atgtgcgaca gcggcaccaa gtacatgatc aacggcatgc cctacctggg caggggcacc 5100
cagaccaacg gcgtgcccct ggccgagtac tacgtgaagg agctgtccaa gcccgtccac 5160
ggcagctgca gaaacatcac ctgcgacaac tggttcacca gcatccccct ggccaagaac 5220
ctgctgcagg agccctacaa gctgaccatc gtgggcaccg tggccagcaa caagagagag 5280
atccccgagg tcctgaagaa cagcaggtcc aggcccgtgg gcaccagcat gttctgcttc 5340
gacggccccc tgaccctggt gtcctacaag cccaagcccg ccaagatggt gtacctgctg 5400
tccagctgcg acgaggacgc cagcatcaac gagagcaccg gcaagcccca gatggtgatg 5460
tactacaacc agaccaaggg cggcgtggac accctggacc agatgtgcag cgtgatgacc 5520
tgcagcagaa agaccaacag gtggcccatg gccctgctgt acggcatgat caacatcgcc 5580
tgcatcaaca gcttcatcat ctacagccac aacgtgagca gcaagggcga gaaggtgcag 5640
agccggaaaa agttcatgcg gaacctgtac atgggcctga cctccagctt catgaggaag 5700
aggctggagg cccccaccct gaagagatac ctgagggaca acatcagcaa catcctgccc 5760
aaagaggtgc ccggcaccag cgacgacagc accgaggagc ccgtgatgaa gaagaggacc 5820
tacgcctact actgtcccag caagatcaga agaaaggcca gcgccagctg caagaagtgt 5880
aagaaggtca tctgccggga gcacaacatc gacatgtgcc agagctgttt c 5931
<210> 113
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> gRNA
<400> 113
tatgtacact tctgacccac 20
<210> 114
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> gRNA SaCas9
<400> 114
gtatcacaat tccagtgggt 20
<210> 115
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> gRNA SaCas9
<400> 115
gtatcacaat tccagtgggt 20
<210> 116
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> gRNA SaCas9
<400> 116
ggacaggatc ggcataaccg 20
<210> 117
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> gRNA SaCas9
<400> 117
gtgctcgggg ccactaggga 20
<210> 118
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Cpf1 gRNA
<400> 118
acttataatt cactgtatca 20
<210> 119
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Cpf1 gRNA
<400> 119
agcttgatat ccatggaatt 20
<210> 120
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Cpf1 gRNA
<400> 120
tgctcggggc cactagggac 20
<210> 121
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Cpf1 gRNA
<400> 121
cttttgtaaa actttatggt 20
<210> 122
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> Cpf1 gRNA
<400> 122
caaaagtaaa tagcccggct 20
<210> 123
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> CjCas9
<400> 123
gccgatcctg tccctagtgg cc 22
<210> 124
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> CjCas9 gRNA
<400> 124
acaattccag tgggtcagaa gt 22
<210> 125
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> CjCas9 gRNA
<400> 125
acacttctga cccactggaa t 21
<210> 126
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> CjCas9 gRNA
<400> 126
gaattccatg gatatcaagc tt 22
<210> 127
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> CjCas9 gRNA
<400> 127
aattccagtg ggtcagaagt gt 22
<210> 128
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CasX gRNA
<400> 128
tcaagcgcgt gtatgtacac 20
<210> 129
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CasX gRNA
<400> 129
ggatcggcat aaccggtgaa 20
<210> 130
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CasX gRNA
<400> 130
tagacatgag gtctatggac 20
<210> 131
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CasX gRNA
<400> 131
taagcttgat atccatggaa 20
<210> 132
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CasX gRNA
<400> 132
tataattcac tgtatcacaa 20
<210> 133
<211> 5
<212> PRT
<213> 人工序列
<220>
<221>位点
<222> 1..5
<223> 其中序列可以重复1-50次
<220>
<223> 接头
<400> 133
Gly Gly Gly Gly Ser
1 5
<210> 134
<211> 5
<212> PRT
<213> 人工序列
<220>
<221>位点
<222> 1..5
<223> 其中序列可以重复1-50次
<220>
<223> 接头
<400> 134
Glu Ala Ala Ala Lys
1 5
<210> 135
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac R275A/N347S/K375A/D450N/S592G
<400> 135
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly
580 585 590
Cys Phe
<210> 136
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac N347S/D450N/T560A/S573A/F594L
<400> 136
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Leu
<210> 137
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac N347A/D450N
<400> 137
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 138
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac R275A/N347S/R372A/D450N/T560A/F594L
<400> 138
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Leu
<210> 139
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
R202K/R275A/N347S/R372A/D450N/T560A/F594L
<400> 139
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Lys Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Leu
<210> 140
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L
<400> 140
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Leu
<210> 141
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac三重突变
D450N/R372A/K375A/R376A
<400> 141
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Ala Ala Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 142
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G
<400> 142
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Ala Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly
580 585 590
Cys Phe
<210> 143
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L
<400> 143
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ala Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly
580 585 590
Cys Leu
<210> 144
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P
<400> 144
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Met Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ala Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Pro Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 145
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
G325A/N347S/K375A/D450N/S573A/M589V/S592G
<400> 145
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Gly
580 585 590
Cys Phe
<210> 146
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac S230N/R277A/N347S/K375A/D450N
<400> 146
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Asn Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Ala Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Ala Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 147
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac T43I/R372A/K375A/A411T/D450N
<400> 147
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Ile Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Ala Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Thr Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser
580 585 590
Cys Phe
<210> 148
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G
<400> 148
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
Tyr Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Ser Trp Phe Thr Ala Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr
545 550 555 560
Tyr Cys Pro Ser Lys Ile Arg Arg Lys Ala Ser Ala Ala Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Val Cys Gln Gly
580 585 590
Cys Phe
<210> 149
<211> 594
<212> PRT
<213> 人工序列
<220>
<223> 修饰的超活性Piggybac
Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G
<400> 149
Met Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln
1 5 10 15
Ser Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp
20 25 30
His Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile
35 40 45
Asp Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu
50 55 60
Asp Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn
65 70 75 80
Arg Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His
85 90 95
Cys Trp Ser Thr Ser Lys Pro Thr Arg Arg Ser Arg Val Ser Ala Leu
100 105 110
Asn Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile
115 120 125
Tyr Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile
130 135 140
Ser Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg
145 150 155 160
Glu Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile
165 170 175
His Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn
180 185 190
His Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr
195 200 205
Val Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu
210 215 220
Arg Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val
225 230 235 240
Phe Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile
245 250 255
Gln Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu
260 265 270
Gly Phe Ala Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro
275 280 285
Ser Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys
290 295 300
Tyr Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn
305 310 315 320
Gly Val Pro Leu Ala Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val
325 330 335
His Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile
340 345 350
Pro Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val
355 360 365
Gly Thr Val Arg Ser Asn Ala Arg Glu Ile Pro Glu Val Leu Lys Asn
370 375 380
Ser Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro
385 390 395 400
Leu Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu
405 410 415
Leu Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys
420 425 430
Pro Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr
435 440 445
Leu Asn Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg
450 455 460
Trp Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn
465 470 475 480
Ser Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val
485 490 495
Gln Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Gly Leu Thr Ser
500 505 510
Ser Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu
515 520 525
Arg Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser
530 535 540
Asp Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Ala
545 550 555 560
Tyr Cys Pro Gly Lys Ile Arg Arg Lys Ala Ser Ala Ser Cys Lys Lys
565 570 575
Cys Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Gly
580 585 590
Cys Phe
<210> 150
<211> 390
<212> DNA
<213> 人工序列
<220>
<223> MCP N55K
<400> 150
atggcttcaa actttactca gttcgtgctc gtggacaatg gtgggacagg ggatgtgaca 60
gtggctcctt ctaatttcgc taatggggtg gcagagtgga tcagctccaa ctcacggagc 120
caggcctaca aggtgacatg cagcgtcagg cagtctagtg cccagaagag aaagtatacc 180
atcaaggtgg aggtccccaa agtggctacc cagacagtgg gcggagtcga actgcctgtc 240
gccgcttgga ggtcctacct gaacatggag ctcactatcc caattttcgc taccaattct 300
gactgtgaac tcatcgtgaa ggcaatgcag gggctcctca aagacggtaa tcctatccct 360
tccgccatcg ccgctaactc aggtatctac 390
<210> 151
<211> 130
<212> PRT
<213> 人工序列
<220>
<223> MCP N55K
<400> 151
Met Ala Ser Asn Phe Thr Gln Phe Val Leu Val Asp Asn Gly Gly Thr
1 5 10 15
Gly Asp Val Thr Val Ala Pro Ser Asn Phe Ala Asn Gly Val Ala Glu
20 25 30
Trp Ile Ser Ser Asn Ser Arg Ser Gln Ala Tyr Lys Val Thr Cys Ser
35 40 45
Val Arg Gln Ser Ser Ala Gln Lys Arg Lys Tyr Thr Ile Lys Val Glu
50 55 60
Val Pro Lys Val Ala Thr Gln Thr Val Gly Gly Val Glu Leu Pro Val
65 70 75 80
Ala Ala Trp Arg Ser Tyr Leu Asn Met Glu Leu Thr Ile Pro Ile Phe
85 90 95
Ala Thr Asn Ser Asp Cys Glu Leu Ile Val Lys Ala Met Gln Gly Leu
100 105 110
Leu Lys Asp Gly Asn Pro Ile Pro Ser Ala Ile Ala Ala Asn Ser Gly
115 120 125
Ile Tyr
130
<210> 152
<211> 162
<212> DNA
<213> 人工序列
<220>
<223> gRNA-MS2(四环)-AAVS1-3间隔物
<400> 152
ggggccacta gggacaggat gttttagagc taggccaaca tgaggatcac ccatgtctgc 60
agggcctagc aagttaaaat aaggctagtc cgttatcaac ttggccaaca tgaggatcac 120
ccatgtctgc agggccaagt ggcaccgagt cggtgctttt tt 162
<210> 153
<211> 162
<212> RNA
<213> 人工序列
<220>
<223> gRNA-MS2(四环)-AAVS1-3 间隔物
<400> 153
ggggccacua gggacaggau guuuuagagc uaggccaaca ugaggaucac ccaugucugc 60
agggccuagc aaguuaaaau aaggcuaguc cguuaucaac uuggccaaca ugaggaucac 120
ccaugucugc agggccaagu ggcaccgagu cggugcuuuu uu 162

Claims (22)

1.组合物，其包含：
(i)第一蛋白或编码所述第一蛋白的核酸构建体，所述第一蛋白包含能够结合和切割靶核酸序列的位点特异性DNA结合蛋白或由其组成；和
(ii)第二蛋白或编码所述第二蛋白的核酸构建体，所述第二蛋白包含转座酶或由其组成；
其中所述转座酶是修饰的高活性PiggyBac，其与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变。
2.根据权利要求1所述的组合物，其中所述第一蛋白和所述第二蛋白融合在一起以形成融合蛋白，任选地通过接头融合。
3.根据权利要求2所述的组合物，其中所述第一蛋白与所述第二蛋白的C末端融合，任选地通过接头融合。
4.根据权利要求1至3中任一项所述的组合物，其中所述转座酶是修饰的高活性PiggyBac，其与未修饰的超活化PiggyBoc相比包含增加切除活性的一个或多个氨基酸突变，和/或与未修饰的超活化PiggyBoc相比包含降低DNA结合活性的一个或多种氨基酸突变。
5.根据权利要求1至4中任一项所述的组合物，其中所述一个或多个氨基酸突变不由R372A、K375A和D450N组成。
6.根据权利要求1至5中任一项所述的组合物，其中所述一个或多个氨基酸突变选自在M194、D450、T560、S564、S573、S592或F594位置处的增加切除活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代M194V和/或D450N。
7.根据权利要求1至6中任一项所述的组合物，其中所述一个或多个氨基酸突变选自在M194或D450位置处的增加切除活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代M194V和/或D450N。
8.根据权利要求1至7中任一项所述的组合物，其中所述一个或多个氨基酸突变选自在位置R275、R277、R347、R372、K375、R376、E377和/或E380处的降低DNA结合活性的氨基酸取代，所述位置编号对应于SEQ IDNO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代R275A、R277、R347S、R372A、K375A、R376A、E377A和/或E380A。
9.根据权利要求1至8中任一项所述的组合物，其中所述一个或多个氨基酸突变选自在位置R372、K375、R376、E377和/或E380处的降低DNA结合活性的氨基酸取代，所述位置编号对应于SEQ ID NO:9的未修饰的高活性PiggyBac的氨基酸编号，优选选自氨基酸取代R372A、K375A、R376A、E377A和/或E380A。
10.根据权利要求1至9中任一项所述的组合物，其中所述修饰的高活性PiggyBac包含双突变N347S和D450N，所述位置编号对应于SEQ ID NO:9的未修饰的超活化PiggyBoc的氨基酸编号。
11.根据权利要求1至10中任一项所述的组合物，其中所述修饰的高活性PiggyBac突变包含一个以下氨基酸取代或氨基酸取代的组合：R372A/K375A/R376A/D450N、K375A/R376A/E377A/E380A/D450N、R372A/K375A/R376A/E377A/E380A/D450N、M194V、R376A、E377A、E380A、M194V/R372A/K375A、S351A/R372A/K375A/R388A/D450N/W465A/S573A/M589V/S592G/F594L、R245A/R275A/R277A/R372A/W465A/M589V、R275A/325A/R372A/T560A、N347A/D450N、N347S/D450N/T560A/S573A/F594L、R202K/R275A/N347S/R372A/D450N/T560A/F594L、R275A/N347S/K375A/D450N/S592G、R275A/N347S/R372A/D450N/T560A/F594L、R275A/R277A/N347S/R372A/D450N/T560A/S564P/F594L、R245A/N347S/R372A/D450N/T560A/S564P/S573A/S592G、R277A/G325A/N347A/K375A/D450N/T560A/S564P/S573A/S592G/F594L、V34M/R275A/G325A/N347S/S351A/R372A/K375A/D450N/T560A/S564P、G325A/N347S/K375A/D450N/S573A/M589V/S592G、S230N/R277A/N347S/K375A/D450N、T43I/R372A/K375A/A411T/D450N、G325A/N347S/S351A/K375A/D450N/S573A/M589V/S592G、Y177H/R275A/G325A/K375A/D450N/T560A/S564P/S592G；所述位置编号对应于SEQ ID NO:9的高活性PiggyBac的氨基酸编号，典型地，所述修饰的转座酶具有选自SEQ ID NO:2-8、10-18和135-149中任一个的氨基酸序列。
12.根据权利要求1至11中任一项所述的组合物，其进一步包含第三蛋白或编码所述第三蛋白的核酸构建体，所述第三蛋白包含第二转座酶或由其组成；其中所述第二转座酶是SEQ ID NO:9的高活性PiggyBac或与SEQ ID NO:9的高活性PiggyBac相比包含一个或多个氨基酸突变的修饰的高活性PiggyBac。
13.根据权利要求12所述的组合物，其中所述第一、第二和第三蛋白融合在一起以形成三重融合蛋白，任选地通过接头融合。
14.根据权利要求1至13中任一项所述的组合物，其中所述第一蛋白包含RNA引导的核酸酶或内切酶或锌指核酸酶，或由其组成。
15.根据权利要求1至14中任一项所述的组合物，其中所述第一蛋白是核酸酶蛋白，所述核酸酶蛋白包含活性DNA切割结构域和引导RNA结合结构域，并且与SEQ ID NO:31的酿脓链球菌(Streptococcus pyogenes)Cas9(SpCas9)、SEQ ID NO:72的金黄色葡萄球菌(Staphylococcus aureus)Cas9(SaCas9)、SEQ ID NO:74的Cpf1、SEQ ID NO:29的空肠弯曲杆菌(Campylobacter jejuni)Cas9(CjCas9)、SEQ ID NO:70的酿脓链球菌Cas9内切酶(nCas9)、SEQ ID NO:75的CasX或SEQ ID NO:76的金黄色葡萄球菌Cas9内切酶具有至少80％、90％、95％、99％或至少100％的同一性；
优选地，其中所述第一蛋白是选自SEQ ID NO:72的金黄色葡萄球菌Cas9(SaCas9)和SEQ ID NO:31的酿脓链球菌Cas9(SpCas9)的Cas9蛋白。
16.根据权利要求1至15中任一项所述的组合物，其进一步包含引导RNA和用于插入基因组的外源核酸。
17.根据权利要求16中任一项所述的组合物，其中所述转座酶与RNA结合蛋白融合，所述RNA结合蛋白能够结合包含在所述引导RNA中的至少一种特异性RNA序列；
任选地，其中所述RNA结合蛋白是MS2噬菌体外壳蛋白(MCP)，并且其中所述引导RNA包含MS2 RNA四环结合序列，优选与SEQ ID NO:153具有至少75％的同一性。
18.根据权利要求16或17所述的组合物，其中所述外源核酸是大DNA片段，其通常具有5kb至25kb的大小，更优选8kb至20kb的大小。
19.根据权利要求1至18中任一项所述的组合物，其中所述组合物包含在纳米颗粒中。
20.核酸，其编码如权利要求2至17中任一项所述的融合蛋白，所述核酸通常为信使RNA(mRNA)。
21.将外源核酸序列位点特异性整合到细胞基因组中的体外方法，所述方法包括向所述细胞递送权利要求1至19中任一项所述的组合物、引导RNA和所述外源核酸。
22.根据权利要求1至19中任一项所述的组合物、引导RNA和外源核酸，用于通过将外源核酸序列位点特异性整合到细胞基因组中来治疗疾病。
CN202180093884.8A 2020-12-16 2021-12-16 可编程转座酶及其用途 Pending CN116940673A (zh)

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