CN113249407B

CN113249407B - Vector for homologous recombination and application thereof

Info

Publication number: CN113249407B
Application number: CN202110610723.3A
Authority: CN
Inventors: 孙瑞林; 陈臣; 周宇
Original assignee: Guangdong Nanmo Biotechnology Co ltd; Shanghai Dishi Biotechnology Co Ltd; Shanghai Model Organisms Center Inc
Current assignee: Guangdong Nanmo Biotechnology Co ltd; Shanghai Dishi Biotechnology Co Ltd; Shanghai Model Organisms Center Inc
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2021-10-26
Anticipated expiration: 2041-06-02
Also published as: CN113862299A; CN113862299B; CN113249407A

Abstract

The invention discloses a vector for homologous recombination and application thereof, wherein the vector for homologous recombination comprises: a homologous left arm, primer P2, an insert, primer P3, and a homologous right arm, wherein the primers P2 and P3 are capable of amplifying a sequence comprising a site of homologous recombination of a gene. The invention further provides a homologous recombination PCR identification method based on the homologous recombination vector. By using the method, no matter whether the target fragment is integrated to the correct position of the genome or not, the primer pair can amplify the PCR product, the result can be quickly judged, the occurrence of false negative condition is effectively reduced, the identification efficiency is improved, and the identification accuracy is improved. The identification of the mouse embryonic stem cell line prepared by homologous recombination and the knock-in mouse model shows that the vector has high-efficiency identification advantage and is suitable for establishing the homologous recombination cell line and the animal model.

Description

Vector for homologous recombination and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a homologous recombination vector, and a preparation method and application thereof.

Background

Polymerase Chain Reaction (PCR) is a nucleic acid synthesis technique for replicating a specific DNA fragment in vitro using the principle of DNA double strand replication, and is a conventional molecular biology technique that can amplify a large amount of a target DNA fragment in a short time. A PCR reaction system needs to comprise: DNA polymerase, primers, deoxynucleoside triphosphate (dNTP), a buffer system, a DNA template, H2O and the like, and the reaction needs to be carried out in a thermal cycling device, particularly at an annealing temperature, and each pair of primers has a specific annealing temperature. In the PCR reaction process, the deletion or the mixture ratio error of each component of the reaction system and the improper annealing temperature can cause the PCR failure, the target band can not be amplified, or the nonspecific product is amplified, so that the false negative or false positive condition is caused.

The precise genetic modification of cells or animals by homologous recombination techniques has great application prospects and practicality in basic research and clinical applications, such as the construction of genetically modified animal models and gene therapy for correcting gene mutations. Because the PCR technology is simple and rapid, in the process of preparing gene modification cell lines and gene modification animal models mediated by homologous recombination such as gene knock-in, conditional knock-out and the like, the PCR method is usually utilized to identify whether correct homologous recombination occurs. In identifying knock-in homologous recombination, a common identification method is to design a PCR primer on the Genome outside the homology arm and a PCR primer on the knocked-in DNA fragment, and theoretically only positive samples with correct recombination can amplify the PCR product of interest (as shown in FIG. 1, panel A and B in FIG. 5. Wenning Qin, et. al, Generation Mouse Models Using CRISPR-Cas9 media Genome Editing, Current protocol Mouse biol.;6(1): 39-66. doi: 10.1002/97800942390. mo 150178.). The method cannot search for the proper annealing temperature of the PCR primer pair because of lack of positive sample control, and if the PCR product with the target size is not amplified (as shown in a blank lane of an A' diagram in figure 1), the method cannot judge that the PCR reaction temperature is not proper, the configuration of a PCR system is wrong, or all identification samples are truly negative, so that false negative misjudgment is easily caused, and particularly, when the homologous arm is long and the PCR product amplification of a long fragment is required, or the knock-in fragment sequence is complex and has no proper available primer sites, the occurrence probability of false negative is greatly increased. Although nuclease technology represented by CRISPR/Cas9 improves the efficiency of homologous recombination by initiating double-stranded DNA breaks, the efficiency with which homologous recombination occurs is still low, especially for large fragments (DNA fragments of over 1000 bp) of DNA. Therefore, in the case that the positive ratio of homologous recombination is low, the occurrence of false negative situation can bring great uncertainty to the work. In the current conventional identification strategy, if the occurrence of false negative condition is to be avoided, a plurality of pairs of PCR primers are often required to be designed, different annealing temperatures are explored, and the sample is repeatedly identified, so that the identification workload is increased, the identification time is prolonged, and the cost is increased.

Therefore, a PCR method capable of improving the efficiency of identifying homologous recombination is urgently needed, and can quickly judge the negative result of PCR and eliminate the false negative condition.

Disclosure of Invention

Aiming at the defects and the actual requirements of the existing PCR identification homologous recombination method.

The invention discloses a homologous recombination vector, which comprises: a homologous left arm, primer P2, an insert, primer P3, and a homologous right arm, wherein the primers P2 and P3 are capable of amplifying a sequence comprising a site of homologous recombination of a gene.

Preferably, the gene homologous recombination site is an insertion site of the insert in the genome.

Preferably, the homologous recombinant vector comprises the following elements in sequence: homologous left arm, primer P2, insert, primer P3 and homologous right arm.

Preferably, the homologous recombination vector further comprises a selection marker.

Preferably, the selectable marker is a positive selectable marker or a negative selectable marker.

The invention discloses a construction method of a homologous recombination vector, which comprises the following steps:

(1) designing primers P2 and P3 according to a gene recombination site to be inserted, wherein the primers P2 and P3 are capable of amplifying a sequence comprising a gene homologous recombination site;

(2) homologous recombination vectors were constructed, and the primers P2 and P3 were ligated to the homologous left arm and the homologous right arm, respectively.

Preferably, primer P2 is inserted between the homologous left arm and the insert and primer P3 is inserted between the homologous right arm and the insert.

The invention discloses a homologous recombination vector, which is prepared by adopting the method.

The invention discloses a gene homologous recombination method, which adopts the homologous recombination vector for operation.

Preferably, the gene is an animal gene, a plant gene or a microbial gene.

Preferably, the animal gene is a mouse gene.

The invention discloses a gene homologous recombination kit, which comprises the homologous recombination vector.

The invention discloses application of the homologous recombination vector in gene homologous recombination.

Preferably, the application comprises mutation of a gene, insertion of a gene.

Preferably, the invention discloses an Il1rl1 gene humanized mouse embryonic stem cell model.

Preferably, the invention discloses a mouse embryonic stem cell model prepared by using the homologous recombination vector.

Preferably, the invention discloses a mouse model prepared by using the homologous recombination vector.

Preferably, the invention discloses a primer sequence related to establishing an Il1rl1 gene humanized mouse embryonic stem cell model.

Preferably, the invention discloses a Pax7-IRES-CreERT2 mouse model.

Preferably, the invention discloses primer sequences involved in establishing a Pax7-IRES-CreERT2 mouse model.

Furthermore, the invention provides a method for constructing a homologous recombination vector, wherein before constructing the homologous recombination vector, PCR amplification is carried out on genomes on the upstream and downstream of a homologous recombination occurrence site through a pre-experiment to obtain a primer pair (such as a B picture in figure 1, a P1/P2 primer pair and a P3/P4 primer pair) with a good amplification effect; when constructing a homologous recombination vector, a pre-experimental primer is introduced into the constructed homologous recombination fragment (such as P2 and P3 primers placed in the recombination fragment in a B diagram in figure 1) to serve as a primer site for identifying whether the recombination is correct or not in the subsequent PCR, and the primer site is paired and amplified with a primer outside a homology arm of a pre-experiment to identify a sample.

The primer pair has mature conditions of early PCR amplification, and if a sample is correctly subjected to homologous recombination, a band with a target size appears; if no correct homologous recombination occurs and no positive target size band appears, a wild-type band should be amplified, so that whether the sample is negative can be quickly judged according to the size of the amplified PCR product (for example, a band lane is shown in a B' diagram in FIG. 1); if neither wild-type nor target size bands are amplified (e.g., no band in panel B' of FIG. 1), then there should be a problem with the PCR amplification system, PCR operation, or PCR reaction process, and the experiment should be repeated until wild-type or target size bands appear, thereby avoiding false negative situations.

The method of the invention establishes a homologous recombination PCR identification verification system, and has no problem in the verification process, PCR products are always amplified in the PCR reaction, and whether the sample is positive or not can be quickly judged by using the size of the products; if no wild type or target product is amplified in the PCR reaction, the experimental process is problematic, and the sample is repeatedly identified, so that the identification accuracy is improved, and the identification efficiency is accelerated. The identification of the mouse embryonic stem cell line prepared by homologous recombination and the knock-in mouse model shows that the method has high-efficiency identification advantage and is suitable for the PCR identification of the homologous recombination cell line and the animal model.

Drawings

FIG. 1 shows a schematic representation of the strategy for identifying homologous recombination events by PCR. FIG. 1 is a diagram A showing a conventional homologous recombination vector construction, generation and PCR identification strategy, and FIG. 1 is a diagram A' showing the electrophoresis results according to the scheme A in FIG. 1; FIG. 1B shows the homologous recombination vector construction, generation and PCR identification strategy of the present invention, and FIG. 1B' shows the electrophoresis schematic result according to the strategy of FIG. 1B.

FIG. 2 shows the construction strategy of the humanized mouse embryonic stem cell model of Il1rl1 gene, the PCR pre-identification strategy and electrophoresis result, and the homologous recombination vector structure. Wherein A in figure 2 is a strategy diagram for constructing an Il1rl1 gene humanized mouse embryonic stem cell model by means of homologous recombination; FIG. 2B is a schematic representation of PCR pre-identification for homologous recombination 5 'and 3' arms and a schematic representation of primer relative positions and product sizes; FIG. 2C 1 map-FIG. 2C 4 map sequentially shows the results of gradient PCR amplification electrophoresis of primer pairs Il1rl1-PreID1, Il1rl1-PreID2, Il1rl1-PreID3 and Il1rl1-PreID4 (M is 1kb DNA marker; 1-8 are corresponding 8 different annealing temperatures); FIG. 2, panel D, shows the structure of the finally constructed homologous recombination vector, in which the Il1rl1-PreID1-Rev (ID 1-R) primer is embedded between the 5 'arm and the knock-in fragment, and the Il1rl1-PreID3-For (ID 3-F) primer is embedded between the knock-in fragment and the 3' arm.

FIG. 3 shows the result of PCR identification of IL1rl1 gene humanized mouse embryonic stem cell resistance clone. FIG. 3A is the diagram of Il1rl1 wild type gene structure and recombined gene structure and the size of each primer pair aiming at different genotype amplification products; the B picture in FIG. 3 is the identification result of 3' arm homologous recombination of 96 resistant clones; panel C of FIG. 3 is a 3' arm homologous recombination duplicate identification of the positive clones identified for panel B of FIG. 3; panel D of FIG. 3 shows the results of 5' arm homologous recombination identification for positive clones identified in panel B of FIG. 3.

FIG. 4 shows the construction strategy, PCR pre-identification strategy and electrophoresis result, and homologous recombination vector structure of the Pax7-IRES-CreERT2 mouse model. FIG. 4A is a schematic diagram of a strategy for constructing a mouse model of Pax7-IRES-CreERT2 by homologous recombination; FIG. 4B is a schematic representation of PCR pre-identification for homologous recombination 5 'and 3' arms and a schematic representation of primer relative positions and product sizes; the C1-4C4 diagrams in FIG. 4 are the results of gradient PCR amplification electrophoresis by primer pairs Pax7-PreID1, Pax7-PreID2, Pax7-PreID3 and Pax7-PreID4 in sequence (M is 1kb DNA marker; 1-8 are corresponding 8 different annealing temperatures); FIG. 4, panel D, shows the structure of the finally constructed homologous recombination vector, in which a Pax7-PreID4-For (ID 4-F) primer and a Pax7-PreID1-Rev (ID 1-R) primer are embedded between the knock-in fragment and the 3' arm in this order.

FIG. 5 shows the result of genotyping F0 generation in the mouse model Pax7-IRES-CreERT 2. FIG. 5A is a diagram showing the structure of the wild-type gene and the structure of the gene after recombination in Pax7, and the sizes of amplification products of the respective primer pairs for different genotypes; the B diagram in FIG. 5 shows the identification result of homologous recombination in the 5' arm of 18 mice of generation F0; FIG. 5C is a graph showing the identification result of homologous recombination in the 3' arm of a mouse positive for the B graph in FIG. 5 (M is 1kb DNA marker; the number is the mouse number; WT is a wild-type control).

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The process and application of the method for constructing the homologous recombination vector are divided into the following steps:

1) firstly, determining the structure of a DNA fragment to be site-specific integrated into a genome according to homologous recombination as a pre-buried primer (such as primers P2 and P3 in the B diagram in FIG. 1, abbreviated as: pre-embedded primers) "are placed at the positions of the knocked-in fragments, and the positions of the pre-embedded primers are selected on the basis that the functions of target genes or fragments are not influenced after the primer sequences are pre-embedded; meanwhile, because the efficiency of amplifying small fragments in the PCR reaction is higher under the same condition, the length of the PCR product with positive targets amplified by the pre-buried primer at the position of the pre-buried primer on the knock-in fragment (such as the P1 primer in the B picture in figure 1) and the post-recombination arm outer primer (such as the P2 primer in the B picture in figure 1) is optimally smaller than the size of the product amplified by the primer pair in the wild type;

2) design and screening principle of pre-identified primer:

(1) designing a pre-identified primer: firstly, determining the length of an expected homologous arm according to the homologous recombination difficulty of a project, determining the design position of an out-of-arm primer of a pre-identified primer according to the length, and taking the design of the pre-identified primer of a 5' arm (5 ' homologous recombination arm, abbreviated as 5' arm) as an example: the forward primer identified by the 5' arm (such as the P1 primer in the B picture in figure 1) needs to be positioned outside the homologous arm; the reverse primer for 5 'arm identification can be positioned on the 3' homologous arm (3 'arm for short) or outside the 3' arm; ③ because the efficiency of amplifying small fragments in PCR reaction is higher under the same condition, the size of the PCR product of positive purpose amplified by the recombined 5' arm outer primer (such as the P1 primer in the B picture in figure 1) and the pre-buried primer (such as the P2 primer in the B picture in figure 1) is smaller than that amplified by the primer pair in the wild type;

(2) screening of pre-identified primers: the method comprises the steps of taking a wild genome as a template, carrying out PCR amplification on a plurality of designed pre-identified primer pairs through temperature gradient PCR, detecting a PCR result through DNA gel electrophoresis, selecting a primer pair with a large annealing temperature range, high amplification efficiency and good amplification product specificity as a primer pair for carrier construction pre-embedding and subsequent homologous recombination PCR identification, and selecting an annealing temperature with high amplification efficiency and good amplification product specificity as a primer pair annealing temperature for subsequent homologous recombination PCR identification. The pre-identified primers designed according to the principle can not amplify the PCR products meeting the requirement, so that the length requirement on the homology arms can be shortened, or the length of the homology arms can be determined according to the length of the PCR products which can be amplified;

(3) constructing a homologous recombination vector of the pre-buried identification primer: integrating the pre-buried primers into the knock-in segment by means of PCR amplification or gene synthesis according to the position of the determined pre-buried primers in the knock-in segment and a conventional vector construction method to obtain a final homologous recombination vector;

4) the PCR identification result of the recombinant sample obtained based on the homologous recombination vector constructed by the method is interpreted as follows: as described above, the primer pair and PCR amplification conditions obtained by early pre-identification by the method of the invention can be used for detecting the occurrence of bands with target sizes if the sample is correctly subjected to homologous recombination; if correct homologous recombination does not occur and no positive target size band appears, a wild type band is amplified, so that whether the sample is negative or not can be quickly judged according to the size condition of the amplified PCR product; if neither the wild-type band nor the band of the desired size is amplified, the PCR amplification system, the PCR operation, or the PCR reaction process should be problematic, and the experiment should be repeated until either the wild-type band or the band of the desired size appears. The interpretation of the results is illustrated by taking the schematic of the diagram B in fig. 1 and the diagram B' in fig. 1 as an example: as shown in Panel B of FIG. 1, the primer pair P1/P2 for 5 'arm pre-identification amplified a zkb size fragment on the wild-type genome, and the primer pair P3/P4 for 3' arm pre-identification amplified a wkb size fragment on the wild-type genome; according to the pre-identification result, a P2 primer is pre-embedded at the 5' end of the knock-in segment, a P3 primer is pre-embedded in the middle region of the knock-in segment, a P1/P2 primer pair is utilized to identify the occurrence condition of 5' arm homologous recombination (the size of a positive PCR product is xkb), and a P3/P4 primer pair is utilized to identify the occurrence condition of 3' arm homologous recombination (the size of the positive PCR product is ykb). The identification results are shown in B ' diagram in FIG. 1, and 4 recombination samples were obtained, and 5' arm homologous recombination identification and 3' arm homologous recombination identification were performed, respectively: 1) in the 5 'homologous arm recombination identification, only xkb-mesh positive products are amplified in the sample 1, and the amplification is positive in 5' arm recombination; sample 2 amplified only zkb size wild type product, which was negative for 5' arm recombination; sample 3 has no positive product or wild product amplified, and needs repeated detection for error of the experiment; sample 4 has higher amplification efficiency in PCR reaction, and besides the xkb-mesh large and small positive products, also zkb large and small wild products are 5' arm recombination positive; 2) in 3 'homologous arm recombinant identification, sample 1 has wkb wild-type products besides ykb-mesh positive products due to higher amplification efficiency, and is 3' arm recombinant positive; sample 2 has no positive product or wild product amplified, and needs repeated detection for error of the experiment; sample 3 amplified wkb size wild type product, which was negative for 3' arm recombination; sample 4 amplified only ykb mesh positive products, positive for 3' arm recombination. Therefore, according to the PCR electrophoresis result, a qualitative result can be rapidly given to each sample condition.

In the following specific examples, the inventors will describe the specific implementation of the method for knock-in mouse embryonic stem cell line construction, knock-in mice and conditional knock-out mouse construction.

Example 1 construction of Il1rl1 Gene humanized mouse embryonic Stem cell model

Through a homologous recombination mode, a partial region of an intron 1, a region of exons 2 to 7 and a partial region of an intron 7 of a mouse Il1rl1 gene are replaced by a region corresponding to a human Il1rl1 gene in a mouse embryonic stem cell, the mouse embryonic stem cell is humanized and transformed, a recombination strategy is shown in a diagram A in a diagram 2, and the specific construction process is as follows:

1) homologous recombination vector construction

Screening pre-buried primers: according to the project situation, the length of two homologous recombination arms (5 'arm and 3 arm') is designed to be 3kb, two pairs of PCR primers are respectively designed aiming at the positions to be recombined of the two homologous arms, the positions of the primers are schematically shown in a B diagram in figure 2, and the primer information is shown in the following tables 1 and 2.

Table 1: primer information table related to Il1rl1 gene humanized mouse embryonic stem cell model construction

The primer pairs respectively use mouse embryonic stem cell genomes as templates, and use DNA polymerase capable of carrying out long-fragment PCR amplification to carry out PCR product amplification, wherein a reaction system and a reaction program are shown as follows:

and (3) PCR reaction system:

PCR reaction composition	Volume (mu l)
		ddH2O	31
PCR Buffer	10
		2.5 mM dNTP	4
Forward primer	1
		Reverse primer	1
DNA Polymerase	2
		genomic DNA	1
Total of	50

PCR reaction procedure:

step (ii) of	Temperature (. degree.C.)	Time	Remarks for note
				1	98	2 min
2	98	15 sec
				3	68	15 sec
4	Temperature gradient	1 min	Repeat steps 2-4 for a total of 34 cycles
				5	68	1 min
6	12	10 min

Set up a total of 8 temperature gradients as: 51.5 deg.C, 53.4 deg.C, 55.7 deg.C, 58.3 deg.C, 61.0 deg.C, 63.7 deg.C, 66.1 deg.C, and 68.0 deg.C (which in turn correspond to numbers 1-8 in the C1-C4 diagrams in FIG. 2).

The electrophoresis results of PCR products respectively carrying out PCR pre-identification on two homologous arms are shown as C1-C4 in figure 2, according to the electrophoresis results, a primer pair Il1rl1-PreID1-For/Rev For 5' arm pre-identification has stronger amplification efficiency, a Rev primer of the primer pair is selected as an embedded primer For homologous recombination 5' arm identification, the primer pair is used as an identification primer For subsequent 5' arm homologous recombination, and 61.0 ℃ is selected as the annealing temperature For subsequent PCR identification; aiming at the primer pair Il1rl1-PreID3-For/Rev of 3' arm pre-identification, the amplification efficiency is better, the For primer of the primer pair is selected as an embedded primer For homologous recombination 3' arm identification, the primer pair is used as an identification primer For subsequent 3' arm homologous recombination, and 61.0 ℃ is selected as the annealing temperature For subsequent PCR identification.

Constructing a homologous recombination vector: respectively designing primer pairs shown in the following table, and taking a PCR product obtained by amplification of the Il1rl1-PreID1-For/Rev primer pair as a template to obtain a 5' arm by amplification; amplifying by using a PCR product obtained by amplifying the Il1rl1-PreID3-For/Rev primer pair as a template to obtain a 3' arm; taking a knock-in fragment obtained by whole gene synthesis as a template, and amplifying to obtain a knock-in fragment; and (3) taking the PBR322 plasmid as a template, and amplifying to obtain a skeleton vector. The 5 'arm, the knock-In fragment, the 3' arm and the skeleton vector are recombined In one step by an In-Fusion mode to construct an Il1rl1 gene humanized homologous recombinant vector (Il 1rl1-donor vector for short). The sequence information of the Il1rl1-donor vector, the 5 'arm, the knock-in fragment, the 3' arm and the skeleton vector is SEQ ID NO 17-21 in sequence.

Table 2: primer information table related to Il1rl1 gene humanized mouse embryonic stem cell model construction

Primer name	Sequence information (5')>3'）
		5' arm forward primer	CGCGGTCGACAAGCTactgtaatcccaaatcaggg (SEQ ID NO:9 )
5' arm reverse primer	CTTGGCGGCTTTTTATGTATGGTG atgttcgtgtactgtgttaa (SEQ ID NO: 10) underlined and oblique font is 5' arm pre-embedded primer
		Knock-in fragment forward primer	TAAAAAGCCGCCAAGggatgtttattgactgacac (SEQ ID NO:11 )
Knock-in fragment reverse primer	GTGGAGAAAGGATTAGAGGCT acctaggattttctcactga (SEQ ID NO: 12) underlined and italicized font is 3' arm pre-embedded primer
		3' arm forward primer	TAATCCTTTCTCCACtcaatgaagcaagggaagag (SEQ ID NO:13 )
3' arm reverse primer	CGACTCTAGAGGATCctattagtgtctattatcat (SEQ ID NO:14 )
		Backbone carrier forward primer	GATCCTCTAGAGTCGAGCAG (SEQ ID NO:15 )
Reverse primer of skeleton carrier	AGCTTGTCGACCGCGGCCTT (SEQ ID NO:16 )

In the construction process of the Il1rl1-donor vector, the 5' arm embedded primer (Il 1rl1-PreID 1-Rev) which is pre-identified and screened is added at the 5' end of the 5' arm reverse primer, so that the purpose of adding the 5' arm embedded primer between the 5' arm and the knock-in fragment is realized; the aim of adding the 3 'arm pre-embedded primer between the 3' arm and the knock-in fragment is realized by adding the 3 'arm pre-embedded primer (Il 1rl1-PreID 3-For) pre-identified and screened at the 5' end of the reverse primer of the knock-in fragment, and the structure of the final recombinant vector is shown as a diagram D in figure 2.

2) Cloning and screening of mouse embryonic Stem cells (ES cells)

And (3) after the Il1rl1-donor vector is confirmed to be correct by sequencing, transforming through escherichia coli, carrying out amplification extraction purification, carrying out mouse embryonic stem cell electrotransformation, and screening through Neomycin resistance after electrotransformation to obtain 96 resistant ES cell clones in total. After the genomic DNA of the resistant ES cell clone is extracted, the resistant ES cell clone is identified by long-fragment PCR using PCR amplification conditions obtained by early-stage pre-identification, and the identification strategy is schematically shown in A in FIG. 3.

The identification process specifically comprises the following steps: firstly, carrying out PCR identification on 3' arm homologous recombination conditions by using an Il1rl1-PreID3-For/Rev primer pair (the PCR reaction system is the same as the above; the PCR reaction program is the same as the above except that the annealing temperature is 61.0 ℃), wherein as shown in a picture A in figure 3, a positive ES cell clone can amplify a PCR product with the size of 3.2kb, a negative ES cell clone can only amplify a wild-type band with the size of 6.5kb, and the identification results of 96 resistant clones are shown (as a picture B in figure 3): 24. clones No. 35, 36, 50, 81 and 83 amplified a positive PCR product of 3.2kb (6.5 kb wild type band was amplified due to the higher amplification efficiency of DNA polymerase), clones No. 37, 49, 61, 73, 85 and 86 did not amplify any band, and the rest clones amplified only a wild type band of 6.5 kb. Therefore,

clones

24, 35, 36, 50, 81, 83 were 3' arm homologous recombination positive clones (duplicate confirmation of these clones is shown in panel C of fig. 3); 37.

clones

49, 61, 73, 85 and 86 are PCR products amplified, and need secondary identification for experimental errors; the remaining clones were 3' arm homologous recombination negative clones. ② aiming at the 3 'arm homologous recombination positive clone No. 24, 35, 36, 50, 81 and 83, using Il1rl1-PreID1-For/Rev primer pair (PCR reaction system is the same as above; PCR reaction program, except that the annealing temperature is 61.0 ℃, the rest is the same as above) to make PCR identification to determine whether the 5' arm has the correct homologous recombination, and the result is shown in D picture in figure 3, the clones No. 24, 35, 36, 50, 81 and 83 all amplify the positive PCR product of 3.6kb (because the amplification efficiency of DNA polymerase is higher, at the same time, 4.9kb wild type band is amplified), the wild type control can only amplify 4.9kb wild type band.

The embodiment shows that the PCR primers with good amplification conditions are obtained by pre-identifying PCR screening of the recombination positions in the early stage, and when the homologous recombination vector is constructed, the homologous recombination vector is pre-embedded into the recombination fragments, so that the identification conclusion of cloning can be quickly obtained according to the PCR result in the subsequent identification of the homologous recombination cell line, and the identification efficiency is improved.

Example 2 construction of Pax7-IRES-CreERT2 Gene knock-in mouse model

By utilizing CRISPR/Cas9 technology and through a homologous recombination mode, an IRES-CreERT2 DNA fragment is inserted into the position of 3' UTR (ultrasuspended region) of a Pax7 gene, a recombination strategy is shown as a diagram A in a figure 4, and the specific construction process is as follows:

1) homologous recombination vector construction

Screening pre-buried primers: according to the project situation, the length of two homologous recombination arms (5 'arm and 3' arm ') is designed to be about 3kb, two pairs of PCR primers are respectively designed aiming at the position of 3' UTR to be inserted into IRES-CreERT2, PCR pre-identification is carried out, in order to avoid the influence of pre-embedded primer insertion on the expression of IRES-CreERT2 knock-in fragment, after the PCR primers identified by the 5 'arm and the 3' arm are both placed in the IRES-CreERT2 knock-in fragment, the positions of the primers are shown in a B diagram in FIG. 4, and the sequence information of the primers is shown in the following tables 3 and 4.

Table 3: primer information table constructed by Pax7-IRES-CreERT2 knock-in mouse model

The primer pairs respectively use wild type C57BL/6 mouse genome as a template, and DNA polymerase capable of performing long-fragment PCR amplification is used for performing PCR product amplification, and the reaction system and the reaction program are as follows:

and (3) PCR reaction system:

PCR reaction procedure:

Set up a total of 8 temperature gradients as: 51.5 deg.C, 53.4 deg.C, 55.7 deg.C, 58.3 deg.C, 61.0 deg.C, 63.7 deg.C, 66.1 deg.C, and 68.0 deg.C (which in turn correspond to numbers 1-8 in the C1-C4 diagrams in FIG. 4).

The electrophoresis results of PCR products pre-identified by PCR on two homology arms are shown in FIG. 4, panels C1-C4: according to the electrophoresis result, aiming at the primer pair Pax7-PreID1-For/Rev pre-identified by the 5' arm, the primer pair Pax7-PreID1-For/Rev has better specificity and stronger amplification efficiency, the Rev primer of the primer pair is selected as the pre-embedded primer identified by the homologous recombination 5' arm, the primer pair is used as the identification primer For the subsequent homologous recombination of the 5' arm, and 63.7 ℃ is selected as the annealing temperature of the subsequent PCR identification; aiming at the primer pair Pax7-PreID4-For/Rev pre-identified by the 3' arm, the amplification efficiency is better, the For primer of the primer pair is selected as a pre-buried primer For homologous recombination 3' arm identification, the primer pair is used as an identification primer For subsequent 3' arm homologous recombination, and 63.7 ℃ is selected as the annealing temperature For subsequent PCR identification.

Constructing a homologous recombination vector: respectively designing primer pairs shown in the following table For constructing a recombinant vector, and obtaining a 5' arm by amplification by using a PCR product obtained by amplification of the Pax7-PreID1-For/Rev primer pair as a template; amplifying by using a PCR product obtained by amplifying the Pax7-PreID4-For/Rev primer pair as a template to obtain a 3' arm; amplifying to obtain a knock-in fragment by taking an IRES-CreERT2 fragment obtained by whole gene synthesis as a template; and (3) taking the PBR322 plasmid as a template, and amplifying to obtain a skeleton vector. By means of In-Fusion, the Pax7-IRES-CreERT2 homologous recombination vector (short for Pax7-donor vector) is constructed by means of one-step recombination of the 5 'arm, the IRES-CreERT2 fragment, the 3' arm and the skeleton vector. The sequence information of the Pax7-donor vector, the 5 'arm, the knock-in fragment and the 3' arm is SEQ ID NO 36-39 in sequence, and the sequence information of the framework vector is SEQ ID NO 21.

Table 4: primer information table constructed by Pax7-IRES-CreERT2 knock-in mouse model

Primer name	Sequence information (5')>3') and description
		5' arm forward primer	CGCGGTCGACAAGCTagttctacatttcaccaaac (SEQ ID NO:30 )
5' arm reverse primer	GGACCCTAGTAGGCTTGTCCCG (SEQ ID NO:31 )
		Knock-in fragment forward primer	AGCCTACTAGGGTCCGCCCCTCTCCCTCCCCCCCCCCT (SEQ ID NO:32 )
Knock-in fragment reverse primer	TGTGGTATCAGCATCTTGGCAAT TCATCAAGCTGTGGCAGGGAAAC (SEQ ID NO:33 ) Pre-embedded primer with underline and oblique font as 3' arm
		3' arm forward primer	GATGCTGATACCACA TTCAAACCACTGCAAGCCCTTAGC GGTCCCTGGGGCAACTTGCCCCATCCAGTGG (SEQ ID NO: 34) underlined and italicized font is 5' arm pre-embedded primer
3' arm reverse primer	CGACTCTAGAGGATCTTCCTAATTTCTGTTTCCTG (SEQ ID NO:35 )

In the construction process of a Pax7-donor vector, the aim of adding the pre-embedded primer of the 3 'arm between the knock-in segment and the 3' arm is fulfilled by adding the pre-embedded primer of the 3 'arm (Pax 7-PreID 4-For) which is pre-identified and screened at the 5' end of the reverse primer of the knock-in segment; the purpose of adding the 5 'arm pre-buried primer between the knock-in fragment and the 3' arm is realized by adding the 5 'arm pre-buried primer (Pax 7-PreID 1-Rev) pre-identified and screened in the 3' arm forward primer, and the structure of the finally constructed vector is shown as a diagram D in fig. 4.

2) F0 mouse PCR identification screening

Pax7-donor vector is sequenced to confirm that the Pax7-donor vector is correct, then is converted by escherichia coli, amplified, extracted and purified, then is mixed with Cas9 and gRNA to carry out mouse fertilized egg microinjection, and the injected fertilized egg is transplanted to an oviduct of a receptor mother mouse after being temporarily cultured by an incubator to obtain a genetically modified mouse F0 generation mouse, wherein 18F 0 generation mice are born in total.

After mouse tail genome extraction, the genotype of the F0 mouse is identified, and the identification strategy is shown as A picture in figure 5, and concretely comprises the following steps: firstly, carrying out PCR identification on the 5 'arm homologous recombination condition by using a Pax7-PreID1-For/Rev primer pair (the PCR reaction system is the same as the above; the PCR reaction program is the same as the above except that the annealing temperature is 63.7 ℃), wherein the homologous recombination positive F0 generation mouse can amplify a PCR product with the size of 6.3kb, the negative mouse can only amplify a wild-type band with the size of 9.8kb, and the identification result of the 5' arm of 18F 0 generation mice is shown as a B picture in figure 5): the mice No. 1 and No. 6 amplified positive PCR products of 6.3kb (due to the higher amplification efficiency of DNA polymerase, 9.8kb wild type band was amplified at the same time), and the other mice amplified only 9.8kb wild type band. Thus, mice nos. 1 and 6 are 5' arm homologous recombination positive mice; the remaining mice were 5' arm homologous recombination negative mice. Secondly, aiming at the 3 'arm homologous recombination positive mice No. 1 and No. 6, carrying out PCR (polymerase chain reaction) by using a Pax7-PreID4-For/Rev primer pair (the PCR reaction system is the same as the above; the PCR reaction program is the same as the above except that the annealing temperature is 63.7 ℃), identifying whether the 3' arm has correct homologous recombination, and obtaining the result as shown in a C diagram in figure 5, wherein the mice No. 1 and No. 6 both amplify a positive PCR product of 5.6kb (because the amplification efficiency of DNA polymerase is higher, and simultaneously an 8.7kb wild type band is amplified), and the wild type control only can amplify a wild type band of 8.7 kb.

The embodiment shows that the PCR primers with good amplification conditions are obtained by pre-identifying PCR screening of the recombination positions in the early stage, and are pre-embedded into the recombination fragments when the homologous recombination vector is constructed, so that the identification conclusion of cloning can be quickly obtained according to the PCR result in the identification of subsequent homologous recombination animal models, and the identification efficiency is improved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shanghai's Square model Biotech Co., Ltd

Shanghai Yushi Biological Technology Co., Ltd.

Guangdong Nanmo Biological Technology Co., Ltd.

<120> a vector for homologous recombination and use thereof

<130> 2021

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cttattgttg gggcagagtg a 21

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cttggcggct ttttatgtat ggtg 24

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcctgccaca tatatggagg agg 23

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccttcaactg tgaatgatct ggt 23

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcctctaat cctttctcca c 21

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggctttcacc cctctcatac tcta 24

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctactagat tttattttac 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctaaaaccaa gagatatcag 20

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgcggtcgac aagctactgt aatcccaaat caggg 35

<210> 10

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cttggcggct ttttatgtat ggtgatgttc gtgtactgtg ttaa 44

<210> 11

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

taaaaagccg ccaagggatg tttattgact gacac 35

<210> 12

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtggagaaag gattagaggc tacctaggat tttctcactg a 41

<210> 13

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

taatcctttc tccactcaat gaagcaaggg aagag 35

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgactctaga ggatcctatt agtgtctatt atcat 35

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gatcctctag agtcgagcag 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agcttgtcga ccgcggcctt 20

<210> 17

<211> 19183

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttctcatgtt tgacagctta tcatcgataa gctgcggccg caaaggccgc ggtcgacaag 60

ctactgtaat cccaaatcag gggaactggt gcaaaattac gttagatatt tgtaaggttg 120

attttttttt ttttttagaa tgaggtagtt tgaagacata aaagggtatt tttttttttc 180

agaaaagttg gcagatgcta gctggattct gaaggacttg gctactttaa aactcctatt 240

tggttcattg attggaggcc gtccttgctc agatgtgcct cctgagctgt gttctatgta 300

ttctcaaatg gctgcattcc atgtctacaa agcccaaaca gaggctgaga tggcttattg 360

tttcttgaag aacctggtta agggacagat cttactttaa taactcagtc atggtgatca 420

gagaaagggg ttgaccatga cagtttctct ctctcatttt ttagtcgacc tgtggaaaaa 480

gagaagaatg catgtttcct tgcttcaacc acttggtttt aaccccagcc acatctgagt 540

gagcttggtt gaattattta agcattatta ttgctaatga tagtactaaa taacgtagcc 600

ttggattttg ctcaaactca tgctgcatgc aatcctgata ataagagtca agagcagcaa 660

ggaacacact caaggtcaag ctcccagaca gcaaaaggct tgctttttct gccatgtaaa 720

gtgtatcacc agtggctaag agctggctaa gccactgggc agagaagata tatcaggagc 780

attccagccc ctataaggag ctggctcaag ctgtgttctt agctagcaat acttccatgc 840

ttgtctttct atgcctggct cacttggatt cggtggctag cctttagagc cagcctgctt 900

gggttctaat tctcttagct ctgtgttgtg tggatccctt acttaatctc tgtgcctctg 960

ggtcatcatt tggaccatgg agagaacaac agcatggcat tgactgactt ggggaatgaa 1020

gaattgatat gggagtcaca gagagccaca taaagcatag aggagatact gcataaagat 1080

ttcaattata tttttctatt ggtgctgggg tggggatggt ccttcaatcc ttcctatgca 1140

tgaagacata aaaggaagtc tcataataga gttaagacaa ggatgcttgg cacaactcaa 1200

aaccaaccgt taacattgtt tcatttgaag ccccaaaaga tgatggcctc cttggaaagg 1260

cttggttatc ttcatcctag atgagattat agatcaaaat ctgaggtact agactatact 1320

ttggacctga gatgctcttt ccattatcca acacatctaa gcacccaagg aacataaaca 1380

tcgtttgcct ctcagggctc atctccatgc ccctcctgca ctctccctaa catgtgatgg 1440

tttgtgagat tgtccacagc acaggaaaca attgtgcccc ctgacagtat cagcagaggt 1500

catgaacagc ctggaggaca gcctggtgcg tgtgtgtgtt tgagatttgg gaatggacat 1560

tatggtaaac agcattgttt atctcagctt cactgaccca gcaaagacag gggcgggtga 1620

tttaattggt tcccctgagg tggattatga cgttgtgctc atggttaaat atttatgtgg 1680

taagtttagg aatttccagt aagtgagaca gcagcatttt tgaacaagtc atggattttg 1740

gctaaaacta aaattctatg atgggcgggt taaaaaaaaa tgccaagaca tctgtgtatt 1800

taaaaagaaa gagggagcat gcccacgtgg gtcgtctgca gaaatgagac gaaggagcgc 1860

caagtagcct cacggctctg agcttattct ctccagccct tcatctgggg taagaatcaa 1920

tgactgtctg aagcttgtaa aagaaaaaag tgcctgcaaa tcaaagaaac gtgcactctt 1980

gaaactacta aggagttaaa tatttattcc tagctttggg gatgtctgta tccctcctct 2040

gactggtgaa ggggaaagcc acagtaatag attctttaaa agaaaaagga ttatttattg 2100

tgcacagacc tctggcaagc ctccgtggct tttgagaaca agcctaggca gttactattc 2160

tttgaaagtt tgggggaaca gtcttggatg gcctcctggc taatgtttta gttcaaattt 2220

ttccttcctt agttttttat aatgatgcct atctgtgagt actggcatgt tgctctagcc 2280

tgctacacgg ctgtggcttt gattgacact gtgctttcta gtatctactc aattttatgg 2340

ttctaagttt tacaaatgca aaaatttgtt tcctttcctc ttaatttagt ttttttttgt 2400

ttgttttttg ttttttgttt tttatttttt ttgaggaaat gttcaagaga ctggtaaggt 2460

ataatcaatc atggaaaaaa aatctagtca ttgtaaggac tgctgtctgg tttcacgttc 2520

agtagggatg gatgcaatat tgtatacctg aaacctacta ttatccatat gtgaatttaa 2580

taaatacgtg tcaaccgact gtcctgtatg gtaagaacaa atcaaaacaa gtaagtaaaa 2640

agcgatacaa attaattatt ttacatggtt tatttttcag tatctacagt gatttctctt 2700

ctggacccta cctcagagag cacttgtcaa ccgcctagtg aacacaccat tactatcctg 2760

tgccattgcc atagagagac ctcagccatc aatcactagc acatgattga cagacagaga 2820

atgggacttt gggctttggc aattctgaca cttcccatgt atttgacagt tacggagggc 2880

agtaagtacc gattctcaga tgagtggtat ggtttgaact taagtaattc tgggaatatt 2940

tggcctcatt taagctgctt gcttcctgtt gttttgggct gaattttatg ttaggaaact 3000

atttcctctt cattcattct ttatcgtaaa tctctcagaa ggaattctct atagggagta 3060

gatcataatt tttgagttat gtaaattaag acttaacaca gtacacgaac atcaccatac 3120

ataaaaagcc gccaagggat gtttattgac tgacacacag tttgtttaga tagataaggc 3180

tgcttttcca ttggataact gtgggggatt tagctccaaa ttaattcatt aacatttcat 3240

aagagatctt aacagttact tattctttcc caaggtatca cagtatatga ccggcttcta 3300

aatttaatcc tagaagaaaa tattgagagt ataagaatta tgatcttttc atttgatcat 3360

ttcaggattg tctttatatc ttttatttgc aggtaaacaa tcatggggcc tggaaaatga 3420

ggctttaatt gtaagatgtc ctagacaagg aaaacctagt tacaccgtgg attggtatta 3480

ctcacaaaca aacaaaagta ttcccactca ggaaagaaat cgtgtgtttg cctcaggcca 3540

acttctgaag tttctaccag ctgcagttgc tgattctggt atttatacct gtattgtcag 3600

aaggtattat gcagaaggct cccatcttct ttcaccctgc tcccctttct tcagtggttg 3660

attgcctgag ctgcccttgc tttcattcct tccctagtcc tttctggaac agttaaattt 3720

ataaaatgat ttgaataaaa gtgatttgga taaacttcta ggaatactat caggttgagg 3780

tctagctcat tctgagctat ttggatttac agttgcaggg attgatttgt agctgactta 3840

gagaaaaacc tagctttcct agtgaccaag ataactgaga gcaattgctt acttttggct 3900

ggaattaaga acaaactagc acagaaaata gtaatctgga tgttttccat ctcagggggc 3960

ctctagtagg tgaaaagggg cttctaacct tcaagttaag ccacagaagg ctgatgagat 4020

tgtgtgccta aaaattaatt acttttgttc acaaattgtt aaatgttttg attttgtggc 4080

tgtatttggc acacacaaaa tgtcaaatga attaaatcaa aaagcaggat gtattagtta 4140

agggcctagg tctgagacta gacaagtgtc aaattcatgc tctgccacta gttaactgtg 4200

cgatgccagc aagttacttc tctgcccttc agtttccttc tctgtaaaat gcatataatg 4260

taataatata tttcatctca aagcattatt gtgaaaataa actaaggtaa tgtgtgaaaa 4320

gtgctcatca tcatcactgg tacagagtaa actctgaata aatagtaatt atctttatta 4380

cactgggatt acagcattac agcagatgta gtgtatgaat aaatggtgaa gaagtcattg 4440

ttagggctac tatgggggct atgtttttag ctcaagtgta acaaaaaagt atttaaactc 4500

tagattttaa tgtttatttt taaataataa aataaccatt atgtatattg atggttttta 4560

agaagaaaag caatattaag taaaggctga atttagatta agttatttca caatgctaag 4620

tgactctttt aattgtctga cttattttaa cagtcccaca ttcaatagga ctggatatgc 4680

gaatgtcacc atatataaaa aacaatcaga ttgcaatgtt ccagattatt tgatgtattc 4740

aacagtatct ggatcagaaa aaaattccaa aatttattgt cctaccattg acctctacaa 4800

ctggacagca cctcttgagt ggtttaaggt aagaagaaat ttggaaggaa atagatgaaa 4860

attacacaat taaaatagac acaagtggcc gggcacagtg gctcatgcct gtaatcctag 4920

cactttggga ggccaaggca ggcagatcac ttgaggccag gagtttgaga ccagcctggc 4980

caacatgagg aaaccccatc tctactaaaa atataaaaat cagctgggtg tggtggcaca 5040

cacctgtaat cccagcagct tgggaggctg aggtatgaaa atcacttgaa cctagaaggc 5100

agaggttaca gcgagcccag attgcaccac tcactccagc ctaggcaaca aacattctgt 5160

caacagataa ataaataaaa gtgaagaatt actgagaagg aaatggaatt tcttatttca 5220

gaattgtcag gctcttcaag gatcaaggta cagggcgcac aagtcatttt tggtcattga 5280

taatgtgatg actgaggacg caggtgatta cacctgtaaa tttatacaca atgaaaatgg 5340

agccaattat agtgtgacgg cgaccaggtc cttcacggtc aagggtaagc tactgacatt 5400

aatgagatag aatactacgt gaaagaagtc gaagtgggaa cagcggtgcc cttctggttg 5460

ggtttcttgc acttctccct cctcccttta cttcctcctg ctccatctta tcttatacat 5520

tctgaactat gacgcaaaga ggttttctga acacactatc aagatttaag aaatttcagg 5580

gggaaattac attactaatt caaagccaca tctgttcttt attctttttt tgtgacttaa 5640

ttttccaaag ataaagcaat ctgaatgcta acttaactta ctttttttga atggcaatac 5700

aactatttgg agagcaaaac cagctttttt tttttttttc tagtttggtg tcagagtttc 5760

tgcaaattaa aaaagagctt aatctttagt aatactcatt ggattcaaag tctaatgaga 5820

ggctttgtga tggtatacta tggtgtacat aaatgttgtc gagtggtttt taatctttgt 5880

ttgcaatact ttcaacatca tcaatggcct tgagtaagtc acttcattct aaaaatgtgt 5940

tttccaagtt attttaaatt ttataaaagc ttatttaagg gaaagatttc acaatcatag 6000

cttatcaatc tacaaaggat tggggtctcc ttagcacaag tcgatctaca gacgtagatg 6060

taatagcccc tttacgtatg aatcgataag cttgatatcg aattccgaag ttcctattct 6120

ctagaaagta taggaacttc aggtctgaag aggagtttac gtccagccaa gctagcttgg 6180

ctgcaggtcg tcgaaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc 6240

atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac 6300

acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc 6360

accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag 6420

gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga 6480

gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt 6540

ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg 6600

cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac 6660

gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcctgttgac 6720

aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc 6780

atgggatcgg ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 6840

aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 6900

cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 6960

aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 7020

gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 7080

ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 7140

gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 7200

aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 7260

ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 7320

atgcccgacg gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 7380

gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 7440

tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 7500

gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 7560

cgccttcttg acgagttctt ctgaggggat caattctcta gagctcgctg atcagcctcg 7620

actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 7680

ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 7740

ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 7800

tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa 7860

agaaccagct ggggctcgac tagagcttgc ggaacccttc gaagttccta ttctctagaa 7920

agtataggaa cttcatcagt caggtacata atggatccac agttttttca gggcaaagca 7980

atattggaga caaatttttg gagtttttca atactccacc gctgtaaaat aagcatcacc 8040

agaccacatt cctatcagtg cctctttctg tttaatatca acccttacag tggtccttaa 8100

tcacactgtc attaaataaa tgagcatgaa gggatggagg attgcagcag tgctccataa 8160

gcactgcccg tctttcagcc ttagtggtca caggagtcag aattccgtac ggggaagatt 8220

tcactgagga tgggccaccc tagtggagaa ctgcgagcaa atctgtggac tcatccattt 8280

attattttca tgggtctttt gaaatcttct ctgtagtctt attcttattc tgtagaagag 8340

tagttttcta acaactacta ggtcatgtaa ttagttttat gggttggatc tgcatttgtt 8400

taagtgatat cagagaataa tgatattaaa gagcatcata ggtaataaaa gaaagtttta 8460

tttaagtgcc tttctgtttc gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 8520

gtttgtcatt atgggttatt gtcagaagaa cttgaaaaac attgctatga aatagaatag 8580

aaacatgaaa atacaagctt tatattgact agcattcaat gctctcctaa tatttatatt 8640

tctttttgtc tttaagatga gcaaggcttt tctctgtttc cagtaatcgg agcccctgca 8700

caaaatgaaa taaaggaagt ggaaattggt aagaaaattt atcagaatgc tgtaaatatt 8760

gcctggaaaa atccttccat atgacccctg ttctgaattc ccttagcagg ggtcaggcaa 8820

ttagcataag gaaccttgag gagtaagtga ggtgacatcc ctgaaagcac ctgccccaag 8880

catttgctaa tattgggaac agggacacag caattgcagt gtttacattt gtttattgta 8940

ctttgtaatt catgatgctt tcatgtatgc atctaatttc atcttcatct ctatcccaga 9000

gcttgggatg gagacctgca gggtgttcat tctgggcaat ggtagccaga tccggtaaaa 9060

catgtttatc ttcaaagtag cttatggaga gatgaagaga gttctgtaga aagatgtgga 9120

agagggcagt tggaaagaaa ctctaatttc tagtagaggg caatcctttt actagaaatc 9180

ctttgtaatg tggggttggt gaaggcagaa tcattggcct tgttagtttc ccatgcagat 9240

gagaatatag tgggagctga gcttcaaacc cagctgggtg aatgaaggta atggaagcag 9300

ggaggaggca agagaggaca tagaaagagg aaggtgctag agatgaggga gggaggtcct 9360

ggtggggtgc atactaagtg ttcagtaagg tttttttttt acattaaatg ggataaaatg 9420

ccagtcgcag aagttaattt tattggtgaa tgtccttact cccctctagg aaaaaacgca 9480

aacctaactt gctctgcttg ttttggaaaa ggcactcagt tcttggctgc cgtcctgtgg 9540

cagcttaatg gaacaaaaat tacagacttt ggtgaaccaa gaattcaaca agaggaaggg 9600

caaaatcaaa ggtattttta tattgaagag aaccatcctc ttccccttgc acatggtttg 9660

cacctgcaaa gtaggcatta aaagtaacag gttgctttct tagtttcagc aatgggctgg 9720

cttgtctaga catggtttta agaatagctg acgtgaagga agaggattta ttgctgcagt 9780

acgactgtct ggccctgaat ttgcatggct tgagaaggca caccgtaaga ctaagtagga 9840

aaaatccaag taaggagtgt ttctgagact ttgatcacct gaactttctc tagcaagtgt 9900

aagcagaatg gagtgtggtt ccaagagatc catcaagaca atgggaatgg cctgtgccat 9960

aaaatgtgct tctcttcttc gggatgttgt ttgctgtctg atctttgtag actgttcctg 10020

tttgctggga gcttctctgc tgcttaaatt gttcgtcctc ccccactccc tcctatcgtt 10080

ggtttgtcta gaacactcag ctgcttcttt ggtcatcctt gttttctaac tttatgaact 10140

ccctctgtgt cactgtatgt gaaaggaaat gcaccaacaa ccgtaaactg aacgtgttct 10200

tttgtgctct tttataactt gcattacatg ttgtaagcat ggtccgttct ataccttttt 10260

ctggtcataa tgaacactca ttttgttagc gagggtggta aagtgaacaa aaaggggaag 10320

tatcaaacta ctgccatttc agtgagaaaa tcctaggtag cctctaatcc tttctccact 10380

caatgaagca agggaagagt gggcctaagg ccttgccaag aactgtaatt ctagacccag 10440

gatgcaacac agacaacatt ttcctgttta taaacactgc caagaagcat ttgcgtctgg 10500

tcattgacgt gaaaaccact tgtagtttac aaagtcataa gcacgtagat agatgttgag 10560

catcaacttc ctgggtcacc ttgaatcaca gtcttgccta gggtaaacat ttcagccacc 10620

cacacaggta cttcacccac agcagttcct ataagaagtc tatcttccat accctctgta 10680

cctccctagc acgcatccgg tgggactttt agacaatttt tctgaacaag ctgcttggga 10740

agttcttact taaacctaaa gaagtgaaga cagcaagctc tggccagttt tgagatcttt 10800

tctcattttt ttgggtcgca agactttagt gtcttgagtt tctttttcct ggatttagag 10860

agaaggtgaa ctgtcaagga tggtagagga atttgtcact actagcaggg atttcacgcc 10920

aggtcttgtg gcctatgcgt ggggatttac aaataaaata aaaaagaact atttacccgg 10980

tctgaccgag gaatctcctc atattcatgg ataaacaaag tctatgagga tcatatggtt 11040

ccaacaggca agcagaccag gaatggtagc agttcctgtg tccaacctgt acatgtgtct 11100

tacaagttcc tgaaggtgaa agccatgttt cttgcgtgtt ttgtgaaaca agggatgtgg 11160

gactcggctg agagatagtt gaaggaggat ctcaatagca gaaggcatca ggatgataga 11220

acccagcaat ttgatgtctc catcacagca ctctgtgacc tgcaactgtg cgtgtatcct 11280

atattcccca ccccctagat gcaaccattt tcctgatcta ctgactcaca tgccttaggg 11340

ctgcccataa acaccgacct taccaatcga attcctatca tttcacaagt tccaagcctc 11400

atttcccccc acaaataccc tgttatattc cttgaacacg tcatcccttc ctatacaaat 11460

gaacgttcca ctactgtcag attaatctta ctgaaaagca ctttcatctc taataccctt 11520

tcacatcagg acacattccc tagcaagagt gtctgatcat gcatagccct gaccacgcct 11580

tccacgaatt ccccaaacgc agttactaca gttcttagaa cacacacttc cacctctgca 11640

catgaactca gcattctctc tggagagggg agtgttcttt ttttttttcc tagaccgttc 11700

tgagtcttct ttgtccttta gtaatccatt ctatagtaag ccttccttcc ttcctctaat 11760

atagagcagt ttctgtttaa gatggctgtc agctgaaatc ccttttgcct cctatcttag 11820

ctgggttttc ctgtatctat gagcttcctt acctttaagc acactctgtg ttactaagcc 11880

ttaagacttg tgccgagtat gtgctaagaa aattgatttg ttgattttga aagtattgtt 11940

gaaaattcag tattggttta ttgggtagga atgccttgtg gttggtttct agggatgtcg 12000

gagttcatgc aaccatccat tctgtctcat tttgctatta ctaatgcccg tttcccccat 12060

ctccccatct cttgatagtt gatcaccgaa gcatctacta catagttgct ggatgtagtt 12120

tattgctaat gtttatcaat gtcttggtga tagtcttaaa agtgttctgg attgaggttg 12180

ctctgttctg gagagatata gtgacacctt acaaaacccg gaacggtgag tatctgtttt 12240

caagattctc ttcacaagga aaagtcccac tgactcttga ttatttcttt aatgatattt 12300

tgctcctaaa cccaatctcc gtttcctttt aacctcatct gtgagatttt ggagtctaca 12360

cggatcattt tccatgcctc tgtgtcactg cttatacttc tgacattcag acattcatct 12420

ccaagattct ccttgccgag attgctattc ctagtgagcc cccataactc tgcctgagac 12480

ccgaacaggt tctgctgttt tgaaaaatgc atgttaataa cattttacaa caggacaata 12540

gaatcatacc tgcttttcaa gtgtgagcag gttatggaga gagttggaac acatgctcat 12600

tgcccttgaa tcctcattcc atgagtacac acttgcacaa actttacaca tgaaaaagaa 12660

cagtcccagt gaagtaggaa ctttgaagcc tgtttgtgca cgaagcaatc aattaactgt 12720

agattgcttg atttgtggga aatacagacc ccttaagaag tcctacttct ttgccactat 12780

ttgccattgg catattacct tccgaggaag tagtatggtt gccatatgcc ctttctgggg 12840

tttcaattgt ctaaggtcaa ctgaagtttg taaagattaa ataagcaaaa caaatgtgag 12900

ttttaagttg agcatgagtc tgagatatgt gattgtcctg cctgggatgt gaattgtctg 12960

tttgttcagt gtatctatgt tgtataatgt ataataacct agcactaatc agttacccta 13020

ttagttaaca ggctcactgc cgaagttccg tggtgcctgt gtttgaggga cagttatttt 13080

atgttgacgt cactgccttc actgctggag tagtgatgcc atggattttg ttgtagtcca 13140

ttgtcctatg tttattacta gctatggttg ttaaactctt actgtgacaa gtgtattagg 13200

tctgtgtgtg cagaagtaag tgttgaatgg agggcacagt attatctgtg gcttcaggta 13260

ctcactggat gtctttgaac ctgtgctctg tggctaaatg aggaatgttg tgtgggaaaa 13320

ctgtaacagt caatagacag agaagaccct tccccaccca tgataataga cactaatagg 13380

atcctctaga gtcgagcagt gtggttttca agaggaagca aaaagcctct ccacccaggc 13440

ctggaatgtt tccacccaat gtcgagcagt gtggttttgc aagaggaagc aaaaagcctc 13500

tccacccagg cctggaatgt ttccacccaa tgtcgagcaa accccgccca gcgtcttgtc 13560

attggcgaat tcgaacacgc agatgcagtc ggggcggcgc ggtcccaggt ccacttcgca 13620

tattaaggtg acgcgtgtgg cctcgaacac cgagcgaccc tgcagcgacc cgcttaacag 13680

cgtcaacagc gtgccgcaga tcttggtggc gtgaaactcc cgcacctctt cggccagcgc 13740

cttgtagaag cgcgtatggc ttcgtacccc ggccatcagc acgcgtctgc gttcgaccag 13800

gctgcgcgtt ctcgcggcca tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa 13860

gaagccacgg aagtccgccc ggagcagaaa atgcccacgc tactgcgggt ttatatagac 13920

ggtccccacg ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc 13980

gacgatatcg tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag 14040

acaatcgcga acatctacac cacacaacac cgccttgacc agggtgagat atcggccggg 14100

gacgcggcgg tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc 14160

gacgccgttc tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg 14220

gccctcaccc tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg 14280

cgatacctta tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg 14340

ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc cggaggacag acacatcgac 14400

cgcctggcca aacgccagcg ccccggcgag cggcttgacc tggctatgct ggccgcgatt 14460

cgccgcgttt acgggctgct tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg 14520

cgggaggatt ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag 14580

agcaacgcgg gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc 14640

gagttgctgg cccccaacgg cgacctgtac aacgtgtttg cctgggcctt ggacgtcttg 14700

gccaaacgcc tccgtcccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc 14760

tgccgggacg ccctgctgca acttacctcc gggatgatcc agacccacgt caccacccca 14820

ggctccatac cgacgatctg cgacctggcg cgcacgtttg cccgggagat gggggaggct 14880

aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag 14940

acagaataaa acgcacgggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg 15000

gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt 15060

ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg 15120

gggcggcagg ccctgccata gccacgggcc ccgtgggtta gggacggggt cccccatggg 15180

gaatggttta tggttcgtgg gggttattat tttgggcgtt gcgtggggtc agtccacgac 15240

tggactgagc agacagaccc atggtttttg gatggcctgg gcatggaccg catgtactgg 15300

cgcgacacga acaccgggcg tctgtggctg ccaaacaccc ccgaccccca aaaaccaccg 15360

cgcggatttc tggcgccgcc ggacgaacta aacctgacta cggcatctct gccccttctt 15420

cgctggtacg aggagcgctt ttgttttgta ttggtcacca cggccgagtt tcctcgaccg 15480

atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc 15540

gtcgccgcac ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg 15600

ctctgggtca ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg 15660

cttgcggtat tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc 15720

aaacgtttcg gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac 15780

gtcttgctgg cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct 15840

tccggcggca tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac 15900

catcagggac agcttcaagg atcgctcgcg gctcttacca gcctaacttc gatcactgga 15960

ccgctgatcg tcacggcgat ttatgccgcc tcggcgagca catggaacgg gttggcatgg 16020

attgtaggcg ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc 16080

cgggccacct cgacctgaat ggaagccggc ggcacctcgc taacggattc accactccaa 16140

gaattggagc caatcaattc ttgcggagaa ctgtgaatgc gcaaaccaac ccttggcaga 16200

acatatccat cgcgtccgcc atctccagca gccgcacgcg gcgcatctcg ggcagcgttg 16260

ggtcctggcc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc 16320

ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact 16380

gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt 16440

cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 16500

caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt 16560

gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac 16620

aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc 16680

gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga 16740

ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc 16800

ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc 16860

acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa 16920

acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 16980

gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 17040

cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 17100

tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata 17160

ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 17220

gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 17280

taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 17340

cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 17400

ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 17460

aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 17520

tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 17580

gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 17640

cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 17700

ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 17760

cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 17820

gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 17880

cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 17940

tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 18000

ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta 18060

aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca 18120

atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc 18180

ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc 18240

tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 18300

agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat 18360

taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 18420

tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc 18480

cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag 18540

ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt 18600

tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 18660

tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg 18720

cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat 18780

tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc 18840

gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc 18900

tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 18960

atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg 19020

tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 19080

cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac 19140

ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa gaa 19183

<210> 18

<211> 3050

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

actgtaatcc caaatcaggg gaactggtgc aaaattacgt tagatatttg taaggttgat 60

tttttttttt ttttagaatg aggtagtttg aagacataaa agggtatttt tttttttcag 120

aaaagttggc agatgctagc tggattctga aggacttggc tactttaaaa ctcctatttg 180

gttcattgat tggaggccgt ccttgctcag atgtgcctcc tgagctgtgt tctatgtatt 240

ctcaaatggc tgcattccat gtctacaaag cccaaacaga ggctgagatg gcttattgtt 300

tcttgaagaa cctggttaag ggacagatct tactttaata actcagtcat ggtgatcaga 360

gaaaggggtt gaccatgaca gtttctctct ctcatttttt agtcgacctg tggaaaaaga 420

gaagaatgca tgtttccttg cttcaaccac ttggttttaa ccccagccac atctgagtga 480

gcttggttga attatttaag cattattatt gctaatgata gtactaaata acgtagcctt 540

ggattttgct caaactcatg ctgcatgcaa tcctgataat aagagtcaag agcagcaagg 600

aacacactca aggtcaagct cccagacagc aaaaggcttg ctttttctgc catgtaaagt 660

gtatcaccag tggctaagag ctggctaagc cactgggcag agaagatata tcaggagcat 720

tccagcccct ataaggagct ggctcaagct gtgttcttag ctagcaatac ttccatgctt 780

gtctttctat gcctggctca cttggattcg gtggctagcc tttagagcca gcctgcttgg 840

gttctaattc tcttagctct gtgttgtgtg gatcccttac ttaatctctg tgcctctggg 900

tcatcatttg gaccatggag agaacaacag catggcattg actgacttgg ggaatgaaga 960

attgatatgg gagtcacaga gagccacata aagcatagag gagatactgc ataaagattt 1020

caattatatt tttctattgg tgctggggtg gggatggtcc ttcaatcctt cctatgcatg 1080

aagacataaa aggaagtctc ataatagagt taagacaagg atgcttggca caactcaaaa 1140

ccaaccgtta acattgtttc atttgaagcc ccaaaagatg atggcctcct tggaaaggct 1200

tggttatctt catcctagat gagattatag atcaaaatct gaggtactag actatacttt 1260

ggacctgaga tgctctttcc attatccaac acatctaagc acccaaggaa cataaacatc 1320

gtttgcctct cagggctcat ctccatgccc ctcctgcact ctccctaaca tgtgatggtt 1380

tgtgagattg tccacagcac aggaaacaat tgtgccccct gacagtatca gcagaggtca 1440

tgaacagcct ggaggacagc ctggtgcgtg tgtgtgtttg agatttggga atggacatta 1500

tggtaaacag cattgtttat ctcagcttca ctgacccagc aaagacaggg gcgggtgatt 1560

taattggttc ccctgaggtg gattatgacg ttgtgctcat ggttaaatat ttatgtggta 1620

agtttaggaa tttccagtaa gtgagacagc agcatttttg aacaagtcat ggattttggc 1680

taaaactaaa attctatgat gggcgggtta aaaaaaaatg ccaagacatc tgtgtattta 1740

aaaagaaaga gggagcatgc ccacgtgggt cgtctgcaga aatgagacga aggagcgcca 1800

agtagcctca cggctctgag cttattctct ccagcccttc atctggggta agaatcaatg 1860

actgtctgaa gcttgtaaaa gaaaaaagtg cctgcaaatc aaagaaacgt gcactcttga 1920

aactactaag gagttaaata tttattccta gctttgggga tgtctgtatc cctcctctga 1980

ctggtgaagg ggaaagccac agtaatagat tctttaaaag aaaaaggatt atttattgtg 2040

cacagacctc tggcaagcct ccgtggcttt tgagaacaag cctaggcagt tactattctt 2100

tgaaagtttg ggggaacagt cttggatggc ctcctggcta atgttttagt tcaaattttt 2160

ccttccttag ttttttataa tgatgcctat ctgtgagtac tggcatgttg ctctagcctg 2220

ctacacggct gtggctttga ttgacactgt gctttctagt atctactcaa ttttatggtt 2280

ctaagtttta caaatgcaaa aatttgtttc ctttcctctt aatttagttt ttttttgttt 2340

gttttttgtt ttttgttttt tatttttttt gaggaaatgt tcaagagact ggtaaggtat 2400

aatcaatcat ggaaaaaaaa tctagtcatt gtaaggactg ctgtctggtt tcacgttcag 2460

tagggatgga tgcaatattg tatacctgaa acctactatt atccatatgt gaatttaata 2520

aatacgtgtc aaccgactgt cctgtatggt aagaacaaat caaaacaagt aagtaaaaag 2580

cgatacaaat taattatttt acatggttta tttttcagta tctacagtga tttctcttct 2640

ggaccctacc tcagagagca cttgtcaacc gcctagtgaa cacaccatta ctatcctgtg 2700

ccattgccat agagagacct cagccatcaa tcactagcac atgattgaca gacagagaat 2760

gggactttgg gctttggcaa ttctgacact tcccatgtat ttgacagtta cggagggcag 2820

taagtaccga ttctcagatg agtggtatgg tttgaactta agtaattctg ggaatatttg 2880

gcctcattta agctgcttgc ttcctgttgt tttgggctga attttatgtt aggaaactat 2940

ttcctcttca ttcattcttt atcgtaaatc tctcagaagg aattctctat agggagtaga 3000

tcataatttt tgagttatgt aaattaagac ttaacacagt acacgaacat 3050

<210> 19

<211> 7222

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

ggatgtttat tgactgacac acagtttgtt tagatagata aggctgcttt tccattggat 60

aactgtgggg gatttagctc caaattaatt cattaacatt tcataagaga tcttaacagt 120

tacttattct ttcccaaggt atcacagtat atgaccggct tctaaattta atcctagaag 180

aaaatattga gagtataaga attatgatct tttcatttga tcatttcagg attgtcttta 240

tatcttttat ttgcaggtaa acaatcatgg ggcctggaaa atgaggcttt aattgtaaga 300

tgtcctagac aaggaaaacc tagttacacc gtggattggt attactcaca aacaaacaaa 360

agtattccca ctcaggaaag aaatcgtgtg tttgcctcag gccaacttct gaagtttcta 420

ccagctgcag ttgctgattc tggtatttat acctgtattg tcagaaggta ttatgcagaa 480

ggctcccatc ttctttcacc ctgctcccct ttcttcagtg gttgattgcc tgagctgccc 540

ttgctttcat tccttcccta gtcctttctg gaacagttaa atttataaaa tgatttgaat 600

aaaagtgatt tggataaact tctaggaata ctatcaggtt gaggtctagc tcattctgag 660

ctatttggat ttacagttgc agggattgat ttgtagctga cttagagaaa aacctagctt 720

tcctagtgac caagataact gagagcaatt gcttactttt ggctggaatt aagaacaaac 780

tagcacagaa aatagtaatc tggatgtttt ccatctcagg gggcctctag taggtgaaaa 840

ggggcttcta accttcaagt taagccacag aaggctgatg agattgtgtg cctaaaaatt 900

aattactttt gttcacaaat tgttaaatgt tttgattttg tggctgtatt tggcacacac 960

aaaatgtcaa atgaattaaa tcaaaaagca ggatgtatta gttaagggcc taggtctgag 1020

actagacaag tgtcaaattc atgctctgcc actagttaac tgtgcgatgc cagcaagtta 1080

cttctctgcc cttcagtttc cttctctgta aaatgcatat aatgtaataa tatatttcat 1140

ctcaaagcat tattgtgaaa ataaactaag gtaatgtgtg aaaagtgctc atcatcatca 1200

ctggtacaga gtaaactctg aataaatagt aattatcttt attacactgg gattacagca 1260

ttacagcaga tgtagtgtat gaataaatgg tgaagaagtc attgttaggg ctactatggg 1320

ggctatgttt ttagctcaag tgtaacaaaa aagtatttaa actctagatt ttaatgttta 1380

tttttaaata ataaaataac cattatgtat attgatggtt tttaagaaga aaagcaatat 1440

taagtaaagg ctgaatttag attaagttat ttcacaatgc taagtgactc ttttaattgt 1500

ctgacttatt ttaacagtcc cacattcaat aggactggat atgcgaatgt caccatatat 1560

aaaaaacaat cagattgcaa tgttccagat tatttgatgt attcaacagt atctggatca 1620

gaaaaaaatt ccaaaattta ttgtcctacc attgacctct acaactggac agcacctctt 1680

gagtggttta aggtaagaag aaatttggaa ggaaatagat gaaaattaca caattaaaat 1740

agacacaagt ggccgggcac agtggctcat gcctgtaatc ctagcacttt gggaggccaa 1800

ggcaggcaga tcacttgagg ccaggagttt gagaccagcc tggccaacat gaggaaaccc 1860

catctctact aaaaatataa aaatcagctg ggtgtggtgg cacacacctg taatcccagc 1920

agcttgggag gctgaggtat gaaaatcact tgaacctaga aggcagaggt tacagcgagc 1980

ccagattgca ccactcactc cagcctaggc aacaaacatt ctgtcaacag ataaataaat 2040

aaaagtgaag aattactgag aaggaaatgg aatttcttat ttcagaattg tcaggctctt 2100

caaggatcaa ggtacagggc gcacaagtca tttttggtca ttgataatgt gatgactgag 2160

gacgcaggtg attacacctg taaatttata cacaatgaaa atggagccaa ttatagtgtg 2220

acggcgacca ggtccttcac ggtcaagggt aagctactga cattaatgag atagaatact 2280

acgtgaaaga agtcgaagtg ggaacagcgg tgcccttctg gttgggtttc ttgcacttct 2340

ccctcctccc tttacttcct cctgctccat cttatcttat acattctgaa ctatgacgca 2400

aagaggtttt ctgaacacac tatcaagatt taagaaattt cagggggaaa ttacattact 2460

aattcaaagc cacatctgtt ctttattctt tttttgtgac ttaattttcc aaagataaag 2520

caatctgaat gctaacttaa cttacttttt ttgaatggca atacaactat ttggagagca 2580

aaaccagctt tttttttttt tttctagttt ggtgtcagag tttctgcaaa ttaaaaaaga 2640

gcttaatctt tagtaatact cattggattc aaagtctaat gagaggcttt gtgatggtat 2700

actatggtgt acataaatgt tgtcgagtgg tttttaatct ttgtttgcaa tactttcaac 2760

atcatcaatg gccttgagta agtcacttca ttctaaaaat gtgttttcca agttatttta 2820

aattttataa aagcttattt aagggaaaga tttcacaatc atagcttatc aatctacaaa 2880

ggattggggt ctccttagca caagtcgatc tacagacgta gatgtaatag cccctttacg 2940

tatgaatcga taagcttgat atcgaattcc gaagttccta ttctctagaa agtataggaa 3000

cttcaggtct gaagaggagt ttacgtccag ccaagctagc ttggctgcag gtcgtcgaaa 3060

ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 3120

ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc 3180

ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc 3240

ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa 3300

gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta 3360

ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg 3420

ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga 3480

aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc 3540

tcctcttcct catctccggg cctttcgacc tgcagcctgt tgacaattaa tcatcggcat 3600

agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggga tcggccattg 3660

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 3720

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 3780

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg 3840

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 3900

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 3960

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 4020

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 4080

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 4140

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgatg 4200

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 4260

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 4320

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 4380

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 4440

tcttctgagg ggatcaattc tctagagctc gctgatcagc ctcgactgtg ccttctagtt 4500

gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 4560

ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 4620

ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 4680

ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 4740

cgactagagc ttgcggaacc cttcgaagtt cctattctct agaaagtata ggaacttcat 4800

cagtcaggta cataatggat ccacagtttt ttcagggcaa agcaatattg gagacaaatt 4860

tttggagttt ttcaatactc caccgctgta aaataagcat caccagacca cattcctatc 4920

agtgcctctt tctgtttaat atcaaccctt acagtggtcc ttaatcacac tgtcattaaa 4980

taaatgagca tgaagggatg gaggattgca gcagtgctcc ataagcactg cccgtctttc 5040

agccttagtg gtcacaggag tcagaattcc gtacggggaa gatttcactg aggatgggcc 5100

accctagtgg agaactgcga gcaaatctgt ggactcatcc atttattatt ttcatgggtc 5160

ttttgaaatc ttctctgtag tcttattctt attctgtaga agagtagttt tctaacaact 5220

actaggtcat gtaattagtt ttatgggttg gatctgcatt tgtttaagtg atatcagaga 5280

ataatgatat taaagagcat cataggtaat aaaagaaagt tttatttaag tgcctttctg 5340

tttcgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtttgt cattatgggt 5400

tattgtcaga agaacttgaa aaacattgct atgaaataga atagaaacat gaaaatacaa 5460

gctttatatt gactagcatt caatgctctc ctaatattta tatttctttt tgtctttaag 5520

atgagcaagg cttttctctg tttccagtaa tcggagcccc tgcacaaaat gaaataaagg 5580

aagtggaaat tggtaagaaa atttatcaga atgctgtaaa tattgcctgg aaaaatcctt 5640

ccatatgacc cctgttctga attcccttag caggggtcag gcaattagca taaggaacct 5700

tgaggagtaa gtgaggtgac atccctgaaa gcacctgccc caagcatttg ctaatattgg 5760

gaacagggac acagcaattg cagtgtttac atttgtttat tgtactttgt aattcatgat 5820

gctttcatgt atgcatctaa tttcatcttc atctctatcc cagagcttgg gatggagacc 5880

tgcagggtgt tcattctggg caatggtagc cagatccggt aaaacatgtt tatcttcaaa 5940

gtagcttatg gagagatgaa gagagttctg tagaaagatg tggaagaggg cagttggaaa 6000

gaaactctaa tttctagtag agggcaatcc ttttactaga aatcctttgt aatgtggggt 6060

tggtgaaggc agaatcattg gccttgttag tttcccatgc agatgagaat atagtgggag 6120

ctgagcttca aacccagctg ggtgaatgaa ggtaatggaa gcagggagga ggcaagagag 6180

gacatagaaa gaggaaggtg ctagagatga gggagggagg tcctggtggg gtgcatacta 6240

agtgttcagt aaggtttttt ttttacatta aatgggataa aatgccagtc gcagaagtta 6300

attttattgg tgaatgtcct tactcccctc taggaaaaaa cgcaaaccta acttgctctg 6360

cttgttttgg aaaaggcact cagttcttgg ctgccgtcct gtggcagctt aatggaacaa 6420

aaattacaga ctttggtgaa ccaagaattc aacaagagga agggcaaaat caaaggtatt 6480

tttatattga agagaaccat cctcttcccc ttgcacatgg tttgcacctg caaagtaggc 6540

attaaaagta acaggttgct ttcttagttt cagcaatggg ctggcttgtc tagacatggt 6600

tttaagaata gctgacgtga aggaagagga tttattgctg cagtacgact gtctggccct 6660

gaatttgcat ggcttgagaa ggcacaccgt aagactaagt aggaaaaatc caagtaagga 6720

gtgtttctga gactttgatc acctgaactt tctctagcaa gtgtaagcag aatggagtgt 6780

ggttccaaga gatccatcaa gacaatggga atggcctgtg ccataaaatg tgcttctctt 6840

cttcgggatg ttgtttgctg tctgatcttt gtagactgtt cctgtttgct gggagcttct 6900

ctgctgctta aattgttcgt cctcccccac tccctcctat cgttggtttg tctagaacac 6960

tcagctgctt ctttggtcat ccttgttttc taactttatg aactccctct gtgtcactgt 7020

atgtgaaagg aaatgcacca acaaccgtaa actgaacgtg ttcttttgtg ctcttttata 7080

acttgcatta catgttgtaa gcatggtccg ttctatacct ttttctggtc ataatgaaca 7140

ctcattttgt tagcgagggt ggtaaagtga acaaaaaggg gaagtatcaa actactgcca 7200

tttcagtgag aaaatcctag gt 7222

<210> 20

<211> 3000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tcaatgaagc aagggaagag tgggcctaag gccttgccaa gaactgtaat tctagaccca 60

ggatgcaaca cagacaacat tttcctgttt ataaacactg ccaagaagca tttgcgtctg 120

gtcattgacg tgaaaaccac ttgtagttta caaagtcata agcacgtaga tagatgttga 180

gcatcaactt cctgggtcac cttgaatcac agtcttgcct agggtaaaca tttcagccac 240

ccacacaggt acttcaccca cagcagttcc tataagaagt ctatcttcca taccctctgt 300

acctccctag cacgcatccg gtgggacttt tagacaattt ttctgaacaa gctgcttggg 360

aagttcttac ttaaacctaa agaagtgaag acagcaagct ctggccagtt ttgagatctt 420

ttctcatttt tttgggtcgc aagactttag tgtcttgagt ttctttttcc tggatttaga 480

gagaaggtga actgtcaagg atggtagagg aatttgtcac tactagcagg gatttcacgc 540

caggtcttgt ggcctatgcg tggggattta caaataaaat aaaaaagaac tatttacccg 600

gtctgaccga ggaatctcct catattcatg gataaacaaa gtctatgagg atcatatggt 660

tccaacaggc aagcagacca ggaatggtag cagttcctgt gtccaacctg tacatgtgtc 720

ttacaagttc ctgaaggtga aagccatgtt tcttgcgtgt tttgtgaaac aagggatgtg 780

ggactcggct gagagatagt tgaaggagga tctcaatagc agaaggcatc aggatgatag 840

aacccagcaa tttgatgtct ccatcacagc actctgtgac ctgcaactgt gcgtgtatcc 900

tatattcccc accccctaga tgcaaccatt ttcctgatct actgactcac atgccttagg 960

gctgcccata aacaccgacc ttaccaatcg aattcctatc atttcacaag ttccaagcct 1020

catttccccc cacaaatacc ctgttatatt ccttgaacac gtcatccctt cctatacaaa 1080

tgaacgttcc actactgtca gattaatctt actgaaaagc actttcatct ctaataccct 1140

ttcacatcag gacacattcc ctagcaagag tgtctgatca tgcatagccc tgaccacgcc 1200

ttccacgaat tccccaaacg cagttactac agttcttaga acacacactt ccacctctgc 1260

acatgaactc agcattctct ctggagaggg gagtgttctt tttttttttc ctagaccgtt 1320

ctgagtcttc tttgtccttt agtaatccat tctatagtaa gccttccttc cttcctctaa 1380

tatagagcag tttctgttta agatggctgt cagctgaaat cccttttgcc tcctatctta 1440

gctgggtttt cctgtatcta tgagcttcct tacctttaag cacactctgt gttactaagc 1500

cttaagactt gtgccgagta tgtgctaaga aaattgattt gttgattttg aaagtattgt 1560

tgaaaattca gtattggttt attgggtagg aatgccttgt ggttggtttc tagggatgtc 1620

ggagttcatg caaccatcca ttctgtctca ttttgctatt actaatgccc gtttccccca 1680

tctccccatc tcttgatagt tgatcaccga agcatctact acatagttgc tggatgtagt 1740

ttattgctaa tgtttatcaa tgtcttggtg atagtcttaa aagtgttctg gattgaggtt 1800

gctctgttct ggagagatat agtgacacct tacaaaaccc ggaacggtga gtatctgttt 1860

tcaagattct cttcacaagg aaaagtccca ctgactcttg attatttctt taatgatatt 1920

ttgctcctaa acccaatctc cgtttccttt taacctcatc tgtgagattt tggagtctac 1980

acggatcatt ttccatgcct ctgtgtcact gcttatactt ctgacattca gacattcatc 2040

tccaagattc tccttgccga gattgctatt cctagtgagc ccccataact ctgcctgaga 2100

cccgaacagg ttctgctgtt ttgaaaaatg catgttaata acattttaca acaggacaat 2160

agaatcatac ctgcttttca agtgtgagca ggttatggag agagttggaa cacatgctca 2220

ttgcccttga atcctcattc catgagtaca cacttgcaca aactttacac atgaaaaaga 2280

acagtcccag tgaagtagga actttgaagc ctgtttgtgc acgaagcaat caattaactg 2340

tagattgctt gatttgtggg aaatacagac cccttaagaa gtcctacttc tttgccacta 2400

tttgccattg gcatattacc ttccgaggaa gtagtatggt tgccatatgc cctttctggg 2460

gtttcaattg tctaaggtca actgaagttt gtaaagatta aataagcaaa acaaatgtga 2520

gttttaagtt gagcatgagt ctgagatatg tgattgtcct gcctgggatg tgaattgtct 2580

gtttgttcag tgtatctatg ttgtataatg tataataacc tagcactaat cagttaccct 2640

attagttaac aggctcactg ccgaagttcc gtggtgcctg tgtttgaggg acagttattt 2700

tatgttgacg tcactgcctt cactgctgga gtagtgatgc catggatttt gttgtagtcc 2760

attgtcctat gtttattact agctatggtt gttaaactct tactgtgaca agtgtattag 2820

gtctgtgtgt gcagaagtaa gtgttgaatg gagggcacag tattatctgt ggcttcaggt 2880

actcactgga tgtctttgaa cctgtgctct gtggctaaat gaggaatgtt gtgtgggaaa 2940

actgtaacag tcaatagaca gagaagaccc ttccccaccc atgataatag acactaatag 3000

<210> 21

<211> 5866

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gatcctctag agtcgagcag tgtggttttc aagaggaagc aaaaagcctc tccacccagg 60

cctggaatgt ttccacccaa tgtcgagcag tgtggttttg caagaggaag caaaaagcct 120

ctccacccag gcctggaatg tttccaccca atgtcgagca aaccccgccc agcgtcttgt 180

cattggcgaa ttcgaacacg cagatgcagt cggggcggcg cggtcccagg tccacttcgc 240

atattaaggt gacgcgtgtg gcctcgaaca ccgagcgacc ctgcagcgac ccgcttaaca 300

gcgtcaacag cgtgccgcag atcttggtgg cgtgaaactc ccgcacctct tcggccagcg 360

ccttgtagaa gcgcgtatgg cttcgtaccc cggccatcag cacgcgtctg cgttcgacca 420

ggctgcgcgt tctcgcggcc atagcaaccg acgtacggcg ttgcgccctc gccggcagca 480

agaagccacg gaagtccgcc cggagcagaa aatgcccacg ctactgcggg tttatataga 540

cggtccccac gggatgggga aaaccaccac cacgcaactg ctggtggccc tgggttcgcg 600

cgacgatatc gtctacgtac ccgagccgat gacttactgg cgggtgctgg gggcttccga 660

gacaatcgcg aacatctaca ccacacaaca ccgccttgac cagggtgaga tatcggccgg 720

ggacgcggcg gtggtaatga caagcgccca gataacaatg ggcatgcctt atgccgtgac 780

cgacgccgtt ctggctcctc atatcggggg ggaggctggg agctcacatg ccccgccccc 840

ggccctcacc ctcatcttcg accgccatcc catcgccgcc ctcctgtgct acccggccgc 900

gcgatacctt atgggcagca tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc 960

gccgaccttg cccggcacaa acatcgtgtt gggggccctt ccggaggaca gacacatcga 1020

ccgcctggcc aaacgccagc gccccggcga gcggcttgac ctggctatgc tggccgcgat 1080

tcgccgcgtt tacgggctgc ttgccaatac ggtgcggtat ctgcagggcg gcgggtcgtg 1140

gcgggaggat tggggacagc tttcggggac ggccgtgccg ccccagggtg ccgagcccca 1200

gagcaacgcg ggcccacgac cccatatcgg ggacacgtta tttaccctgt ttcgggcccc 1260

cgagttgctg gcccccaacg gcgacctgta caacgtgttt gcctgggcct tggacgtctt 1320

ggccaaacgc ctccgtccca tgcacgtctt tatcctggat tacgaccaat cgcccgccgg 1380

ctgccgggac gccctgctgc aacttacctc cgggatgatc cagacccacg tcaccacccc 1440

aggctccata ccgacgatct gcgacctggc gcgcacgttt gcccgggaga tgggggaggc 1500

taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa 1560

gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag 1620

ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct 1680

tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc 1740

ggggcggcag gccctgccat agccacgggc cccgtgggtt agggacgggg tcccccatgg 1800

ggaatggttt atggttcgtg ggggttatta ttttgggcgt tgcgtggggt cagtccacga 1860

ctggactgag cagacagacc catggttttt ggatggcctg ggcatggacc gcatgtactg 1920

gcgcgacacg aacaccgggc gtctgtggct gccaaacacc cccgaccccc aaaaaccacc 1980

gcgcggattt ctggcgccgc cggacgaact aaacctgact acggcatctc tgccccttct 2040

tcgctggtac gaggagcgct tttgttttgt attggtcacc acggccgagt ttcctcgacc 2100

gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat 2160

cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc 2220

gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc 2280

gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac 2340

caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta 2400

cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc 2460

ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga 2520

ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg 2580

accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg 2640

gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag 2700

ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt caccactcca 2760

agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa cccttggcag 2820

aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc gggcagcgtt 2880

gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg 2940

cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac 3000

tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt 3060

tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc 3120

gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat 3180

tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca 3240

caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct 3300

cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg 3360

accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg 3420

cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt 3480

cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa 3540

aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 3600

agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg 3660

acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga 3720

ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 3780

accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 3840

tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 3900

ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 3960

ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 4020

gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 4080

gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 4140

ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 4200

tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 4260

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 4320

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 4380

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 4440

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 4500

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4560

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4620

gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 4680

aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 4740

aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 4800

cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 4860

ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 4920

cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 4980

ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 5040

ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 5100

ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 5160

gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 5220

ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 5280

ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 5340

gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 5400

ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 5460

cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 5520

ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 5580

aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 5640

gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 5700

gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 5760

cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattctca tgtttgacag 5820

cttatcatcg ataagctgcg gccgcaaagg ccgcggtcga caagct 5866

<210> 22

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

accagcaggc atgttgtcta tt 22

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gctaagggct tgcagtggtt tgaa 24

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

tgcctcgggt tttatttttc tcaa 24

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

cccccaaccc cactcgctca g 21

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cggaataact gtagtggtcg tgtc 24

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ttggtcttgg cttggtgctt ttat 24

<210> 28

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

attgccaaga tgctgatacc aca 23

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gcgctacgat gcacacctga c 21

<210> 30

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cgcggtcgac aagctagttc tacatttcac caaac 35

<210> 31

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ggaccctagt aggcttgtcc cg 22

<210> 32

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

agcctactag ggtccgcccc tctccctccc ccccccct 38

<210> 33

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

tgtggtatca gcatcttggc aattcatcaa gctgtggcag ggaaac 46

<210> 34

<211> 70

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gatgctgata ccacattcaa accactgcaa gcccttagcg gtccctgggg caacttgccc 60

catccagtgg 70

<210> 35

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cgactctaga ggatcttcct aatttctgtt tcctg 35

<210> 36

<211> 14498

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ttctcatgtt tgacagctta tcatcgataa gctgcggccg caaaggccgc ggtcgacaag 60

ctagttctac atttcaccaa acagggacag gggacagtcc cactatctgg cttatatgac 120

tggaaaaata cctaaaagta ccctgagtac agcattggga gcaggatagc acagggctaa 180

atctctgggg tttctgtttc tatcccttca gccctctaag agggtggctt actgatccat 240

ggaccctgac acccaaggag aaatgacagt gtgaccagag ctttaaagtt ttccttcagc 300

aagagttggc aactccccac tccccattca cagccaggca agggtggcca tagctgcctc 360

tgctctgcta ccttggagcc tgaggctgag tgagtgacgt caccagccca ggaactcagc 420

atagatcttc ccctccactc acccactatg agcctggctg gagaggcaca cacacgattc 480

aaagacaccc caggacattt tcatagcctt taggagtggg gaggcttgtc ctgagccttc 540

tatgacagcg tgttttaaaa cagtgtctgt accccagctt tcaagtagat aaactaacat 600

aaaggaagac cagtttgctc aaaaccatat gactgggaat gtacccagga atggccaccc 660

ttttctccat gccaccgcta aaggtcacat ttcttctagg cctgcgctca caagcaccac 720

actctacctt cacactatgt gtgtggattc tttgcctgca gttctttggt tcatatgctc 780

catcccatga tatagacaag gaaactgagg ctgaaatgct atggcccagg tcacactcaa 840

ggttgggagg caactattga gattgtccct ttggactccc tggtctcacc atcaagctgt 900

gtcccctgcc tcctggctct acctccccct gccctgcttt aagcagaaga tgccacgctt 960

ggggaagcac agagatagtc tacagggagc cctgaaaggc gggtaacaga gggagccttg 1020

tgactctccg tgcccagctt tcacctctct atgcatgggg gttctgtggg ggcacactgg 1080

ttatagtcac aagacctaaa ggccctggga ggccctggac ctttgcctgt ccagtgcctt 1140

agtcaggaaa gatgtctgct agagcttcaa ttaggacaga tccacatgct tcgcaggcca 1200

gccattgtag ttgcacatga cagacatctg tgtcttcctc aggtgagctc ttatgcttgc 1260

aggacctgtg tgtgcatagg tatctgttta caaggagcaa ggggctcaca caggtgtgtg 1320

tgtctgtatg tgtgtgtgtc tcaaatgggc atgggtaggt attgttatgt atgaatattc 1380

atgctatgct cagcattgcc ttgtgaagac actcctttcc cctacacaca gacacacaca 1440

cacacacaca cacacataca cacacacaca catacacacc actcattttc acacgcatcc 1500

ccaccaatgc tcacaggact tctctggagc tggcaggctg tgggtttcag ctctcatgtt 1560

ttctctcaaa ggccctcttg gctcacttgc aaaagccact ttggctcttg tgcaaggacc 1620

ctgttgtgcc tcatgcatac ctgcactcag ggtccagaaa aggtagagag ccttcatcct 1680

cagtgttaac ctggactcac tcagccgggt tcactggcca ggatatgcaa atggactgag 1740

ggagggaagc ttaattctct actgaaatct gggatgtgta cgtgtacttc cctttagagc 1800

cagaaaaaga gcagacaaat aataaacact gaggaaatat ttgctgagag ggtgggaaag 1860

agatttataa gggaactatg ggtggaagaa ggatggaggg tgttctcatt ttatttctgt 1920

tactgtgcca aataccctga ccaaaagtaa cttagggaag gaatgggttc attttactta 1980

caatgacatg tgttacagtt catgataaca ggaagtcaag gcaggaactt gaattggtca 2040

catcaaaccc acactgaaaa gcagagagaa acaatgtatc catgctgtct acttacttgc 2100

ctttggctgg cttcattctt aatacacctt agggttccct gcctagggaa tggtgtcacc 2160

cacagtgggc tgtgtctctt aataaccaat tagcagtcaa gacagttccc ccacagacat 2220

gtccacaagc caacctgatc tagacaattc ttcaactgat gctcacctcc caggtcattt 2280

taggctgtgt gaagttgaca ataaaaatta gccaacaaag gaaagaggga ctgatggata 2340

gatggtcaga aggatggaca tatagacaga agattaatga acagaagaat gggtagatga 2400

atagtgtggg tggaagaaca gatgagaaga ttggcaggtg tgtaaaggga agacattgta 2460

tgtggatgat tgtgaagaag aatggatggt ggaaatggat ggatagatca taaggtttga 2520

tgggtaatta gacagaaatg gatggatgga tggatggatg gatggatgga tggatgggtg 2580

ggtgggtggg tgggtgggtg ggtggatgga tggatggatg gatggataga tggatggatg 2640

gatggatgga tgagtagata gaaggagaga aggatacatg gctggaagaa gggatggagt 2700

agaagatgga taaatagatt tgtgaatgtg cagaagaatg gaagaaaagg aggctggatg 2760

gatagattag taagatgcat gggtaaacag atgaatagat ggatgttata ggtggatggg 2820

tggatggaca agagctggtt aggatggatg gaacggtggt tggatggaca gatggacaga 2880

tggatgaagg ttaagtggaa ggatgtatga aaagaacctc agtttctaga agcttctctc 2940

tctttccagc tgctgttgat tacctggcca aaaacgtgag cctgtccaca cagcgccgta 3000

tgaagcttgg ggaacactcc gctgtgctgg gacttcttcc tgtggaaacg ggacaagcct 3060

actagggtcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa 3120

taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat 3180

gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct 3240

ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct 3300

tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc 3360

gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa 3420

ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc 3480

gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg 3540

gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc 3600

ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat 3660

gggctccaat ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag 3720

tgatgaggtt cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca 3780

tacctggaaa atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa 3840

ccggaaatgg tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc 3900

gcgcggtctg gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg 3960

tcggtccggg ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat 4020

ccgaaaagaa aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac 4080

tgatttcgac caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa 4140

tctggcattt ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat 4200

cagggttaaa gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac 4260

gaaaacgctg gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact 4320

ggtcgagcga tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg 4380

ccgggtcaga aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc 4440

cctggaaggg atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg 4500

tcagagatac ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc 4560

ccgcgctgga gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat 4620

tgtcatgaac tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga 4680

agatggcgat ctcgagccat ctgctggaga catgagagct gccaaccttt ggccaagccc 4740

gctcatgatc aaacgctcta agaagaacag cctggccttg tccctgacgg ccgaccagat 4800

ggtcagtgcc ttgttggatg ctgagccccc catactctat tccgagtatg atcctaccag 4860

acccttcagt gaagcttcga tgatgggctt actgaccaac ctggcagaca gggagctggt 4920

tcacatgatc aactgggcga agagggtgcc aggctttgtg gatttgaccc tccatgatca 4980

ggtccacctt ctagaatgtg cctggctaga gatcctgatg attggtctcg tctggcgctc 5040

catggagcac ccagtgaagc tactgtttgc tcctaacttg ctcttggaca ggaaccaggg 5100

aaaatgtgta gagggcatgg tggagatctt cgacatgctg ctggctacat catctcggtt 5160

ccgcatgatg aatctgcagg gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa 5220

ttctggagtg tacacatttc tgtccagcac cctgaagtct ctggaagaga aggaccatat 5280

ccaccgagtc ctggacaaga tcacagacac tttgatccac ctgatggcca aggcaggcct 5340

gaccctgcag cagcagcacc agcggctggc ccagctcctc ctcatcctct cccacatcag 5400

gcacatgagt aacaaaggca tggagcatct gtacagcatg aagtgcaaga acgtggtgcc 5460

cctctatgac ctgctgctgg aggcggcgga cgcccaccgc ctacatgcgc ccactagccg 5520

tggaggggca tccgtggagg agacggacca aagccacttg gccactgcgg gctctacttc 5580

atcgcattcc ttgcaaaagt attacatcac gggggaggca gagggtttcc ctgccacagc 5640

ttgatgaatt gccaagatgc tgataccaca ttcaaaccac tgcaagccct tagcggtccc 5700

tggggcaact tgccccatcc agtggcccag ccaacccttc ccaagccctg agtctcctca 5760

cctcagtccc cttatccccc tggggttgca ggagaccaag ggaaaaaaac cctttccctt 5820

cctacaggaa accctctgga gacggaaaac cagtgtgcca tctacccatg cttagtgacc 5880

cagagtggcc ccttgccttc ccctctttct tcagaggggt tcctaggcat cctgcagtga 5940

cctccagctc acatccacct tctctgtatc gtggcctcgg tcctgtctca gtgcagagat 6000

tgaggctcaa tttgaaccaa gcacctagtt atcagaagaa aatggtgcca aagacaaggc 6060

cctggagtcc ttgacctctg agtcgtgggt gccctggcta tgggtgtagg tggagcccat 6120

gggtgtcctc agtcacagag ctgggagctc tctctcgctc gcttggcatc aggactgcag 6180

cctctttcac tggacactga gatgagtccc cagggtgttc ccaggggaga aagcaggtaa 6240

catcccagct ttacctagga atccagagga ctttaggact gtccctatgc accctgcagg 6300

gcatcaggag accaggaagg gattctagca gagggtaggg ggcacagagg cagagctgat 6360

tgcccatggg ctatcccaga atgcctggtc ctgaatctag catcaggagg tgcaggattc 6420

ctaggctgca atctgacaga ggcttgccca ctgtgtcagg cctgggcagc ccacagaacc 6480

tgtcactctc ctcaattggt aggagaagag gtcttgaggt gacaggaggc agaaggcagg 6540

ctcagacagt cagagagcac caagctttca agtccgcacc cctggggttc ggcataccat 6600

cttgctggca gctggaaacc tggttccctg aaagggggcc tccatcctcc agaatgtaag 6660

gctcttgatg ccaccggatg cagagagcct tctcgggcca gacaaaattg ctgctccacc 6720

ccagagaaga tgttccagcc ttcttggcat cttagaggaa ggccatgtcg ctgtcctttt 6780

cagagtagca tatttttcag tgatggctgc tcagtcagga ggcttctgtc gccttacaaa 6840

gcacagtgcg ctctgggcac tgtttctaag ccaccccatc ccacccccac ccccgccacc 6900

ccggggacag aggaagatgc taaaagtccc agcaaagagg acaaagcacc tttcttaagc 6960

actccagagt cttccttgta ccccgccctc tcttagagct gggtcttttg agggaaacgg 7020

attgctgagc cctccccccc ccccaatcct ctcctctgtg gagctgttta tcatcctcta 7080

tttatcaaaa tcgcatccat ctttaccctc tccttcgcta tagcctactt ctggatcacc 7140

ctcatccagt gctggtaccc cacagcacta aatccaggaa ccctgggctt gatcacctgt 7200

tgccacctgt acacatgaaa ccacctgctg gcccggccca tgtctcctgc cctcagccag 7260

caagacattc ctagagagag gaactatggg cttaaaagcc ccaactgact tccttttgcc 7320

tggggacctg aaccgacaag acaccaggga cacttgtcta catgaacatg tgaccaatgt 7380

acaccgattt ctcatctcta gacctattat ctgaagcctg tcccgggcca tgactagaat 7440

ggcttgtatc tgtggtttag agaagtctaa taataactga gggcaaactg actctctggt 7500

agcatggagc accaggcgga tggagctcac cagctctgtc caggtttcaa aggaggagac 7560

tgttgggctc ttcaaggtct ggacaagagg aaagccacat tgcccccttg ggaacccagg 7620

ttctcctttt gaacttctca cagctgcaag cacccctttc aaagaccaaa tgcatcctcc 7680

tccacattcc ttgctccctg gaggcctggc tctggataca cctgagtctt cgttcaccta 7740

ctacacttta ggagcaggaa cttcaagcag gtgacatcca cagggcccag tcccagccaa 7800

gggagcaaca ttccaacgct tggaccaatc ataatgatct gcccgtgagg gtaaccgcaa 7860

ctagagacct gcttgggaga aaacaaaatg acttctcatt ccatgccatg cctctgaatg 7920

ctcccccaag ctgccatctt ggtataaaat gggacttgtg ttgtggggaa ccccttgacc 7980

ccaacaggtt ttcccaactg tctcatgctt ttgtgaatct gtctgctttg atctgtaaaa 8040

ctcagccttg tttgggcagc ttgtaatttc aacagtgagg cgacatcgat tagatgagag 8100

gcaccaggcc tctccgccgc cgtccctctg tggccgtccc tctggggttg agcagaacct 8160

agaagaaggc cgatttccag tggccagact ggaccagaaa cagcccccac cccaatccct 8220

gtaaatagag tcaatagcaa aataagaggg gcgccctcca tgtcacctca agtagctact 8280

ggttcttctg tggaggcccc tctgaactca ttgtctggta gttgaaaatg tgatgttgtg 8340

ctgtttgttt atagaacatt ggctttttat atataaatct atatacttaa aaacaaaaac 8400

aaaaacaaaa aaaatggaaa gaaaccccca cagtttgtgt cgtgctagtg ccggggacca 8460

tctgtaggga tctcattgtg tgtttggatt atgtatgcca gcctttctcg ggttcctcct 8520

tctttttttt tttctttgtc atttccctac tcccgatttg gtttttctct ctcgctctga 8580

aaatgaaaga gatggcttga agagccaagc tcctactcct ttactttata ggcgaattaa 8640

ctttgccctt cttttagggg accaggaaca agcacaggaa acagaaatta ggaagatcct 8700

ctagagtcga gcagtgtggt tttcaagagg aagcaaaaag cctctccacc caggcctgga 8760

atgtttccac ccaatgtcga gcagtgtggt tttgcaagag gaagcaaaaa gcctctccac 8820

ccaggcctgg aatgtttcca cccaatgtcg agcaaacccc gcccagcgtc ttgtcattgg 8880

cgaattcgaa cacgcagatg cagtcggggc ggcgcggtcc caggtccact tcgcatatta 8940

aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag cgacccgctt aacagcgtca 9000

acagcgtgcc gcagatcttg gtggcgtgaa actcccgcac ctcttcggcc agcgccttgt 9060

agaagcgcgt atggcttcgt accccggcca tcagcacgcg tctgcgttcg accaggctgc 9120

gcgttctcgc ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc 9180

cacggaagtc cgcccggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc 9240

ccacgggatg gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga 9300

tatcgtctac gtacccgagc cgatgactta ctggcgggtg ctgggggctt ccgagacaat 9360

cgcgaacatc tacaccacac aacaccgcct tgaccagggt gagatatcgg ccggggacgc 9420

ggcggtggta atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc 9480

cgttctggct cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct 9540

caccctcatc ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata 9600

ccttatgggc agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac 9660

cttgcccggc acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct 9720

ggccaaacgc cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg 9780

cgtttacggg ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga 9840

ggattgggga cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa 9900

cgcgggccca cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt 9960

gctggccccc aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa 10020

acgcctccgt cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg 10080

ggacgccctg ctgcaactta cctccgggat gatccagacc cacgtcacca ccccaggctc 10140

cataccgacg atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg 10200

aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga 10260

ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg 10320

cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt 10380

tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg 10440

gcaggccctg ccatagccac gggccccgtg ggttagggac ggggtccccc atggggaatg 10500

gtttatggtt cgtgggggtt attattttgg gcgttgcgtg gggtcagtcc acgactggac 10560

tgagcagaca gacccatggt ttttggatgg cctgggcatg gaccgcatgt actggcgcga 10620

cacgaacacc gggcgtctgt ggctgccaaa cacccccgac ccccaaaaac caccgcgcgg 10680

atttctggcg ccgccggacg aactaaacct gactacggca tctctgcccc ttcttcgctg 10740

gtacgaggag cgcttttgtt ttgtattggt caccacggcc gagtttcctc gaccgatgcc 10800

cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc 10860

cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg 10920

ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc 10980

ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg 11040

tttcggcgag aagcaggcca ttatcgccgg catggcggcc gacgcgctgg gctacgtctt 11100

gctggcgttc gcgacgcgag gctggatggc cttccccatt atgattcttc tcgcttccgg 11160

cggcatcggg atgcccgcgt tgcaggccat gctgtccagg caggtagatg acgaccatca 11220

gggacagctt caaggatcgc tcgcggctct taccagccta acttcgatca ctggaccgct 11280

gatcgtcacg gcgatttatg ccgcctcggc gagcacatgg aacgggttgg catggattgt 11340

aggcgccgcc ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc 11400

cacctcgacc tgaatggaag ccggcggcac ctcgctaacg gattcaccac tccaagaatt 11460

ggagccaatc aattcttgcg gagaactgtg aatgcgcaaa ccaacccttg gcagaacata 11520

tccatcgcgt ccgccatctc cagcagccgc acgcggcgca tctcgggcag cgttgggtcc 11580

tggccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt 11640

tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc 11700

tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa 11760

agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga 11820

tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc 11880

tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt 11940

tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc 12000

atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa 12060

caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg 12120

gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac 12180

cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc 12240

tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 12300

caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag 12360

tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac 12420

tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 12480

tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 12540

gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 12600

caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 12660

tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 12720

gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 12780

ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 12840

cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 12900

tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 12960

tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 13020

cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 13080

agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 13140

agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 13200

gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 13260

aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 13320

ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 13380

gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 13440

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 13500

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 13560

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 13620

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 13680

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 13740

ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 13800

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 13860

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 13920

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 13980

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 14040

cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 14100

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 14160

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 14220

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 14280

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 14340

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 14400

ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 14460

aaaataggcg tatcacgagg ccctttcgtc ttcaagaa 14498

<210> 37

<211> 3008

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

agttctacat ttcaccaaac agggacaggg gacagtccca ctatctggct tatatgactg 60

gaaaaatacc taaaagtacc ctgagtacag cattgggagc aggatagcac agggctaaat 120

ctctggggtt tctgtttcta tcccttcagc cctctaagag ggtggcttac tgatccatgg 180

accctgacac ccaaggagaa atgacagtgt gaccagagct ttaaagtttt ccttcagcaa 240

gagttggcaa ctccccactc cccattcaca gccaggcaag ggtggccata gctgcctctg 300

ctctgctacc ttggagcctg aggctgagtg agtgacgtca ccagcccagg aactcagcat 360

agatcttccc ctccactcac ccactatgag cctggctgga gaggcacaca cacgattcaa 420

agacacccca ggacattttc atagccttta ggagtgggga ggcttgtcct gagccttcta 480

tgacagcgtg ttttaaaaca gtgtctgtac cccagctttc aagtagataa actaacataa 540

aggaagacca gtttgctcaa aaccatatga ctgggaatgt acccaggaat ggccaccctt 600

ttctccatgc caccgctaaa ggtcacattt cttctaggcc tgcgctcaca agcaccacac 660

tctaccttca cactatgtgt gtggattctt tgcctgcagt tctttggttc atatgctcca 720

tcccatgata tagacaagga aactgaggct gaaatgctat ggcccaggtc acactcaagg 780

ttgggaggca actattgaga ttgtcccttt ggactccctg gtctcaccat caagctgtgt 840

cccctgcctc ctggctctac ctccccctgc cctgctttaa gcagaagatg ccacgcttgg 900

ggaagcacag agatagtcta cagggagccc tgaaaggcgg gtaacagagg gagccttgtg 960

actctccgtg cccagctttc acctctctat gcatgggggt tctgtggggg cacactggtt 1020

atagtcacaa gacctaaagg ccctgggagg ccctggacct ttgcctgtcc agtgccttag 1080

tcaggaaaga tgtctgctag agcttcaatt aggacagatc cacatgcttc gcaggccagc 1140

cattgtagtt gcacatgaca gacatctgtg tcttcctcag gtgagctctt atgcttgcag 1200

gacctgtgtg tgcataggta tctgtttaca aggagcaagg ggctcacaca ggtgtgtgtg 1260

tctgtatgtg tgtgtgtctc aaatgggcat gggtaggtat tgttatgtat gaatattcat 1320

gctatgctca gcattgcctt gtgaagacac tcctttcccc tacacacaga cacacacaca 1380

cacacacaca cacatacaca cacacacaca tacacaccac tcattttcac acgcatcccc 1440

accaatgctc acaggacttc tctggagctg gcaggctgtg ggtttcagct ctcatgtttt 1500

ctctcaaagg ccctcttggc tcacttgcaa aagccacttt ggctcttgtg caaggaccct 1560

gttgtgcctc atgcatacct gcactcaggg tccagaaaag gtagagagcc ttcatcctca 1620

gtgttaacct ggactcactc agccgggttc actggccagg atatgcaaat ggactgaggg 1680

agggaagctt aattctctac tgaaatctgg gatgtgtacg tgtacttccc tttagagcca 1740

gaaaaagagc agacaaataa taaacactga ggaaatattt gctgagaggg tgggaaagag 1800

atttataagg gaactatggg tggaagaagg atggagggtg ttctcatttt atttctgtta 1860

ctgtgccaaa taccctgacc aaaagtaact tagggaagga atgggttcat tttacttaca 1920

atgacatgtg ttacagttca tgataacagg aagtcaaggc aggaacttga attggtcaca 1980

tcaaacccac actgaaaagc agagagaaac aatgtatcca tgctgtctac ttacttgcct 2040

ttggctggct tcattcttaa tacaccttag ggttccctgc ctagggaatg gtgtcaccca 2100

cagtgggctg tgtctcttaa taaccaatta gcagtcaaga cagttccccc acagacatgt 2160

ccacaagcca acctgatcta gacaattctt caactgatgc tcacctccca ggtcatttta 2220

ggctgtgtga agttgacaat aaaaattagc caacaaagga aagagggact gatggataga 2280

tggtcagaag gatggacata tagacagaag attaatgaac agaagaatgg gtagatgaat 2340

agtgtgggtg gaagaacaga tgagaagatt ggcaggtgtg taaagggaag acattgtatg 2400

tggatgattg tgaagaagaa tggatggtgg aaatggatgg atagatcata aggtttgatg 2460

ggtaattaga cagaaatgga tggatggatg gatggatgga tggatggatg gatgggtggg 2520

tgggtgggtg ggtgggtggg tggatggatg gatggatgga tggatagatg gatggatgga 2580

tggatggatg agtagataga aggagagaag gatacatggc tggaagaagg gatggagtag 2640

aagatggata aatagatttg tgaatgtgca gaagaatgga agaaaaggag gctggatgga 2700

tagattagta agatgcatgg gtaaacagat gaatagatgg atgttatagg tggatgggtg 2760

gatggacaag agctggttag gatggatgga acggtggttg gatggacaga tggacagatg 2820

gatgaaggtt aagtggaagg atgtatgaaa agaacctcag tttctagaag cttctctctc 2880

tttccagctg ctgttgatta cctggccaaa aacgtgagcc tgtccacaca gcgccgtatg 2940

aagcttgggg aacactccgc tgtgctggga cttcttcctg tggaaacggg acaagcctac 3000

tagggtcc 3008

<210> 38

<211> 2577

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat gggctccaat 600

ttactgaccg tacaccaaaa tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt 660

cgcaagaacc tgatggacat gttcagggat cgccaggcgt tttctgagca tacctggaaa 720

atgcttctgt ccgtttgccg gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg 780

tttcccgcag aacctgaaga tgttcgcgat tatcttctat atcttcaggc gcgcggtctg 840

gcagtaaaaa ctatccagca acatttgggc cagctaaaca tgcttcatcg tcggtccggg 900

ctgccacgac caagtgacag caatgctgtt tcactggtta tgcggcggat ccgaaaagaa 960

aacgttgatg ccggtgaacg tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac 1020

caggttcgtt cactcatgga aaatagcgat cgctgccagg atatacgtaa tctggcattt 1080

ctggggattg cttataacac cctgttacgt atagccgaaa ttgccaggat cagggttaaa 1140

gatatctcac gtactgacgg tgggagaatg ttaatccata ttggcagaac gaaaacgctg 1200

gttagcaccg caggtgtaga gaaggcactt agcctggggg taactaaact ggtcgagcga 1260

tggatttccg tctctggtgt agctgatgat ccgaataact acctgttttg ccgggtcaga 1320

aaaaatggtg ttgccgcgcc atctgccacc agccagctat caactcgcgc cctggaaggg 1380

atttttgaag caactcatcg attgatttac ggcgctaagg atgactctgg tcagagatac 1440

ctggcctggt ctggacacag tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga 1500

gtttcaatac cggagatcat gcaagctggt ggctggacca atgtaaatat tgtcatgaac 1560

tatatccgta acctggatag tgaaacaggg gcaatggtgc gcctgctgga agatggcgat 1620

ctcgagccat ctgctggaga catgagagct gccaaccttt ggccaagccc gctcatgatc 1680

aaacgctcta agaagaacag cctggccttg tccctgacgg ccgaccagat ggtcagtgcc 1740

ttgttggatg ctgagccccc catactctat tccgagtatg atcctaccag acccttcagt 1800

gaagcttcga tgatgggctt actgaccaac ctggcagaca gggagctggt tcacatgatc 1860

aactgggcga agagggtgcc aggctttgtg gatttgaccc tccatgatca ggtccacctt 1920

ctagaatgtg cctggctaga gatcctgatg attggtctcg tctggcgctc catggagcac 1980

ccagtgaagc tactgtttgc tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta 2040

gagggcatgg tggagatctt cgacatgctg ctggctacat catctcggtt ccgcatgatg 2100

aatctgcagg gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg 2160

tacacatttc tgtccagcac cctgaagtct ctggaagaga aggaccatat ccaccgagtc 2220

ctggacaaga tcacagacac tttgatccac ctgatggcca aggcaggcct gaccctgcag 2280

cagcagcacc agcggctggc ccagctcctc ctcatcctct cccacatcag gcacatgagt 2340

aacaaaggca tggagcatct gtacagcatg aagtgcaaga acgtggtgcc cctctatgac 2400

ctgctgctgg aggcggcgga cgcccaccgc ctacatgcgc ccactagccg tggaggggca 2460

tccgtggagg agacggacca aagccacttg gccactgcgg gctctacttc atcgcattcc 2520

ttgcaaaagt attacatcac gggggaggca gagggtttcc ctgccacagc ttgatga 2577

<210> 39

<211> 2995

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ctggggcaac ttgccccatc cagtggccca gccaaccctt cccaagccct gagtctcctc 60

acctcagtcc ccttatcccc ctggggttgc aggagaccaa gggaaaaaaa ccctttccct 120

tcctacagga aaccctctgg agacggaaaa ccagtgtgcc atctacccat gcttagtgac 180

ccagagtggc cccttgcctt cccctctttc ttcagagggg ttcctaggca tcctgcagtg 240

acctccagct cacatccacc ttctctgtat cgtggcctcg gtcctgtctc agtgcagaga 300

ttgaggctca atttgaacca agcacctagt tatcagaaga aaatggtgcc aaagacaagg 360

ccctggagtc cttgacctct gagtcgtggg tgccctggct atgggtgtag gtggagccca 420

tgggtgtcct cagtcacaga gctgggagct ctctctcgct cgcttggcat caggactgca 480

gcctctttca ctggacactg agatgagtcc ccagggtgtt cccaggggag aaagcaggta 540

acatcccagc tttacctagg aatccagagg actttaggac tgtccctatg caccctgcag 600

ggcatcagga gaccaggaag ggattctagc agagggtagg gggcacagag gcagagctga 660

ttgcccatgg gctatcccag aatgcctggt cctgaatcta gcatcaggag gtgcaggatt 720

cctaggctgc aatctgacag aggcttgccc actgtgtcag gcctgggcag cccacagaac 780

ctgtcactct cctcaattgg taggagaaga ggtcttgagg tgacaggagg cagaaggcag 840

gctcagacag tcagagagca ccaagctttc aagtccgcac ccctggggtt cggcatacca 900

tcttgctggc agctggaaac ctggttccct gaaagggggc ctccatcctc cagaatgtaa 960

ggctcttgat gccaccggat gcagagagcc ttctcgggcc agacaaaatt gctgctccac 1020

cccagagaag atgttccagc cttcttggca tcttagagga aggccatgtc gctgtccttt 1080

tcagagtagc atatttttca gtgatggctg ctcagtcagg aggcttctgt cgccttacaa 1140

agcacagtgc gctctgggca ctgtttctaa gccaccccat cccaccccca cccccgccac 1200

cccggggaca gaggaagatg ctaaaagtcc cagcaaagag gacaaagcac ctttcttaag 1260

cactccagag tcttccttgt accccgccct ctcttagagc tgggtctttt gagggaaacg 1320

gattgctgag ccctcccccc cccccaatcc tctcctctgt ggagctgttt atcatcctct 1380

atttatcaaa atcgcatcca tctttaccct ctccttcgct atagcctact tctggatcac 1440

cctcatccag tgctggtacc ccacagcact aaatccagga accctgggct tgatcacctg 1500

ttgccacctg tacacatgaa accacctgct ggcccggccc atgtctcctg ccctcagcca 1560

gcaagacatt cctagagaga ggaactatgg gcttaaaagc cccaactgac ttccttttgc 1620

ctggggacct gaaccgacaa gacaccaggg acacttgtct acatgaacat gtgaccaatg 1680

tacaccgatt tctcatctct agacctatta tctgaagcct gtcccgggcc atgactagaa 1740

tggcttgtat ctgtggttta gagaagtcta ataataactg agggcaaact gactctctgg 1800

tagcatggag caccaggcgg atggagctca ccagctctgt ccaggtttca aaggaggaga 1860

ctgttgggct cttcaaggtc tggacaagag gaaagccaca ttgccccctt gggaacccag 1920

gttctccttt tgaacttctc acagctgcaa gcaccccttt caaagaccaa atgcatcctc 1980

ctccacattc cttgctccct ggaggcctgg ctctggatac acctgagtct tcgttcacct 2040

actacacttt aggagcagga acttcaagca ggtgacatcc acagggccca gtcccagcca 2100

agggagcaac attccaacgc ttggaccaat cataatgatc tgcccgtgag ggtaaccgca 2160

actagagacc tgcttgggag aaaacaaaat gacttctcat tccatgccat gcctctgaat 2220

gctcccccaa gctgccatct tggtataaaa tgggacttgt gttgtgggga accccttgac 2280

cccaacaggt tttcccaact gtctcatgct tttgtgaatc tgtctgcttt gatctgtaaa 2340

actcagcctt gtttgggcag cttgtaattt caacagtgag gcgacatcga ttagatgaga 2400

ggcaccaggc ctctccgccg ccgtccctct gtggccgtcc ctctggggtt gagcagaacc 2460

tagaagaagg ccgatttcca gtggccagac tggaccagaa acagccccca ccccaatccc 2520

tgtaaataga gtcaatagca aaataagagg ggcgccctcc atgtcacctc aagtagctac 2580

tggttcttct gtggaggccc ctctgaactc attgtctggt agttgaaaat gtgatgttgt 2640

gctgtttgtt tatagaacat tggcttttta tatataaatc tatatactta aaaacaaaaa 2700

caaaaacaaa aaaaatggaa agaaaccccc acagtttgtg tcgtgctagt gccggggacc 2760

atctgtaggg atctcattgt gtgtttggat tatgtatgcc agcctttctc gggttcctcc 2820

ttcttttttt ttttctttgt catttcccta ctcccgattt ggtttttctc tctcgctctg 2880

aaaatgaaag agatggcttg aagagccaag ctcctactcc tttactttat aggcgaatta 2940

actttgccct tcttttaggg gaccaggaac aagcacagga aacagaaatt aggaa 2995

Claims

1. A homologous recombination vector is characterized in that the homologous recombination vector is Il1rl1-donor vector, and the sequence of the homologous recombination vector is shown as SEQ ID NO. 17.

2. The method for constructing the homologous recombination vector according to claim 1, comprising: constructing an Il1rl1 gene humanized homologous recombinant vector by a one-step recombination mode of a 5 'arm, a knock-In fragment, a 3' arm and a skeleton vector In an In-Fusion mode, wherein the sequences of the 5 'arm, the knock-In fragment, the 3' arm and the skeleton vector are respectively shown as SEQ ID NO: 18-21.

3. A method of gene homologous recombination, which comprises using the homologous recombination vector of claim 1.

4. A gene homologous recombination kit comprising the homologous recombination vector of claim 1.

5. Use of the homologous recombination vector of claim 1 for homologous recombination of genes.