NZ571952A

NZ571952A - Novel genes encoding insecticidal proteins

Info

Publication number: NZ571952A
Application number: NZ571952A
Authority: NZ
Inventors: Rie Jeroen Van; Frank Meulewaeter; Eldik Gerben Van
Original assignee: Bayer Bioscience Nv
Priority date: 2006-03-21
Filing date: 2007-03-16
Publication date: 2011-06-30
Also published as: ATE511515T1; UA98108C2; CA2924415A1; DK1999141T3

Abstract

Disclosed is a chimeric gene, comprising the following operably-linked sequences: a) a coding region encoding a Cry1C protein, comprising a DNA sequence with at least 98 % sequence identity to the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6, or comprising a DNA sequence hybridizing under stringent hybridization conditions to the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6, wherein said coding region encodes a protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2, and b) a promoter region capable of directing expression in plant cells. Also disclosed is the use of the chimeric gene to control insect pests.

Description

New Zealand Paient Spedficaiion for Paient Number 571 952 1 Novel genes encoding insecticidal proteins.

INTRODUCTION The present invention relates to new gene sequences encoding insecticidal proteins produced by Bacillus thuringiensis strains. Particularly, new chimeric genes encoding a CrylC protein are provided which are useful to protect plants from insect damage. Also included herein are plant cells or plants comprising such genes and methods of making or using them, as well as plant cells or plants comprising such crylC chimeric gene and at least one other gene encoding an insecticidal protein, such as new gene sequences encoding a Cry1 B or Cry1 D protein.

BACKGROUND OF THE INVENTION Strain and proteins derived from Bacillus thuringiensis (abbreviated herein as "Bt") are well known for their specific toxicity to insect pests, and they have been used since almost a century to control insect pests. Some transgenic plant species expressing Bt proteins are now available, and they successfully limit insect damage on plants.

Despite the isolation of quite a number of insecticidal Bt proteins, only a few Bt proteins have been expressed in transgenic plants that have been commercialized, and this only in some crops. Most commercialized transgenic Bt plants belong to the bigger field crops such as corn and cotton. In smaller market crops such as vegetables, only a few plant species have been transformed with Bt genes so as to render them resistant to major Lepidopteran insect pests, but to date no Lepidopteran-resistant vegetable Bt-plant or seed is deregulated and marketed. Zhao et al. (2003) have described transgenic broccoli plants expressing a CrylAc or a CrylC Bt toxin, as well as crosses between these plants so that both the CrylAc and CrylC toxins are expressed in the same plants, but these plants have not been commercialized. NewLeaf ™ potatoes comprising a Cry3A Coleopteran-active gene were briefly commercialized in Northern America, but have been withdrawn from the market in 2001. 2 The current invention provides new genes encoding proteins of the CrylC type of Bt proteins, which ideally are combined with genes encoding proteins of the CrylB or CrylD type Bt proteins.

The DNA sequences of the crylC, crylB or crylD genes of the invention and of the modified transit peptide of the invention (shown in the enclosed sequence listing) are artificial genes, not found in nature, and are different from any known DNA sequence. Indeed, any one of the DNA sequences of SEQ ID Nos. 1, 3, 10, 14 or 16 shows at most 76.6 % sequence identity with the closest known DNA sequences.

OBJECTS AND SUMMARY OF THE INVENTION In the current invention, several new insect control genes derived from Bt are provided for use in plants. Specifically, such genes are useful in vegetables plant crops, particularly Brassicaceae plants such as cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale, broccoli, Brussels sprouts, mustard spinach, and the like. Particularly, in one embodiment of this invention the following Brassica species plants are protected from insects by the new genes of the current invention: B. carinata, B. elongata, B. fruticulosa, B. juncea, B. napus, B. narinosa, B. nigra, B. oleracea, B. perviridis, B. rapa, B. rupestris, B. septiceps, B. tournefortii, and the like, particularly plants of the species Brassica oleraceae or Brassica napus. The plants or seeds comprising at least one of the new genes of the invention can be obtained by transformation of plant cells and production of plants or seed therefrom comprising the genes of the invention. Also included herein are plants or seeds obtained by crossing with a plant transformed to contain at least one of the genes of the invention, and by application of routine breeding steps. Obviously, any plant species to be protected from insect species that are killed or controlled by the Bt proteins encoded by the novel genes of this invention can be transformed with the genes of the invention to obtain transgenic plants and seeds with increased resistance to such insects.

In one embodiment, the current invention also provides a combination of technologies to allow for the most optimal product from a resistance management point of view. Indeed, in one embodiment of this invention the plants of the invention produce at least 2 different Bt proteins and such proteins are encoded by the highly-expressed cry genes of 3 the invention which have been stably integrated, preferably at a single locus in the plant's genome. In one embodiment of the invention, such at least 2 Bt genes include a crylC and a crylB gene, a crylC and a crylD gene, or a combination of a crylC, a cryl B and a cryl D gene of this invention. In one embodiment of the invention a marker gene allowing rapid identification of transgenic plants, preferably a herbicide resistance gene, is located in the same plant, particularly at the same locus in the plant's genome as a cry gene of the invention. In one embodiment of this invention, the marker gene is a gene encoding a phosphinothricin acetyltransferase or a glyphosate-insensitive EPSPS.

In the invention also novel crylB and crylD genes, particularly crylB or crylD chimeric genes, are provided, which can be expressed in plants at high levels, such as the cry1B1 and cry1B2 and the cry1D1 and cry1D2 genes. Also plants cells, plants or seeds comprising any of these genes and methods of producing or using them alone or in combination are provided herein.

Also, the current invention provides novel genes encoding an insecticidal protein comprising a functional plant intron in their coding sequence. The presence of the intron also secures that the gene does not express a functional protein when the gene is in an environment where the intron cannot be spliced, such as a bacteria or another prokaryotic microorganism. The presence of this intron in the gene sequence also allows for high expression levels to be obtained in plants.

Also included herein are variants of the CrylC protein of the invention comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine. Also provided herein are variants of the Cryl B protein of the invention comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at 4 position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine.

Also included in this invention is a novel DNA encoding a chloroplast transit peptide, particularly a DNA comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16, as well as such DNA encoding a variant of the protein of SEQ ID No. 17, such as a chloroplast transit peptide comprising the sequence of SEQ ID No. 17 from amino acid position 3 to amino acid position 124, wherein the Cys amino acid at position 55 is replaced by Tyr and/or wherein a Gly amino acid is added after the Gly amino acid at position 51.

Specifically, the current invention provides a chimeric gene, comprising the following operably-linked sequences: a) a coding region encoding a CrylC protein, comprising the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6 or a variant thereof, and b) a promoter region capable of directing expression in plant cells. In one embodiment, such promoter comprises the sequence of SEQ ID No. 18 or 19. In another embodiment, the chimeric gene further comprises a 3' polyadenylation and transcript termination region, particularly that of the NADP-malic enzyme gene from Flaveria bidentis. In another embodiment, the chimeric gene further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

The current invention also provides a DNA comprising any of the above chimeric genes, further comprising a second chimeric gene, said second chimeric gene comprising the following operably-linked sequences: a) a second coding region encoding a Cryl B protein comprising the DNA of SEQ ID No. 8 or 10, and b) a second promoter region capable of directing expression in plant cells; or a DNA comprising any of the above chimeric genes, further comprising a second chimeric gene, said second chimeric gene comprising the following operably-linked sequences: a) a coding region encoding a CrylD protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter region capable of directing expression in plant cells. In one embodiment, the above DNAs are provided, wherein said second promoter region comprises the sequence of SEQ ID No. 18 or 19 and is different from said first promoter region; or wherein said second chimeric gene further comprises a 3' polyadenylation and transcript termination region, particularly of the NADP-malic enzyme gene from Flaveria bidentis. In one embodiment, the second chimeric gene in these DNAs further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

The current invention also provides the above DNAs, further comprising a third chimeric gene, said third chimeric gene comprising the following operably-linked sequences: a) a coding region encoding a CrylD protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter region capable of directing expression in plants.

Also included in the current invention are a transgenic plant cell or plant, comprising any of the above genes or DNAs stably incorporated in its genome, preferably when the cell or plant is a Brassica species plant or plant cell, particularly of the species Brassica oleraceae, more particularly cabbage or cauliflower.

Also included in this invention is the use of any of the above chimeric genes or DNAs to control insect pests, to obtain plant cells, plants or seeds with increased resistance to insects; the use of any of the above chimeric genes or DNAs to delay or prevent insect resistance development in transgenic plants expressing an insecticidal protein by insects attempting to feed on such plants; or the use of any of the above chimeric genes or DNAs to obtain cabbage, oilseed rape or cauliflower protected from Plutella xylostella. Also included herein are methods for controlling insects, comprising the step of planting or sowing in a field, plants comprising any of the above chimeric genes or DNAs; as well as methods of controlling insects in Brassica species plants, comprising the step of expressing any of the above chimeric genes or DNA in plants; or methods of producing plants or seeds resistant to insects, comprising the steps of: a) obtaining a plant transformed with the gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12, and b) selecting progeny of said plant or seeds thereof, containing said gene or DNA.

Also provided in accordance with this invention is a chimeric gene comprising the following operably-linked sequences: a) a first fragment of a coding sequence encoding an insecticidal protein, b) a plant intron sequence, c) a second fragment of said coding sequence, d) a promoter region capable of directing expression in plant cells, and wherein no insecticidal protein can be produced from such chimeric gene in a given host 6 cell wherein the intron is not spliced; particularly such chimeric gene wherein such intron is the second intron of the ST-LS1 gene of Solanum tuberosum.

Further provided herein is also a microorganism comprising any of the above chimeric genes or DNAs, particularly when such microorganism is of the genus Escherichia, Bacillus or Agrobacterium.

DESCRIPTION In accordance with this invention, a "nucleic acid sequence" refers to a DNA or RNA molecule in single or double stranded form, preferably a DNA molecule. An "isolated DNA", as used herein, refers to a DNA which is not naturally-occurring or no longer in the natural environment wherein it was originally present, e.g., a DNA coding sequence associated with other regulatory elements in a chimeric gene, a DNA transferred into another host cell, such as a plant cell, or an artificial, synthetically-made DNA sequence having a different nucleotide sequence compared to any naturally-occurring DNA sequence.

In accordance with this invention, nucleic acid sequences, particularly DNA sequences, encoding Bt Cry toxins or variants thereof have been constructed. The new DNA sequences are designated herein as cry1C1-4, cry1B1, cry1B2, cry1D1, and cry1D2, and their encoded proteins are designated herein as CrylC (e.g., Cry1C1, Cry1C3, and Cry1C4), CrylB (e.g., Cry1B1 and Cry1B2) and CrylD (e.g., Cry1D1 and Cry1D2) proteins. Also a new DNA sequence encoding a modified chloroplast transit peptide is provided herein, e.g., a DNA comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16, which is designed for optimal expression in plants, particularly vegetables such as Brassicaceae plants, especially cabbage and cauliflower.

In accordance with this invention "CrylC protein" refers to any insecticidal protein comprising the smallest fragment of the amino acid sequence of SEQ ID No. 2 which retains insecticidal activity (hereinafter referred to as "smallest toxic fragment"), particularly any protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2, preferably any insecticidal 7 protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 3 to amino acid position 627. Also included herein is an insecticidal protein comprising the amino acid sequence of SEQ ID No. 2 (also named Cry1C1 protein herein), SEQ ID No. 5 (also named Cry1C3 protein herein) or SEQ ID No. 7 (also named Cry1C4 protein herein).

A CrylC protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2 retains all or most of the insecticidal activity of the entire protein as produced in nature, and addition of protein sequences at the N- or C-terminal part thereof do not disrupt this activity. Hence, any protein characterized by an amino acid sequence containing or including this region is useful and forms part of this invention. This includes insecticidal hybrid or chimeric proteins comprising the smallest toxic protein fragment of the protein of SEQ ID No. 2. Also included in this definition are variants of proteins comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2, such as insecticidal proteins comprising a sequence having a sequence identity of at least 95 %, particularly at least 96 %, 97 %, 98 % or 99 % at the amino acid sequence level with this region of SEQ ID No.2, as determined using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5; for amino acid sequence comparisons, the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted without significantly changing, preferably without changing, the insecticidal activity of the protein. Preferred variants of the CrylC protein of the invention include a protein comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine. Also included herein are any CrylC-based protein variants, hybrids or mutants retaining substantially the same insecticidal activity as that of the CrylC protein of the invention defined above. 8 The terminology DNA or protein "comprising" a certain sequence X, as used herein, refers to a DNA or protein including or containing at least the sequence X, so that other nucleotide or amino acid sequences can be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end, e.g. (the nucleotide sequence of) a selectable marker protein as disclosed in EP 0 193 259, (the nucleotide sequence of) a transit peptide, and/or a 5" or 3' leader sequence.

For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (x100) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970) in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5).

The "smallest toxic fragment" of a Cry protein of the invention, as used herein, is that smallest fragment or portion of a Cry protein retaining insecticidal activity that can be obtained by enzymatic, such as trypsin or chymotrypsin, digestion of the full length Cry protein, or that smallest fragment or portion of a Cry protein retaining insecticidal activity that can be obtained by making nucleotide deletions in the DNA encoding a Cry protein. Such smallest toxic fragment can also be obtained by treatment of a Cry protein with insect gut juice, preferably midgut juice, from an insect species susceptible to (i.e., killed or otherwise negative affected in its growth or feeding by) such Cry protein.

In accordance with this invention, "CrylD protein" refers to any insecticidal protein comprising the smallest toxic fragment of the amino acid sequence of SEQ ID No. 15, particularly any insecticidal protein comprising the amino acid sequence from the amino acid at position 21 or 29 to the amino acid at position 604 in SEQ ID No. 15, preferably any insecticidal protein comprising the amino acid sequence of SEQ ID No. 15 from amino acid position 3 to amino acid position 604. Also included herein is an insecticidal protein comprising the amino acid sequence of SEQ ID No. 13 (also named Cry1D1 protein herein) or SEQ ID No. 15 (also named Cry1D2 protein herein). A CrylD protein 9 comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15 retains all or most of the insecticidal activity of the entire protein as produced in nature, and addition of protein sequences at the N- or C-terminal part thereof do not disrupt this activity. Hence, any protein characterized by an amino acid sequence containing or including this region is useful and forms part of this invention. This includes insecticidal hybrid or chimeric proteins comprising the smallest toxic protein fragment of the protein of SEQ ID No. 15. Also included in this definition are protein variants differing in the amino acid sequence from the amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15, such as proteins with a sequence identity of at least 95 %, particularly at least 97 %, at least 98 % or at least 99 % at the amino acid sequence level in this region of SEQ ID No. 15, as determined using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5, for amino acid sequence comparisons, the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted in the region from the amino acid at position 29 to the amino acid at position 604 in SEQ ID No. 15 without significantly changing, preferably without changing, the insecticidal activity of the protein.

In accordance with this invention, "CrylB protein" refers to any insecticidal protein comprising the smallest toxic fragment of the amino acid sequence of SEQ ID No. 11, particularly any insecticidal protein comprising the amino acid sequence from the amino acid at position 31 to the amino acid at position 648, in SEQ ID No. 11, preferably any insecticidal protein comprising the amino acid sequence of SEQ ID No. 11 from amino acid position 3 to amino acid position 648. Also included herein is any insecticidal protein comprising the amino acid sequence of SEQ ID No. 11 or SEQ ID No. 9. A CrylB protein comprising the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ ID No. 11 retains all or most of the insecticidal activity of the entire protein as produced in nature, and addition of protein sequences at the N- or C-terminal part thereof do not disrupt this activity. Hence, any protein characterized by an amino acid sequence containing or including this region is useful and forms part of this invention. This includes insecticidal hybrids or chimeric proteins comprising the smallest toxic protein fragment of SEQ ID No. 11. Also included in this definition are insecticidal proteins comprising variants of the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ ID No. 11, such as insecticidal proteins having a sequence identity of at least 80 %, particularly at least 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % at the amino acid sequence level in this region of SEQ ID No. 11, as determined using pairwise alignments using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5, for amino acid sequence comparisons, the EBLOSUM62 matrix is used), preferably proteins having some, preferably 5-10, particularly less than 5, amino acids added, replaced or deleted in the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ ID No. 11 without significantly changing, preferably without changing, the insecticidal activity of the protein. Preferred variants of the CrylB protein of the invention include an insecticidal protein comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine.

As used herein, the terms DNA or gene, as in "cry1C1 DNA", refers to any DNA sequence encoding the CrylC, CrylB or CrylD protein, respectively, as defined above. This includes naturally occurring, artificial or synthetic DNA sequences encoding the CrylC, CrylB or CrylD proteins defined above such as any one of SEQ ID Nos. 2, 5, 7, 9, 11, 13, 15. Also included herein are DNA sequences encoding insecticidal proteins which are similar enough to any one of the DNA sequences of SEQ ID No. 1,3,4, 6, 8, , 12, or 14 so that they can (i.e., have the ability to) hybridize to these DNA sequences under stringent hybridization conditions. Stringent hybridization conditions, as used herein, refers particularly to the following conditions: immobilizing the relevant DNA sequences on a filter, and prehybridizing the filters for either 1 to 2 hours in 50 % formamide, 5 % SSPE, 2x Denhardt's reagent and 0.1 % SDS at 42 ° C, or 1 to 2 hours in 6x SSC, 2xDenhardt's reagent and 0.1 % SDS at 68 °C. The denatured dig- or radiolabeled probe is then added directly to the prehybridization fluid and incubation is carried out for 16 to 24 hours at the appropriate temperature mentioned above. After incubation, the filters are then washed for 30 minutes at room temperature in 2x SSC, 0.1 % SDS, followed by 2 washes of 30 minutes each at 68 °C in 0.5 x SSC and 0.1 % 11 SDS. An autoradiograph is established by exposing the filters for 24 to 48 hours to X-ray film (Kodak XAR-2 or equivalent) at -70 °C with an intensifying screen. Of course, equivalent conditions and parameters can be used in this process while still retaining the desired stringent hybridization conditions. Preferred variants of the crylC, crylB or crylD DNA of this invention are a DNA encoding the insecticidal CrylC, CrylB or CrylD protein variants described above.

Also included herein as a CrylC DNA or gene as defined herein are: a) a DNA comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide position 85 to nucleotide position 2073, b) a DNA comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide position 85 to nucleotide position 2073, c) a DNA comprising the nucleotide sequence of SEQ ID No. 1 from nucleotide position 85 to nucleotide position 2073 fused to the DNA sequence of SEQ ID No. 16, d) a DNA comprising the nucleotide sequence of SEQ ID No. 4 from nucleotide position 7 to nucleotide position 2439, e) a DNA comprising the nucleotide sequence of SEQ ID No. 3 from nucleotide position 85 to nucleotide position 2073 fused to the DNA sequence of SEQ ID No. 16, orf) a DNA comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 7 to nucleotide position 2439.

Also included herein as a CrylD DNA or gene as defined herein are: a) a DNA comprising the nucleotide sequence of SEQ ID No. 14 from nucleotide position 85 to nucleotide position 1812, or b) a DNA comprising the nucleotide sequence of SEQ ID No. 12 from nucleotide position 7 to nucleotide position 2178.

Also included herein as a CrylB DNA or gene as defined herein are: a) a DNA comprising the nucleotide sequence of SEQ ID No. 8 from nucleotide position 7 to nucleotide position 2310, or b) a DNA comprising the nucleotide sequence of SEQ ID No. 10 from nucleotide position 91 to nucleotide position 1944.

The DNA sequences of the cry 1C, cryl B or cryl D genes of the invention (as shown in the sequence listing, without transit peptide sequence) show at most only 76.6 % sequence identity with the closest previously known DNA sequences available in databases. Available sequence databases were checked for the sequences with closest sequence identity using the well-known BLAST algorithm, and then the Needleman- 12 Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) was used to find the optimum alignment between the closest sequences and the sequences of the invention (considering their entire length, using default settings (gap opening penalty 10, gap extension penalty 0.5). For the Cryl D DNA, a fragment of the prior art sequence (of equal length) was selected to secure optimal alignment, but even then only 72.5 % sequence identity was the closest sequence identity with any known DNA sequence listed in the available databases.

Hence, also included herein as crylC, crylB orcrylD genes are DNA sequences encoding an insecticidal protein with at least 80 %, 90 %, preferably at least 93 to 97 %, particularly at least 98 % or at least 99 %, sequence identity to any one of the coding sequences of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 or DNA sequences encoding an insecticidal protein hybridizing to any one of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 under stringent hybridization conditions, preferably hybridizing stringently to that part of the DNA sequence of any one of SEQ ID No. 1, 3, 4, 6, 8, 10, 12, or 14 which is required to encode the smallest toxic protein fragment of the proteins of this invention. The DNA sequence identities referred to herein are calculated using the Needleman-Wunsch global alignment algorithm in EMBOSS (Rice et al., 2000) to find optimum alignment over the entire length of the sequences, using default settings (gap opening penalty 10, gap extension penalty 0.5; for DNA sequence comparisons, the EDNAFULL matrix is used), the stringent hybridization conditions are as defined above.

"Insecticidal activity" of a protein, as used herein, means the capacity of a protein to kill insects, inhibit their growth or cause a reduction in insect feeding when such protein is ingested by insects, preferably by expression in a recombinant host such as a plant cell. It is understood that activity to insects of one insect species, preferably the larvae thereof, is sufficient for a protein to have insecticidal activity as used herein, although often insects of different insect species are affected by the proteins of the invention. The recombinant hosts expressing at least one of the CrylC, CrylB or CrylD proteins of the invention are typically developed for or targeted to a specific major insect pest species for a certain crop or region where such insect species is a pest, e.g., the diamondback moth for Brassica plant species, but other insects will often also be controlled by the recombinant hosts of the invention, such as by the transgenic plant cells or plants, e.g., 13 the exemplified transgenic Brassica cauliflower or cabbage plant cells or plants of the invention comprising the crylC and/or crylB gene in accordance with the invention.

"(Insect-)controlling amounts" of or "control" by a protein, or a recombinant host expressing a protein of this invention, as used herein, refers to an amount of protein which is sufficient to limit damage to a plant by insects feeding on such plant, e.g. by killing the insects or by inhibiting the insect development, fertility or growth in such a manner that an insect species provides less damage to a plant. This does not mean that treatment of plants with chemical insecticides will no be longer necessary (e.g., to control insect species not affected by the proteins of the invention, such as (secondary) Coleopteran or Dipteran insect pests), but that treatment by chemical insecticides for the insects targeted by the proteins of the invention can be significantly reduced or avoided, while still obtaining acceptable plant performance in the field and acceptable yield.

In accordance with this invention, insects susceptible to the new Cry proteins of the invention are contacted with these proteins in insect-controlling amounts, preferably insect-killing amounts. In one embodiment of this invention, recombinant hosts of the invention, such as transgenic plant cells or plants of the invention, express a protein or a combination of proteins of the invention at high levels, such that a "high dose" level is obtained. A "high dose level", "high dose insect resistance" or "high dose" expression, as used herein when referring to a recombinant plant cell or plant, refers to a concentration of the insecticidal protein in a plant cell or plant (measured by ELISA as a percentage of the total soluble protein, which total soluble protein is measured after extraction of soluble proteins in an extraction buffer (e.g., the extraction buffer described in Jansens et al., 1997) using Bradford analysis (Bio-Rad, Richmond, CA; Bradford, 1976)) which kills a developmental stage of the target insect which is significantly less susceptible, preferably between 25 to 100 times less susceptible to the toxin than the first larval stage of the insect and can thus can be expected to ensure full control of the target insect. In one embodiment this refers to the obtaining of at least 97 percent, preferably at least 99 percent, most preferably 100 percent, mortality for the fourth larval instar (for insects having 5 larval instars) or the last larval instar (for insects having 4 or less larval instars) of a target insect, as measured 10 to 14 days after insect infestation of such plant cells or plant in routine insect bioassays, preferably whole plant bioassays, using suitable controls. The existence of one target insect species (i.e., an insect 14 species, preferably the larvae thereof, which can cause significant damage to a plant species or variety, and which is typically an insect for which a transgenic Bt plant is designed and developed) for which transformed plant cells or plants according to this invention provide a "high dose" level insect resistance is sufficient for a plant to be designated as giving "high dose" expression in accordance with this invention. Preferred target insects for the proteins of this invention are economically damaging insect pests of plants.

The terms "Cryl protein/DNA" or "Cry protein/DNA of this invention", as used herein, refer to any one of the Cry 1C, Cryl B, or Cryl D proteins or any one of the cryl C, cryl B or crylD DNA sequences as defined herein. A Cry or Cryl protein, as used herein, can be a protein in the full length size, also named a protoxin, or can be in a truncated form as long as the insecticidal activity is retained, or can be a combination of different proteins in a hybrid or fusion protein. A "protoxin" refers to the full length insecticidal crystal protein as it is encoded by the naturally-occurring Bt DNA sequence, a "toxin" refers to an insecticidal fragment thereof, particularly the smallest toxic fragment thereof, typically in the molecular weight range of about 50-65 kD, particularly about 60 kD, as determined by SDS-PAGE electrophoresis compared to routinely-used molecular weight standards.

A "chimeric gene", as used herein, is used to refer to a gene or DNA sequence comprising at least two different DNA fragments (such as a promoter, 5' untranslated leader, coding region, intron, 3' untranslated trailer, and a 3' end transcript formation and polyadenylation region) which are not naturally associated with each other or which originate from different sources. Typically, a plant-expressible chimeric gene, as used herein, is a gene comprising a promoter region operably-linked to a synthetic, man-made coding sequence such as any of the cryl C, cryl B or cryl D genes of the invention.

The DNA sequences encoding the Cryl proteins of the invention can be chemically synthesized using routine techniques, and can be inserted in expression vectors to produce high amounts of Cryl proteins. The Cryl proteins can be used to prepare specific monoclonal or polyclonal antibodies in a conventional manner (Hofte et al., 1988) to develop immuno-assays (e.g., ELISA, Western blotting, antibody-coated dip sticks) to detect the presence of absence of these proteins in any material, such as plant material.

The tools developed to identify transgenic plant cells, plants, or plant materials such as leaves or seeds comprising any one of the cryl genes of the invention integrated in their genome, or DNA-containing products which comprise or are derived from plant material comprising a cryl gene of the invention are based on the specific sequence characteristics of the novel genes of the invention, such as, a specific restriction map of the genomic region comprising the introduced (foreign) cryl gene, molecular markers or the sequence of the foreign DNA integrated in the plant's genome.

Once the sequence of a foreign DNA such as the cryl genes of the invention is known, primers and probes can be developed which specifically recognize these sequences in the nucleic acid (DNA or RNA) of a sample by way of a molecular biological technique. For instance a PCR method can be developed to identify the genes of the invention in biological samples (such as samples of plants, plant material or products comprising plant material). Such a PCR is based on at least two specific "primers", e.g., one recognizing a sequence within the cryl gene and the other recognizing a sequence within the associated transit peptide sequence or within the regulatory regions such as the promoter or 3' end of the chimeric gene comprising said cryl gene of the invention, or both recognizing specifically the cryl gene of the invention. The primers preferably have a sequence of between 15 and 35 nucleotides which under optimized PCR conditions "specifically recognize" a sequence within the cryl chimeric gene of the invention, so that a specific fragment ("integration fragment" or discriminating amplicon) is amplified from a nucleic acid sample comprising a cryl gene of the invention. This means that only the targeted integration fragment, and no other sequence in the plant genome or foreign DNA, is amplified under optimized PCR conditions.

PCR primers suitable for the invention are oligonucleotides ranging in length from 17 nucleotides to about 200 nucleotides, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides selected from the crylC, 16 crylB or crylD chimeric gene sequence as transferred to plant cells or plants of the invention.

The primers may of course be longer than the mentioned 17 consecutive nucleotides, and may, e.g., be 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even longer. The primers may entirely consist of nucleotide sequences selected from the cryl nucleotide sequences. However, the nucleotide sequence of the primers at their 5' end (i.e. outside of the 3'-located 17 consecutive nucleotides) is less critical. Thus, the 5' sequence of the primers may consist of a nucleotide sequence selected from the cryl chimeric gene sequence, as appropriate, but may contain several (e.g. 1, 2, 5, 10) mismatches. The 5' sequence of the primers may even entirely consist of a nucleotide sequence unrelated to the cryl genes of the invention, such as a nucleotide sequence representing one or more restriction enzyme recognition sites. Such unrelated sequences or flanking DNA sequences with mismatches should preferably be no longer than 100, more preferably no longer than 50 or no longer than 25 nucleotides.

Moreover, suitable primers may comprise or consist of a nucleotide sequence at their 3' end spanning the joining region between the cryl gene of the invention and the associated transit peptide sequence or the regulatory elements in the cryl chimeric gene integrated in the plant DNA, such as a promoter sequence, a leader sequence, a trailer sequence or a 3' transcript termination and polyadenylation sequence. It will also be immediately clear to the skilled artisan that properly selected PCR primer pairs should also not comprise sequences complementary to each other.

The term "primer" as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form, though the single-stranded form is preferred. Probes can be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process. 17 The term "recognizing" as used herein when referring to specific primers, refers to the fact that the specific primers specifically hybridize to a nucleic acid sequence in the cryl genes of the invention under a standard PCR identification protocol, whereby the specificity is determined by the presence of positive and negative controls as is well known in the art.

Also included herein is a kit to detect the cryl genes of the invention in biological material, as well as the use of such kit to screen biological material. A "kit" as used herein refers to a set of reagents for the purpose of performing the identification of the cryl genes of the invention in biological samples. More particularly, a preferred embodiment of the kit of the invention comprises at least one or two specific primers, as described above. Optionally, the kit can further comprise any other reagent described herein in the PCR identification protocol. Alternatively, according to another embodiment of this invention, the kit can comprise a specific probe, as described above, which specifically hybridizes with nucleic acid of biological samples to identify the presence of the cryl genes therein. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer, label) for identification of the cryl genes in biological samples, using the specific probe.

Standard PCR protocols are described in the art, such as in 'PCR Applications Manual" (Roche Molecular Biochemicals, 2nd Edition, 1999). The optimal conditions for the PCR, including the sequence of the specific primers, is specified in a PCR identification protocol for each cryl gene-containing plant species. It is however understood that a number of parameters in the PCR identification protocol may need to be adjusted to specific laboratory conditions, and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA may require adjustment of, for instance, the amount of primers, polymerase and annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR identification protocol. These adjustments will however be apparent to a person skilled in the art, and are furthermore detailed in current PCR application manuals such as the one cited above. 18 Examples of suitable primer combinations in accordance with the invention are (sequence 5' - 3') for the cryl B gene of the invention: P1B227 (TAC TTC GAA CAG AAA GAA CGA GAA CGA G, SEQ ID No. 20) and P1B228 (GTC CAG CGA AAG GAA CTC CAA GAA, SEQ ID No. 21), and for the crylC gene of the invention: P1C247 (AAC CTT GAG GGA CTT GGA AAC, SEQ ID No. 22) and P1C252 (AAG ATG AGG GTT TCT GAT AGC AG, SEQ ID No. 23). Hence, any gene encoding an insecticidal CrylB or CrylC protein and specifically recognized by these primers is included herein, as well as any method to detect such genes using such or other specific primers.

Also specific markers or labeled probes can be designed to detect the DNA sequences of this invention, and any use of specific markers or probes directed to any of the crylC, cryl B or cryl D genes of the invention is included herein. In one embodiment of this invention, the specific markers, primers or labeled probes do not detect or recognize any plant, preferably any plant of the same species as the test plant, not containing a cryl DNA sequence of the invention, particularly any such markers, primers or labeled probes do not detect or recognize any plant expressing a CrylC, CrylD or CrylB protein wherein such plant does not contain a DNA sequence of the invention (such as a crylC, crylD or crylB DNA as defined herein, e.g., a DNA comprising the nucleotide sequence of any one of SEQ ID No. 1,3,4, 6, 8, 10, 12, or 14).

The DNA sequences of this invention can be slightly modified to allow for more convenient restriction enzyme sites, or to make small changes without changing the efficacy and without significantly changing, preferably without changing, the protein they encode. Indeed, because of the degeneracy of the genetic code, it is well known that most amino acid codons can be replaced by others without changing the amino acid sequence of the protein. Furthermore, some amino acids can be substituted by other equivalent amino acids without significantly changing, preferably without changing, the insecticidal activity of the protein. Also, changes in amino acid sequence or composition in regions of the molecule, different from those responsible for binding or pore formation are less likely to cause a difference in insecticidal activity of the protein (e.g., the C-terminal part of the Cryl protoxin can be removed or be replaced by another amino acid sequence without affecting the insecticidal activity of the Cryl proteins of the invention). Equivalents of the DNA sequences of the invention include DNA sequences with less 19 than 20, preferably 5-10, nucleotide differences compared to the cryl genes of this invention as defined herein, but which encode an insecticidal Cryl protein of the invention, as defined herein.

Small modifications to a DNA sequence such as described above can be routinely made, i.e., by PCR-mediated mutagenesis (Ho et al., 1989, White et al., 1989). More profound modifications to a DNA sequence can be routinely done by de novo DNA synthesis of a desired coding region using available techniques.

The term "encoding", as used herein, when referring to a gene encoding a protein, refers to the capacity of such gene to produce a protein upon transcription and translation of the coding sequence contained in such gene in a target host cell. Hence, the cry1C1 chimeric gene of the invention encodes the Cry1C1 protein of the invention, even though this gene contains two coding sequences interrupted by a non-coding intron sequence.

With the term "substantially the same", when referring to the amino acid sequence of a Cryl protein of this invention, is meant to include an amino acid sequence that differs in no more than 5 %, preferably no more than 2 %, to the amino acid sequence of the protein compared to; and when referring to toxicity of Cry protein, is meant to include a protein whose LC50 value obtained under the same conditions of bio-assay (preferably in the same bio-assay using insects from the same population and suitable controls) differs no more then 2 times, preferably no more than 50 %, of the LC50 value obtained for the protein compared to.

"Microorganism", as used herein, refers to any living organism that can be observed only with the aid of a microscope, such as bacteria, yeast cells, plant cells, viruses, fungi.

This includes all generally unicellular organisms with dimensions beneath the limits of vision which can be propagated and manipulated in a laboratory, typically prokaryotic or unicellular eukaryotic life forms, including tissue cultures and plasmids.

The cryl DNA sequences of the invention, prepared from total DNA, can be ligated in suitable expression vectors and transformed in suitable host cells which can then be screened by conventional detection tools for presence and expression of the toxin.

A database search with the genes of this invention indicates that the DNA sequences of the invention are significantly different from any previously described genes or DNA sequences encoding toxins with activity against Lepidoptera (see, e.g., the January 26, 2006 version of DNA sequences described in patent applications (Geneseq release 200602), Hofte and Whiteley, 1989; Crickmore et al., 1998; and the August 2, 2005 update on the Bt nomenclature website corresponding to the Crickmore et al. (1998) publication, found at: http://www.lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/index.html).

The closest sequence identity at the DNA level (for the entire length of the sequences of the invention) in available DNA sequence databases (from patent or scientific literature) was 76.60 % for the crylC DNA of SEQ ID No. 1 or 3, 73 % for the cryl B DNA of SEQ ID No. 10, and 72.5 % for the crylD DNA of SEQ ID No. 14, using the above defined Needleman-Wunsch default settings in EMBOSS. Hence, assuming the available DNA sequence databases are representative of all known DNA sequences, the DNA sequences of this invention differ in at least 23 % of their nucleotides from any previously known DNA sequence. Assuming the closest sequences are contained in the available databases, this reflects a difference in about 485 nucleotides for the nucleotide sequence of SEQ ID No. 1 or 3, a difference in about 524 nucleotides for the nucleotide sequence of SEQ ID No. 10, and a difference in about 498 nucleotides for the nucleotide sequence of SEQ ID No. 14 with their respective closest published DNA sequence. This difference will be even more pronounced for the DNA sequences of SEQ ID No. 4, 6, 8, or 12, which encode a fusion protein with a transit peptide. Also the optimized chloroplast transit peptide DNA sequence of this invention (SEQ ID No. 16), which was adapted for expression in the target plants of the invention, was found to have about 76.1 % sequence identity (for that part of equal length to the SEQ ID No. 16 sequence) to the closest DNA sequence identified in available DNA sequence databases and hence is very different. 21 By an "insecticidally effective part (portion or fragment)" of DNA sequences encoding a Cryl protein, also referred to herein as "truncated gene" or "truncated DNA", is meant a DNA sequence encoding a polypeptide which has fewer amino acids than the Cryl protein protoxin form but which is still insecticidal.

In order to express all or an insecticidally effective part of the DNA sequence encoding a Cry protein of this invention in a recombinant host such as E. coli, in other Bt strains or in plants, suitable restriction sites can be introduced, flanking the DNA sequence. This can be done by site-directed mutagenesis, using well-known procedures (Stanssens et al., 1989; White et al., 1989). In order to obtain improved expression in plants, the cryl genes of the invention are artificial genes, wherein the sequence has been adapted for optimal expression by DNA synthesis. In such sequence, replacement of DNA sequences inhibiting optimal expression is achieved by designing DNA sequences comprising codons more preferred by plants, preferably the target plant genus or species.

For obtaining enhanced expression in plants or preventing expression of an insecticidal protein when not present in a plant host cell (such as in a bacterial host cell), in one embodiments of the invention a plant intron is inserted in the chimeric cryl genes of the invention, preferably in the coding sequence of at least one of the cryl genes of the invention. Any of the known plant introns (e.g., Brown, 1986, Brown and Simpson, 1998, Brown et al., 1996) can be used herein as long as it is operably-linked to the coding sequence fragments so as to assure proper splicing. Operable linkage of the intron and the resulting proper splicing is conveniently checked in the target host plant species or cells thereof by RT-PCR or Northern blot or by any other means available in the art. In one embodiment an intron of a dicot plant gene is used in genes to be expressed in dicot plant cells, and a monocot intron is used in genes to be expressed in monocot plants. In one embodiment, the intron of the invention is the second intron of the light-inducible tissue-specific ST-LS1 gene of Solarium tuberosum (potato) as described by Eckes et al. (1986), e.g., the nucleotide sequence of SEQ ID No. 1 between nucleotide position 672 and 862. In one embodiment of this invention a plant intron is introduced into any Bt insecticidal protein coding sequence, particularly the intron of SEQ ID No. 1 between nucleotide position 672 and 862, so that it is effectively spliced in plant cells. Effective 22 splicing in plants cells can be measured using routine techniques, such as RT-PCR, Northern blotting, or the detection of a functional protein produced in plant cells. Of course, for effective splicing the intron needs to be inserted in the correct position of the coding sequence so that functional 5' and 3' splice sites are obtained in the sequence. The two cry genes of the invention, illustrated in SEQ ID Nos. 1 and 3, each containing a plant intron at a different location, were found by RT-PCR analysis to both be effectively spliced in Brassica oleraceae plant cells, and to produce an mRNA encoding the expected Cry protein.

In accordance with one embodiment of this invention, the proteins are targeted to intracellular organelles such as plastids, preferably chloroplasts, mitochondria, or are secreted from the cell, potentially optimizing protein stability and/or expression. For this purpose, the chimeric genes of the invention comprise a coding region encoding a signal or target peptide, linked to the Cry protein coding region of the invention. Particularly preferred peptides to be included in the proteins of this invention are the transit peptides for chloroplast or other plastid targeting, especially duplicated transit peptide regions from plant genes whose gene product is targeted to the plastids, the optimized transit peptide described by Lebrun et al. (1996), or Capellades et al. (US Patent 5,635,618), the transit peptide of ferredoxin-NADP+oxidoreductase from spinach (Oelmuller et al., 1993), the transit peptide described in Wong et al. (1992) and the targeting peptides in published PCT patent application WO 00/26371. In one embodiment of the invention, the chloroplast transit peptide comprises the sequence of SEQ ID No. 17 from amino acid position 3 to amino acid position 124 or variant thereof, such as a chloroplast transit peptide comprising the sequence of SEQ ID No. 17 from amino acid position 3 to amino acid position 124, wherein the Cys amino acid at position 55 is replaced by Tyr in SEQ ID No. 17 and/or wherein a Gly amino acid is added after the Gly amino acid at position 51 in SEQ ID No. 17. Also preferred are peptides signalling secretion of a protein linked to such peptide outside the cell, such as the secretion signal of the potato proteinase inhibitor II (Keil et al., 1986), the secretion signal of the alpha-amylase 3 gene of rice (Sutliff et al., 1991) and the secretion signal of tobacco PR1 protein (Cornelissen et al., 1986). 23 Particularly useful signal peptides in accordance with the invention include the chloroplast transit peptide (e.g., Van Den Broeck et al. (1985), or the optimized chloroplast transit peptide of US patent 5, 510,471 and US patent 5,635,618 causing transport of the protein to the chloroplasts, a secretory signal peptide or a peptide targeting the protein to other plastids, mitochondria, the ER, or another organelle. Signal sequences for targeting to intracellular organelles or for secretion outside the plant cell or to the cell wall are found in naturally targeted or secreted proteins, preferably those described by Klosgen et al. (1989), Klosgen and Weil (1991), Neuhaus & Rogers (1998), Bih et al. (1999), Morris et al. (1999), Hesse et al. (1989), Tavladoraki et al. (1998), Terashima et al. (1999), Park et al. (1997), Shcherban et al. (1995), all of which are incorporated herein by reference, particularly the signal peptide sequences from targeted or secreted proteins of Brassica plant species, corn, cotton, or soybean. A preferred DNA sequence encoding a transit peptide of the invention is a DNA comprising the sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16.

Furthermore, for any target pest insect, the binding properties of the Cry proteins of the invention can be evaluated, using methods known in the art (e.g., Van Rie et al., 1990), to determine if the Cryl proteins of the invention bind to sites on a target insect midgut that are not recognized (or competed for) by other Cry or non-Cry proteins. Other Bt toxins binding to different binding sites in relevant susceptible insects, or other toxins derived from Bt strains or other sources (such as VIP toxins or insect (gut) proteinase inhibitors) with a different mode of action are very valuable to also express in a plant in addition to any one of the cryl genes herein, to prevent or delay the development of insect resistance to a plant expressing insecticidal toxins. Because of the characteristics of the new cryl genes, they are extremely useful for transforming plants, e.g. monocots such as corn or wheat and dicots such as cotton, soybean and Brassica species plants, to protect these plants from insect damage.

Especially for insect resistance management purposes for a specific insect pest, it is preferred to combine a crylC gene of this invention with another gene encoding a different insect control protein, particularly a Bt crystal protein, which does not recognize at least one binding site recognized by such CrylC protein in a target insect. Preferred 24 insect control proteins to combine with the CrylC proteins of this invention, particularly for simultaneous expression in plants, preferably Brassica species plants, particularly cabbage and cauliflower, include the CrylB protein of this invention or the CrylD protein of this invention, the VIP3Aa protein or a toxic fragment thereof as described in Estruch et al., 1996 and US Patent 6,291,156, or insecticidal proteins from Xhenorhabdus, Serratia or Photorhabdus species strains (e.g., Waterfield et al., 2001; ffrench-Constant and Bowen, 2000). In one embodiment, such co-expression is obtained by transforming a plant already expressing an insect control protein with a cryl gene of this invention, or by crossing plants transformed with the insect control protein and plants transformed with the cryl gene of this invention. For Brassica species plants, preferably the cryl gene is used as first gene and as second gene the Cryl B, Cryl D or VIP3Aa protein or variants or derivatives thereof are used. Methods for obtaining expression of different Bt (or similarly, for other insect control proteins) insecticidal proteins in the same plant in an effort to minimize or prevent resistance development to transgenic insect-resistant plants are described in EP patent 0 408 403. In one embodiment of the invention, the crylC gene of the invention is located in one and the same locus as a second insect control gene, such as a CrylB or CrylD gene, in the transgenic plant cells or plants of the invention, so that these genes do not segregate in the progeny of such plant cells or plants.

Preferably, for selection purposes but also for increasing the weed control options, the transgenic plants of the invention are also transformed with a DNA encoding a protein inactivating a broad-spectrum herbicide or encoding a protein which is a variant of the protein target for the herbicide but which protein variant is insensitive to such herbicide, e.g., herbicides based on glufosinate orglyphosate.

The insecticidally effective cryl gene, preferably the cryl chimeric gene, encoding an insecticidally effective portion of the Cry protoxin, can be stably inserted in a conventional manner into the nuclear genome of a plant cell, and the so-transformed plant cell can be used in a conventional manner to produce a transformed plant that is insect-resistant. In this regard, a disarmed Ti-plasmid, containing the insecticidally effective cryl gene part, in Agrobacterium, e.g., Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO 84/02913 and published European Patent application ("EP") 0 242 246 and in De Block et al. (1989). Preferred Ti-plasmid vectors each contain the insecticidally effective cry gene part between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 233 247), pollen mediated transformation (as described, for example in EP 0 270 356, PCT publication WO 85/01856, and US Patent 4,684,611), plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and US Patent 4,407,956), liposome-mediated transformation (as described, for example in US Patent 4,536,475), and other methods such as the methods for transforming certain lines of corn (e.g., US patent 6,140,553; Fromm et al., 1990; Gordon-Kamm et al., 1990) and the method for transforming monocots generally (PCT publication WO 92/09696). For cotton transformation, especially preferred is the method described in PCT patent publication WO 00/71733. For soybean transformation, reference is made to methods known in the art, e.g., Hinchee et al. (1988) and Christou et al. (1990) or the method of WO 00/42207.

Also, besides transformation of the nuclear genome, also transformation of the plastid genome, preferably chloroplast genome, is included in the invention. Kota et al. (1999) have described a method to express a Cry2A protein in tobacco chloroplasts, and Lin et al. (2003) described expression of a crylC gene in transplastomic tobacco plants.

The resulting transformed plant can be used in a conventional plant breeding scheme to produce more transformed plants with the same characteristics or to introduce the insecticidally effective cry gene part in other varieties of the same or related plant species. Seeds, which are obtained from the transformed plants, contain the insecticidally effective cry gene part as a stable genomic insert.

The insecticidally effective cryl gene, preferably the sequence of SEQ ID No. 1, 3, 4 or 6, is inserted in a plant cell genome so that the inserted gene is downstream (i.e., 3') of, and under the control of, a promoter which can direct expression of the gene in a plant 26 cell (herein named a "plant-expressible promoter"). This is preferably accomplished by inserting the cryl chimeric gene comprising a plant-expressible promoter in the plant cell genome, particularly in the nuclear or plastid (e.g., chloroplast) genome. Preferred plant-expressible promoters include: the strong constitutive 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al., 1981), CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al. (1985), promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., 1992, see also Cornejo et al., 1993), the gos2 promoter (de Pater et al., 1992), the emu promoter (Last et al., 1990), Arabidopsis actin promoters such as the promoter described by An et al. (1996), rice actin promoters such as the promoter described by Zhang et al. (1991); promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al. (1998)), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932), particularly the duplicated promoter region derived from the subterranean clover stunt virus genome segment 4 or 7 (referred to as the "S7S7" or "S4S4" promoters herein) described by Boevink et al. (1995) or Schunmann et al. (2003), an alcohol dehydrogenase promoter, e.g., pAdhlS (GenBank accession numbers X04049, X00581), and the TR1' promoter and the TR2' promoter (the "TR11 promoter" and "TR2' promoter", respectively) which drive the expression of the 1' and 2' genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (e.g., leaves and/or roots) whereby the inserted cry gene part is expressed only in cells of the specific tissue(s) or organ(s). For example, the insecticidally effective cry gene part could be selectively expressed in the leaves of a plant (e.g., corn, cotton) by placing the insecticidally effective gene part under the control of a light-inducible promoter such as the promoter of the ribulose-1,5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant such as pea as disclosed in US Patent 5,254,799. Another alternative is to use a promoter whose expression is inducible, preferably by wounding such as insect feeding, e.g., the MPI promoter described by Cordera et al. (1994), or the Agrobacterium TR2' or mannopine synthase promoter (Velten et al., 1984) or a promoter inducible by chemical factors. 27 The insecticidally effective cry gene part is preferably inserted in the plant genome so that the inserted gene part is upstream (i.e., 5') of suitable 3' end transcription regulation signals (i.e., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the cryl chimeric gene in the plant cell genome. Preferred polyadenylation and transcript formation signals include those of the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (Marshall et al., 1996), nopaline synthase gene (Depicker et al., 1982), the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7 (Velten and Schell, 1985), which act as 3-untranslated DNA sequences in transformed plant cells.

In one embodiment of this invention, at least one of the genes of the invention, preferably at least 2, are transformed into plants selected from the group consisting of: corn, cotton, watercress, horseradish, wasabi, arugula, cress, radish, canola, soybean, vegetable plants, Cruciferae plant species, Brassicaceae plant species such as cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale, broccoli, Brussels sprouts, mustard spinach, and the like. Particularly, in one embodiment of this invention the following Brassica species plants are protected from insects by the genes of this invention: B. carinata, B. elongata, B. fruticulosa, B. juncea, S. napus, B. narinosa, B. hirta, B. rosularis, B. nigra, B. oleracea, B. perviridis, B. rapa, B. rupestris, B. septiceps, B. tournefortii, and the like, particularly plants of the species Brassica oleraceae (such as the subspecies botrytis and capitata) or Brassica napus, as well as plants of the following genus: Raphanus (such as R. sativus), Armoracia (such as A. rusticana), Wasabia (such as W. japonica), Eruca (such as E. vesicaria), Nastrurtium (such as N. officinale), and Lepidium (such as L. sativum).

The invention includes the above listed Brassica species plants transformed with at least one or two genes of the invention, such as the crylB and crylC genes of the invention, as well as plants obtained after crossing or breeding with related plants (including plants of a related plant species) that contain the genes of the invention. Such crossing or breeding can be done using traditional breeding techniques known in the art, but may also include known in vitro work such as embryo rescue, protoplast fusion, and the like. The invention hence also relates to Brassicaceae plants such as B. napus, B. rapa, B. juncea or B. carinata, that contain the gene or genes of the invention, such as the crylB and crylC genes of the invention, from crossings with a transformed B. oleracea plant or 28 the progeny thereof, or to B. oleracea plants that contain the gene or genes of the invention, such as the crylB and crylC genes of the invention, from crossings with a transformed B. napus plant, and to uses of such plants.

Transformation of plant cells can also be used to produce the proteins of the invention in large amounts in plant cell cultures, e.g., to produce a Cryl protein that can then be applied onto crops after proper formulation. When reference to a transgenic plant cell is made herein, this refers to a plant cell (or also a plant protoplast) as such in isolation or in tissue culture, or to a plant cell (or protoplast) contained in a plant or in a differentiated organ or tissue, and both possibilities are specifically included herein. Hence, a reference to a plant cell in the description or claims is not meant to refer only to isolated cells in culture, but refers to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present.

All or part of the cryl genes of the invention, encoding an anti-lepidopteran protein, can also be used to transform bacteria, such as a B. thuringiensis which has insecticidal activity against Lepidoptera or Coleoptera. Thereby, a transformed Bt strain can be produced which is useful for combatting a wide spectrum of lepidopteran and coleopteran insect pests or for combatting additional lepidopteran insect pests. Transformation of bacteria, such as bacteria of the genus Pseudomonas, Agrobacterium, Bacillus or Escherichia, with the cryl genes of this invention, incorporated in a suitable cloning vehicle, can be carried out in a conventional manner, preferably using conventional electroporation techniques as described in Mahillon et al. (1989) and in PCT Patent publication WO 90/06999.

Transformed Bacillus species strains containing the cry gene of this invention can be fermented by conventional methods (Dulmage, 1981; Bernhard and Utz, 1993) to provide high yields of cells. Under appropriate conditions which are well understood (Dulmage, 1981), these strains each sporulate to produce crystal proteins containing the Cry protoxin in high yields.

An insecticidal, particularly anti-lepidopteran, composition of this invention can be formulated in a conventional manner using the microorganisms transformed with the cry gene, or preferably their respective Cry proteins or the Cry protoxin, toxin or insecticidally effective protoxin portion as an active ingredient, together with suitable 29 carriers, diluents, emulsifiers and/or dispersants (e.g., as described by Bernhard and Utz, 1993). This insecticide composition can be formulated as a wettable powder, pellets, granules or dust or as a liquid formulation with aqueous or non-aqueous solvents as a foam, gel, suspension, concentrate, etc.

A method for controlling insects, particularly Lepidoptera, in accordance with this invention can comprise applying (e.g., spraying), to a locus (area) to be protected, an insecticidal amount of the Cry proteins or host cells transformed with the cry gene of this invention. The locus to be protected can include, for example, the habitat of the insect pests or growing vegetation or an area where vegetation is to be grown.

In one embodiment of this invention, insects against which the cryl genes or Cryl proteins of the invention can be used include insects selected from the group consisting of: Plutella xylostella, Spodoptera exigua, Spodoptera littoralis, Spodoptera frugiperda, Trichoplusia ni, Heliothis virescens, Mamestra brassicae, Pieris brassicae, Manduca sexta, Choristoneura fumiferana, Choristoneura occidentalis, Choristoneura rosaceana, Pandemis pyrusana, Platynota stultana, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, Lambina fiscellaria, Chilo suppressalis, Chilo partellus, Scirpophaga incertulas, Argyrotaenia citrana, Artogeia rapa, Chrysomela scripta, Ostrinia nubilalis, Pseudoplusia includens, and Thaumetopoea pityocampa. In one embodiment, Plutella xylostella (diamondback moth) is a preferred target insect pest. This is a cosmopolitan species that causes major losses in several Cruciferous plants, particularly Brassicacaea plants. The CrylC, CrylB and CrylD proteins encoded by the genes of this invention are particularly useful to control this insect, e.g., by expression of the genes of the invention in cells of a plant.

Such insects can be controlled by planting or growing plants comprising any one of the crylC genes of the invention in a field, or by securing the presence of a CrylC protein as defined herein in or on plants infested by such insects (e.g., by sowing or planting a Brassica species plant such as a cabbage or cauliflower plant transformed with the cry1C1 or cry1C2 gene of this invention, or spraying a composition containing a CrylC protein of this invention). The invention also relates to the use of the cryl genes of this invention, at least the cry1C1 or cry1C2 genes, in plants to protect them against Lepidopteran insect pests, preferably in combination with a crylB or crylD gene of this invention.

In the current invention, also a modified coding sequence encoding a chloroplast transit peptide is provided. Such coding sequence has a codon usage adapted for high expression in plants, particularly Brassicaceae plants such as Brassica oleracea or Brassica napus, especially cabbage, cauliflower or oilseed rape (canola). In one embodiment of the invention, the modified transit peptide comprises the nucleotide sequence of SEQ ID No. 16 from nucleotide position 7 to nucleotide position 371, particularly the sequence of SEQ ID No. 16. Also plant cells, plants or seeds comprising the modified transit peptide coding sequence of the invention, as well as the use of this transit peptide coding sequence for targeting any protein to the chloroplast, particularly to the chloroplast of vegetable plants, particularly Brassica species plants, are included in this invention.

These and/or other embodiments of this invention are reflected in the wordings of the claims, that form part of the description of the invention.

The following Examples illustrate the invention, and are not provided to limit the invention or the protection sought. Unless otherwise stated, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

The enclosed sequence listing referred to in the Examples, the Claims and the Description is as follows: Sequence Listing: SEQ ID No.1: optimized cry1C1 coding sequence comprising an intron at position 672 SEQ ID No.2: amino acid sequence of the Cry1C1 protein encoded by SEQ ID No. 1 31 SEQ ID No.3: optimized cry1C2 coding sequence, comprising an intron at position 489 SEQ ID No.4: optimized cry1C3 coding sequence, comprising the sequences of SEQ ID No. 1 and SEQ ID No. 16, encoding a fusion protein with a transit peptide SEQ ID No.5: Cry1C3 protein encoded by SEQ ID No. 4 SEQ ID No.6: optimized cry1C4 coding sequence, comprising the sequences of SEQ ID No. 3 and SEQ ID No. 16, encoding a fusion protein with a transit peptide SEQ ID No.7: Cry1C4 protein encoded by SEQ ID No. 6 SEQ ID No.8: optimized cry1B1 coding sequence, including a transit peptide coding sequence SEQ ID No.9: Cry1B1 protein encoded by the sequence of SEQ ID No. 8 SEQ ID No. 10: optimized cry1B2 coding sequence SEQ ID No.11: Cry1B2 protein encoded by the sequence of SEQ ID No. 10 SEQ ID No. 12: optimized cry1D1 coding sequence, including a transit peptide coding sequence SEQ ID No. 13: Cry1D1 protein encoded by the sequence of SEQ ID No. 12 SEQ ID No.14: optimized cry1D2 coding sequence SEQ ID No. 15: Cry1D2 protein encoded by the sequence of SEQ ID No. 14 SEQ ID No. 16: coding sequence encoding an optimized chloroplast transit peptide SEQ ID No. 17: chloroplast transit peptide encoded by the sequence of SEQ ID No. 16 SEQ ID No. 18: duplicated S7 subterranean clover stunt virus promoter sequence (S7S7) SEQ ID No. 19: duplicated S4 subterranean clover stunt virus promoter sequence (S4S4) SEQ ID No. 20: cryl B gene primer P1B227 SEQ ID No. 21: crylB gene primer P1B228 SEQ ID No. 22: crylC gene primer P1C247 SEQ ID No. 23: crylC gene primer P1C252 EXAMPLES 1.Construction of chimeric genes and transformation vectors.

Several cryl genes were designed and assembled using a combination of technologies to achieve genes with optimal performance in plant cells. 32 The cry1C1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 1. This DNA encodes the insecticidal Cry1C1 protein of the invention (SEQ ID No. 2). For transformation of plants, a first chimeric gene (the cry1C1 chimeric gene) is constructed comprising the following operably-linked elements (5' to 3'): a promoter comprising the duplicated promoter region derived from the subterranean clover stunt virus genome segment 7 (S7S7 promoter, Boevink et al., 1995, SEQ ID No. 18), the leader sequence of the tapetum-specific E1 gene (GE1) of Oryza sativa (Michiels et al., 1992), the cry1C1 DNA comprising the second intron of the light-inducible tissue-specific ST-LS1 gene of Solanum tuberosum (Eckes et al., 1986) at position 672 (SEQ ID No. 1), and the sequence including the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3' Me1, Marshall et al., 1996).

A similar crylC chimeric gene was made, wherein the ST-LS1 intron 2 is at position 489 of the crylC DNA (i.e., the cry1C2 DNA), this is the cry1C2 chimeric gene, otherwise constructed exactly like the cry1C1 chimeric gene.

To secure targeting of the CrylC protein to the plant cell chloroplast, variants of the cry1C1 and cry1C2 chimeric genes are constructed which comprise a modified sequence encoding an optimized transit peptide (SEQ ID No.16) as described by Lebrun et al. (1996) operably-linked to the crylC coding region so that a transit peptide fusion protein is expressed in plant cells. These are the cry1C3 and cry1C4 chimeric genes, comprising the cry1C3 and cry1C4 coding sequences, respectively, which each contain the sequence of the modified chloroplast transit peptide of SEQ ID No. 16. The cry1C3 DNA sequence is shown in SEQ ID No. 4, it is a fusion of the cry1C1 sequence of SEQ ID No. 1 with the transit peptide coding sequence of SEQ ID No. 16. The cry1C4 DNA sequence is shown in SEQ ID No. 6, it is a fusion of the cry1C2 sequence of SEQ ID No. 3 with the transit peptide coding sequence of SEQ ID No. 16.

The cry1B1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 8. This DNA encodes the insecticidal Cry1B1 protein of the invention (SEQ ID No. 9). For transformation of plants, a chimeric gene (the cry1B1 chimeric gene) is constructed comprising the following operably-linked elements (5' to 3'): a promoter comprising the duplicated promoter region derived from the subterranean clover stunt virus genome segment 4 (S4S4 promoter, Boevink et al., 1995, SEQ ID No. 33 19), the leader sequence of the E1 gene (GE1) of Oryza sativa (Michiels et al., 1992), the cry1B1 DNA comprising the sequence of the modified chloroplast transit peptide of SEQ ID No. 16, and the sequence including the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3' Me1, Marshall et al., 1996).

A second form of the crylB chimeric gene was also made, using the cry1B2 DNA (SEQ ID No. 10), wherein no sequence encoding an optimized transit peptide is contained, so that cytoplasmic accumulation of the CrylB protein occurs in plant cells. This is the Cryl B2 chimeric gene.

The cry1D1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 12. This DNA encodes the insecticidal Cry1D1 protein of the invention (SEQ ID No. 13). For transformation of plants, a chimeric gene (the cry1D1 chimeric gene) is constructed comprising the following operably-linked elements (5' to 3'): an S4S4 promoter (SEQ ID No. 19), the leader sequence of the E1 gene (GE1) of Oryza sativa (Michiels et al., 1992), the cry1D1 DNA comprising the sequence of the modified chloroplast transit peptide of SEQ ID No. 16, and the sequence including the 3' untranslated region of the NADP-malic enzyme gene from Flaveria bidentis (3' Me1, Marshall et al., 1996).

A second form of the crylD chimeric gene was also made, using the cry1D2 DNA, wherein no sequence encoding an optimized transit peptide is contained, so that cytoplasmic accumulation of the CrylD protein occurs in plant cells. This is the Cry1D2 chimeric gene.

A DNA transformation vector (pT1C4B1) is made comprising between the T-DNA borders the cry1C4 chimeric gene and the cry1B1 chimeric gene in a head-to-tail orientation (3'Me1-cry1C4-GE1 leader-S7S7 -S4S4-GE1 leader-cry1B1-3'Me1), as well as a transfer vector (pT1C2B2) comprising between the T-DNA borders the cry1C2 chimeric gene and the cry1B2 chimeric gene in a head-to-tail orientation (3'Me1-cry1C2-GE1 leader-S7S7 - S4S4-GE1 leader-cry1B2-3'Me1). In such manner, with both T-DNA vectors, the crylC and crylB genes of the invention will be co-transferred to the plant cell and will be located at one locus after successful transformation. 34 Similar T-DNA vectors are constructed which contain the above crylC chimeric genes but which contain as second chimeric gene the cry1D1 or cry1D2 chimeric genes instead of the above cryl B chimeric genes. Also a triple cry gene transformation vector is constructed, comprising both the crylC, crylD and crylB genes (all either with or without modified transit peptide).

The transformation vectors containing the genes of the invention were derived from pGSC1700 (Cornelissen and Vandewiele, 1989). The vector backbone contains the following genetic elements: a) the plasmid core comprising the origin of replication from the plasmid pBR322 (Bolivar et al., 1977) for replication in Escherichia coli and a restriction fragment comprising the origin of replication from the Pseudomonas plasmid pVS1 (Itoh et al., 1984) for replication in Agrobacterium tumefaciens. b) a selectable marker gene conferring resistance to streptomycin and spectinomycin (aadA) for propagation and selection of the plasmid in Escherichia coli and Agrobacterium tumefaciens. c) a DNA region consisting of a fragment of the neomycin phosphotransferase coding sequence of the nptl gene from transposon Tn903 (Oka et al., 1981).

The T-DNA region of each transformation vector also contains a chimeric bar gene that serves as selectable marker gene. Expression of the bar gene enables the production of an enzyme, phosphinothricin-acetyl transferase, that metabolizes the herbicide glufosinate-ammonium, thus rendering it non-herbicidal in the plant. The chimeric bar gene comprises the 35S3 promoter region from the Cauliflower Mosaic Virus 35S transcript (Odell et al.,1985), the bar coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces hygroscopicus as described by Thompson et al. (1987), and a 3' transcript termination and polyadenylation sequence from the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al., 1982).

Similar transformation vectors as those described are also constructed, wherein the cry1C1 or cry1C3 chimeric genes are used (similar as the above crylC genes but having the ST-LS1 intron at a position 489). Also these vectors contain the cry1B1 or cry1B2 chimeric genes, or the cryl D1 or cryl D2 chimeric genes described above.

All constructed plasmids are confirmed to be accurate by restriction enzyme digest analysis and by DNA sequencing, before they are used for plant transformation. 2.Plant transformation and regeneration.

The above transformation vectors pT1C4B1 and pT1C2B2 containing the crylC and cryl B genes of the invention are transferred into Agrobacterium tumefaciens strains for transformation in plants using routine methods.

Cauliflower and cabbage plants are transformed using Agrobacterium transformation. Seeds of Brassica oleracea var. capitata (cabbage) or Brassica oleracea var. botrytis (cauliflower) are sterilized by dipping in 70% ethanol followed by submersion in 6% bleach. The seeds are then rinsed with sterile water and transferred to small Petri-plates containing MS based medium. The Petri-plates are placed in glass containers and incubated for 5-8 days at 24°C. Hypocotyl explants of 0.5-0.7 cm are cut and placed in liquid medium with appropriate hormones. Agrobacterium tumefaciens carrying the genes of interest are added to the medium to make a final concentration of 1x107 bacteria/ml. After the co-cultivation period, the explants are washed in liquid medium with appropriate antibiotics and hormones and blotted dry on filter paper.

The explants are cultured for one week on callus induction medium with 5 mg/l silver nitrate and 250 mg/l of both Triacillin and Carbenicillin and 10 mg/l phosphinothricin for selection of transformation events.

Every two weeks explants are transferred to fresh medium. Every week explants are checked for callus formation. Calli are excised from the explants and transferred to shoot induction medium. Shoots are transferred to plastic containers with rooting medium. Shoots are kept on this medium until they are normalized or rooted. If they are 3-10 cm in size and have a nicely developed root system, they are transferred to the greenhouse.

Oilseed rape plants are also transformed with the crylC and crylB genes using Agrobacterium tumefaciens. Hypocotyl explants of Brassica napus are used in routine transformation and regeneration methods, e.g., the method described by De Block et al. (1989). 36 3. Analysis of transformants.

Once the transformed plants are regenerated, PCR and Southern analysis are used to confirm integration of the transgenes. Immunological analyses such as CrylC- and CrylB-specific ELISA assays or Western blots are used to select those transformed plants showing optimal expression levels of the CrylC and CrylB proteins.

RT-PCR experiments on RNA collected from cauliflower plants shown to be transformed with the crylC genes of SEQ ID No. 1 or 3, comprising a plant intron at a different position, confirmed that splicing occurs correctly and that a functional CrylC protein is produced in these plants. This is also confirmed by Northern blot analysis of these plants.

Also, insect assays using Plutella xylostella larvae under standard insect bio-assay conditions using proper controls with selected transformed cabbage, cauliflower and oilseed rape plants containing the CrylC and CrylB genes confirm the high insecticidal activity and the high dose of these protein expressed, in those transformed plants selected for optimal expression. Also, Plutella xylostella insects that have been selected for resistance to the CrylC or CrylB protein, are still effectively killed by the plants of the invention.

Progeny plants and seeds are also obtained from the transformed, selected plants of the invention, and the genes of the invention are shown to segregate in such progeny in the expected Mendelian fashion. Selection of the transgenic plants in the greenhouse and in the field at multiple locations will result in the identification of plant lines which have optimal stability and expression of the cryl chimeric genes combined with optimal agronomical performance. Crossing of the selected best performing transgenic plants with several different commercial lines, and repeated backcrossing therewith, result in the presence of the (linked) crylB and crylC genes of the invention in different cabbage, cauliflower or oilseed rape genetic backgrounds, optimally adapted to different areas or climatic conditions. 37 Cited references: An et al. (1996) Plant J. 10, 107 Bernhard and Utz (1993) "Production of Bacillus thuringiensis insecticides for experimental and commercial uses", In Bacillus thuringiensis, An Environmental Biopesticide: Theory and Practice, pp.255-267, eds. Entwistle, P.F., Cory, J.S., Bailey, M.J. and Higgs, S., John Wiley and Sons, New York (1993).

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Claims

WO 2007/107302 39 PCT/EP2007/002342 Tavladoraki et al. (1998), FEBS Lett. 426, 62-66. Terashima et al. (1999), Appl. Microbiol. Biotechnol. 52, 516-523. Thompson et al. (1987) The EMBO Journal, 6: 2519-2523. Van Den Broeck et al., 1985, Nature 313, 358. Van Rie et al. (1990) Science 247, 72. Velten et al. (1984) J., EMBO J 3, 2723-2730. Velten and Schell (1985) Nucleic Acids Research 13, 6981-6998. Verdaguer et al. (1998) Plant Mol. Biol. 37, 1055-1067. Waterfield et al. (2001) Trends Microbiol 9, 185-91. White et al. (1989) Trends in Genet. 5, 185-189. Wong et al. (1992) Plant Molec. Biol.20, 81-93. Zambryski (1988) Annual Review of Genetics, 22: 1-30. Zhang et al. (1991) The Plant Cell 3, 1155-1165. Zhao et al. (2003) Nature Biotechnology, 21: 1493 - 1497. All cited references are hereby incorporated by reference into the description. The citation of any of these references is not to be construed as an acknowledgement of the accuracy of every statement contained in such reference, nor as an acknowledgement that such reference is relevant prior art or part of the common general knowledge in any territory. Received at IPONZ on 26 May 2011 40 CLAIMS

1. A chimeric gene, comprising the following operably-linked sequences: a) a coding region encoding a CrylC protein, comprising a DNA sequence with at least 98 % sequence identity to the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6, or comprising a DNA sequence hybridizing under stringent hybridization conditions to the DNA of any one of SEQ ID Nos. 1, 3, 4 or 6, wherein said coding region encodes a protein comprising the amino acid sequence from the amino acid at position 29 to the amino acid at position 627 in SEQ ID No. 2, and b) a promoter region capable of directing expression in plant cells.

2. The chimeric gene of claim 1, wherein said promoter comprises the sequence of SEQ ID No. 18 or 19.

3. The chimeric gene of claim 1 or 2, which also comprises a 3' polyadenylation and transcript termination region.

4. The chimeric gene of claim 3, wherein said 3' polyadenylation and transcript termination region is of the NADP-malic enzyme gene from Flaveria bidentis.

5. The chimeric gene of any one of claims 1 to 4, which further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

6. A DNA comprising the chimeric gene of any one of claims 1 to 5, further comprising a second chimeric gene, said second chimeric gene comprising the following operably-linked sequences: a) a second coding region encoding a CrylB protein comprising a DNA sequence with at least 98 % sequence identity to the DNA of SEQ ID No. 8 or 10, or comprising a DNA sequence hybridizing under stringent hybridization conditions to the DNA of SEQ ID No. 8 or 10, wherein said coding region 3661986-1 Received at IPONZ on 26 May 2011 41 encodes a protein comprising the amino acid sequence from the amino acid at position 31 to the amino acid at position 648 in SEQ ID No. 11, and b) a second promoter region capable of directing expression in plant cells.

7. A DNA comprising the chimeric gene of any one of claims 1 to 5, further comprising a second chimeric gene, said second chimeric gene comprising the following operably-linked sequences: a) a coding region encoding a CrylD protein comprising a DNA sequence with at least 98 % sequence identity to the DNA of SEQ ID No. 12 or 14, or comprising a DNA sequence hybridizing under stringent hybridization conditions to the DNA of SEQ ID No. 12 or 14, wherein said coding region encodes a protein comprising the amino acid sequence from the amino acid at position 21 to the amino acid at position 604 in SEQ ID No. 15, and b) a promoter region capable of directing expression in plant cells.

8. The DNA of claim 6 or 7, wherein said second promoter region comprises the sequence of SEQ ID No. 18 or 19 and is different from said first promoter region.

9. The DNA of claim 6 or 7, wherein said second chimeric gene further comprises a 3' polyadenylation and transcript termination region.

10. The DNA of claim 9, wherein said 3' polyadenylation and transcript termination region is of the NADP-malic enzyme gene from Flaveria bidentis.

11. The DNA of any one of claims 6 to 10, wherein said second chimeric gene further comprises the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding region.

12. The DNA of claim 6, further comprising a third chimeric gene, said third chimeric gene comprising the following operably-linked sequences: 3661986-1 Received at IPONZ on 26 May 2011 42 a) a coding region encoding a CrylD protein comprising the DNA of SEQ ID No. 12 or 14, and b) a promoter region capable of directing expression in plants.

13. A transgenic plant cell, comprising the gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12 stably incorporated in its genome.

14. A plant comprising the gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12 stably incorporated in its genome.

15. The plant of claim 14 or the plant cell of claim 13, which is a Brassica species plant or plant cell.

16. The plant of claim 15 which is of the species Brassica oleraceae, Brassica napus, Brassica rapa, Brassica juncea or Brassica carinata.

17. The plant of claim 16 which is cabbage or cauliflower.

18. Use of the chimeric gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12 to control insect pests.

19. A method for controlling insects, comprising: planting or sowing in a field, plants comprising the chimeric gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12.

20. A method of controlling insects in Brassica species plants, comprising: expressing the chimeric genes of any one of claims 1 to 5, or the DNA of any one of claims 6 to 12 in plants.

21. A method of producing plants or seeds resistant to insects, comprising the steps of: 3661986-1 Received at IPONZ on 26 May 2011 43 a) obtaining a plant transformed with the gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12, and b) selecting progeny of said plant or seeds thereof, containing said gene or DNA.

22. A microorganism comprising the chimeric gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12.

23. The microorganism of claim 22, which is of the genus Escherichia, Bacillus or Agrobacterium.

24. Use of the chimeric gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12 to obtain plant cells, plants or seeds with increased resistance to insects.

25. Use of the DNA of any one of claims 6 to 12 to delay or prevent insect resistance development in transgenic plants expressing an insecticidal protein by insects attempting to feed on such plants.

26. Use of the chimeric gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12 to obtain plants of the species Brassica oleraceae, Brassica napus, Brassica rapa, Brassica juncea or Brassica carinata protected from Plutella xylostella. TI.

27.The chimeric gene of any one of claims 1 to 5 or the DNA of any one of claims 6 to 12, wherein the CrylC protein is a variant comprising the sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, but wherein one, some or all of the following amino acids at the following positions compared to the positions in SEQ ID No. 2 are changed: the amino acid at position 125 is Alanine, the amino acid at position 184 is Valine, the amino acid at position 295 is Arginine, the amino acid at position 454 is Aspartic acid, or the amino acid at position 593 is Arginine. 3661986-1 Received at IPONZ on 26 May 2011 44

28. The DNA of any one of claims 6 to 12, wherein the Cry 1B protein is a variant comprising the sequence of SEQ ID No. 11 from amino acid position 31 to 648, but wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine or the amino acid at position 353 in SEQ ID No. 11 is Arginine, or a protein wherein the amino acid at position 151 in SEQ ID No.11 is Tyrosine and the amino acid at position 353 in SEQ ID No. 11 is Arginine.

29. Use of the chimeric gene of claim 27 or the DNA of claim 28 to obtain plants of the species Brassica oleraceae, Brassica napus, Brassica rapa, Brassica juncea or Brassica carinata protected from Plutella xylostella.

30. A transgenic plant cell, comprising the gene of claim 27 or the DNA of claim 28 stably incorporated in its genome. 3661986-1 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt SEQUENCE LISTING <110> Bayer Bioscience N.V. Van Rie, Jeroen Meulewaeter, Frank Van Eldik, Gerben <120> Novel genes encoding insecticidal proteins <130> BCS06-2003 <160> 23 <170> Patentln version 3.3 <210> 1 <211> 2076 <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> CDS <222> (1)..(672) <220> <221> Intron <222> (673)..(861) <220> <221> CDS <222> (862)..(2073) <400> 1 atg get gag gag aac aac cag aac cag tgt ate cct tac aac tgt ctt 48 Met Ala Glu Glu Asn Asn Gin Asn Gin Cys lie Pro Tyr Asn Cys Leu 15 10 15 teg aac cct gag gag gtt ctt ctt gat gga gag aga ate tct act gga 96 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (1 of 73) [3/06/2011 12:22:37 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg lie Ser Thr Gly 20 25 30 aac tct tct ate gat att tct ctt tct ctt gtt cag ttc ctt gtt tct 144 Asn Ser Ser lie Asp lie Ser Leu Ser Leu Val Gin Phe Leu Val Ser 35 40 45 aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt ate gat ttc gtt 192 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu He Asp Phe Val 50 55 60 tgg gga ate gtt gga cct tct cag tgg gat get ttc ctt gtt cag ate 240 Trp Gly lie Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80 gag cag ctt ate aac gag aga ate get gag ttc get aga aac get get 288 Glu Gin Leu He Asn Glu Arg He Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 ate get aac ctt gag gga ctt gga aac aac ttc aac ate tac gtt gag 336 He Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn He Tyr Val Glu 100 105 110 get ttc aag gag tgg gag gag gat cct aac aac cct gag act aga act 384 Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr 115 120 125 aga gtt ate gat aga ttc aga ate ctt gat gga ctt ctt gag aga gat 432 Arg Val He Asp Arg Phe Arg He Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 att cct tct ttc aga ate tct gga ttc gag gtt cct ctt ctt tct gtt 480 He Pro Ser Phe Arg He Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 tac get cag get get aac ctt cat ctt get ate ctt aga gat tct gtt 528 Tyr Ala Gin Ala Ala Asn Leu His Leu Ala He Leu Arg Asp Ser Val 165 170 175 ate ttc gga gag aga tgg gga ctt act act ate aac gtt aac gag aac 576 He Phe Gly Glu Arg Trp Gly Leu Thr Thr He Asn Val Asn Glu Asn 180 185 190 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (2 of 73) [3/06/2011 12:22:37 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt tac aac aga ctt ate aga cat ate gat gag tac get gat cat tgt get 624 Tyr Asn Arg Leu lie Arg His He Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 aac act tac aac aga gga ctt aac aac ctt cct aag tct act tac cag 672 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin 210 215 220 gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata 732 taatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg 792 tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt 852 gatgtgcag gac tgg ate act tac aac aga ctt aga aga gat ctt act ctt 903 Asp Trp He Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu 225 230 235 act gtt ctt gat att get get ttc ttc cct aac tac gat aac aga aga 951 Thr Val Leu Asp He Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg 240 245 250 tac cct ate cag cct gtt gga cag ctt act aga gag gtt tac act gat 999 Tyr Pro He Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp 255 260 265 270 cct ctt ate aac ttc aac cct cag ctt cag tct gtt get cag ctt cct 1047 Pro Leu He Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro 275 280 285 act ttc aac gtt atg gag tct tct get ate aga aac cct cat ctt ttc 1095 Thr Phe Asn Val Met Glu Ser Ser Ala He Arg Asn Pro His Leu Phe 290 295 300 gat att ctt aac aac ctt act ate ttc act gac tgg ttc tct gtt gga 1143 Asp He Leu Asn Asn Leu Thr He Phe Thr Asp Trp Phe Ser Val Gly 305 310 315 aga aac ttc tac tgg gga gga cat aga gtt ate tct tct ctt ate gga 1191 Arg Asn Phe Tyr Trp Gly Gly His Arg Val He Ser Ser Leu He Gly 320 325 330 gga gga aac ate act tct cct ate tac gga aga gag get aac cag gag 1239 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (3 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Gly Gly Asn lie Thr Ser Pro He Tyr Gly Arg Glu Ala Asn Gin Glu 335 340 345 350 cct cct aga tct ttc act ttc aac gga cct gtt ttc aga act ctt tct 1287 Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser 355 360 365 aac cct act ctt aga ctt ctt cag cag cct tgg cct get cct cct ttc 1335 Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe 370 375 380 aac ctt aga gga gtt gag gga gtt gag ttc tct act cct act aac tct 1383 Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser 385 390 395 ttc act tac aga gga aga gga act gtt gat tct ctt act gag ctt cct 1431 Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro 400 405 410 cct gag gat aac tct gtt cct cct aga gag gga tac tct cat aga ctt 1479 Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu 415 420 425 430 tgt cat get act ttc gtt cag aga tct gga act cct ttc ctt act act 1527 Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr 435 440 445 gga gtt gtt ttc tct tgg act cat aga tct get act ctt act aac act 1575 Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr 450 455 460 ate gat cct gag agg ate aac cag ate cct ctt gtt aag gga ttc aga 1623 He Asp Pro Glu Arg He Asn Gin He Pro Leu Val Lys Gly Phe Arg 465 470 475 gtt tgg gga gga act tct gtt ate act gga cct gga ttc act gga gga 1671 Val Trp Gly Gly Thr Ser Val He Thr Gly Pro Gly Phe Thr Gly Gly 480 485 490 gat att ctt aga aga aac act ttc gga gat ttc gtt tct ctt cag gtt 1719 Asp He Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val 495 500 505 510 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (4 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt aac ate aac tct cct ate act cag aga tac aga ctt aga ttc aga tac 1767 Asn lie Asn Ser Pro He Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr 515 520 525 get tct tct aga gat get aga gtt ate gtt ctt act gga get get tct 1815 Ala Ser Ser Arg Asp Ala Arg Val He Val Leu Thr Gly Ala Ala Ser 530 535 540 act gga gtt gga gga cag gtt tct gtt aac atg cct ctt cag aag act 1863 Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr 545 550 555 atg gag ate gga gag aac ctt act tct aga act ttc aga tac act gat 1911 Met Glu He Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp 560 565 570 ttc tct aac cct ttc tct ttc aga get aac cct gat att ate gga ate 1959 Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp He He Gly He 575 580 585 590 tct gag cag cct ctt ttc gga get gga tct ate tct tct gga gag ctt 2007 Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser He Ser Ser Gly Glu Leu 595 600 605 tac ate gat aaa ate gag ate ate ctt get gat get act ttc gag get 2055 Tyr He Asp Lys He Glu He He Leu Ala Asp Ala Thr Phe Glu Ala 610 615 620 gag tct gat tta gag aga tga 2076 Glu Ser Asp Leu Glu Arg 625 <210> 2 <211> 628 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 2 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (5 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Met Ala Glu Glu Asn Asn Gin Asn Gin Cys lie Pro Tyr Asn Cys Leu 15 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg He Ser Thr Gly 20 25 30 Asn Ser Ser He Asp He Ser Leu Ser Leu Val Gin Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu He Asp Phe Val 50 55 60 Trp Gly He Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80 Glu Gin Leu He Asn Glu Arg He Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 He Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn He Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr 115 120 125 Arg Val He Asp Arg Phe Arg He Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 He Pro Ser Phe Arg He Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gin Ala Ala Asn Leu His Leu Ala He Leu Arg Asp Ser Val 165 170 175 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (6 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt lie Phe Gly Glu Arg Trp Gly Leu Thr Thr He Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu He Arg His He Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin 210 215 220 Asp Trp He Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp He Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 He Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 He Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Ser Ser Ala He Arg Asn Pro His Leu Phe Asp He 290 295 300 Leu Asn Asn Leu Thr He Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val He Ser Ser Leu He Gly Gly Gly 325 330 335 Asn He Thr Ser Pro He Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro 340 345 350 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (7 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr lie Asp 450 455 460 Pro Glu Arg He Asn Gin He Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val He Thr Gly Pro Gly Phe Thr Gly Gly Asp He 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn He 500 505 510 Asn Ser Pro He Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (8 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 515 520 525 Ser Arg Asp Ala Arg Val lie Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu 545 550 555 560 He Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp He He Gly He Ser Glu 580 585 590 Gin Pro Leu Phe Gly Ala Gly Ser He Ser Ser Gly Glu Leu Tyr He 595 600 605 Asp Lys He Glu He He Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser 610 615 620 Asp Leu Glu Arg 625 <210> 3 <211> 2076 <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> exon <222> (1)..(489) file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (9 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <220> <221> Intron <222> (490)..(678) <220> <221> exon <222> (679)..(2073) <400> 3 atg get gag gag aac aac cag aac cag tgt ate cct tac aac tgt ctt 48 Met Ala Glu Glu Asn Asn Gin Asn Gin Cys lie Pro Tyr Asn Cys Leu 15 10 15 teg aac cct gag gag gtt ctt ctt gat gga gag aga ate tct act gga 96 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg He Ser Thr Gly 20 25 30 aac tct tct ate gat att tct ctt tct ctt gtt cag ttc ctt gtt tct 144 Asn Ser Ser He Asp He Ser Leu Ser Leu Val Gin Phe Leu Val Ser 35 40 45 aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt ate gat ttc gtt 192 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu He Asp Phe Val 50 55 60 tgg gga ate gtt gga cct tct cag tgg gat get ttc ctt gtt cag ate 240 Trp Gly He Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin He 65 70 75 80 gag cag ctt ate aac gag aga ate get gag ttc get aga aac get get 288 Glu Gin Leu He Asn Glu Arg He Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 ate get aac ctt gag gga ctt gga aac aac ttc aac ate tac gtt gag 336 He Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn He Tyr Val Glu 100 105 110 get ttc aag gag tgg gag gag gat cct aac aac cct gag act aga act 384 Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr 115 120 125 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (10 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt aga gtt ate gat aga ttc aga ate ctt gat gga ctt ctt gag aga gat 432 Arg Val lie Asp Arg Phe Arg He Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 att cct tct ttc aga ate tct gga ttc gaa gtt cct ctt ctt tct gtt 480 He Pro Ser Phe Arg He Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 tac get cag gtaagtttct gcttctacct ttgatatata tataataatt 529 Tyr Ala Gin atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa agaatgtagt 589 atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat aacttttcta 649 atatatgacc aaaacatggt gatgtgcag get get aac ctt cat ctt get ate 702 Ala Ala Asn Leu His Leu Ala He 165 170 ctt aga gat tct gtt ate ttc gga gag aga tgg gga ctt act act ate 750 Leu Arg Asp Ser Val He Phe Gly Glu Arg Trp Gly Leu Thr Thr He 175 180 185 aac gtt aac gag aac tac aac aga ctt ate aga cat ate gat gag tac 798 Asn Val Asn Glu Asn Tyr Asn Arg Leu He Arg His He Asp Glu Tyr 190 195 200 get gat cat tgt get aac act tac aac aga gga ctt aac aac ctt cct 846 Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro 205 210 215 aag tct act tac cag gac tgg ate act tac aac aga ctt aga aga gat 894 Lys Ser Thr Tyr Gin Asp Trp He Thr Tyr Asn Arg Leu Arg Arg Asp 220 225 230 235 ctt act ctt act gtt ctt gat att get get ttc ttc cct aac tac gat 942 Leu Thr Leu Thr Val Leu Asp He Ala Ala Phe Phe Pro Asn Tyr Asp 240 245 250 aac aga aga tac cct ate cag cct gtt gga cag ctt act aga gag gtt 990 Asn Arg Arg Tyr Pro lie Gin Pro Val Gly Gin Leu Thr Arg Glu Val file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (11 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 255 260 265 tac act gat cct ctt ate aac ttc aac cct cag ctt cag tct gtt get 1038 Tyr Thr Asp Pro Leu lie Asn Phe Asn Pro Gin Leu Gin Ser Val Ala 270 275 280 cag ctt cct act ttc aac gtt atg gag tct tct get ate aga aac cct 1086 Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala He Arg Asn Pro 285 290 295 cat ctt ttc gat att ctt aac aac ctt act ate ttc act gac tgg ttc 1134 His Leu Phe Asp He Leu Asn Asn Leu Thr He Phe Thr Asp Trp Phe 300 305 310 315 tct gtt gga aga aac ttc tac tgg gga gga cat aga gtt ate tct tct 1182 Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val He Ser Ser 320 325 330 ctt ate gga gga gga aac ate act tct cct ate tac gga aga gag get 1230 Leu He Gly Gly Gly Asn He Thr Ser Pro He Tyr Gly Arg Glu Ala 335 340 345 aac cag gag cct cct aga tct ttc act ttc aac gga cct gtt ttc aga 1278 Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg 350 355 360 act ctt tct aac cct act ctt aga ctt ctt cag cag cct tgg cct get 1326 Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala 365 370 375 cct cct ttc aac ctt aga gga gtt gag gga gtt gag ttc tct act cct 1374 Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro 380 385 390 395 act aac tct ttc act tac aga gga aga gga act gtt gat tct ctt act 1422 Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr 400 405 410 gag ctt cct cct gag gat aac tct gtt cct cct aga gag gga tac tct 1470 Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser 415 420 425 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (12 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt cat aga ctt tgt cat get act ttc gtt cag aga tct gga act cct ttc 1518 His Arg Leu Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe 430 435 440 ctt act act gga gtt gtt ttc tct tgg act cat aga tct get act ctt 1566 Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu 445 450 455 act aac act ate gat cct gag agg ate aac cag ate cct ctt gtt aag 1614 Thr Asn Thr lie Asp Pro Glu Arg He Asn Gin He Pro Leu Val Lys 460 465 470 475 gga ttc aga gtt tgg gga gga act tct gtt ate act gga cct gga ttc 1662 Gly Phe Arg Val Trp Gly Gly Thr Ser Val He Thr Gly Pro Gly Phe 480 485 490 act gga gga gat att ctt aga aga aac act ttc gga gat ttc gtt tct 1710 Thr Gly Gly Asp He Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser 495 500 505 ctt cag gtt aac ate aac tct cct ate act cag aga tac aga ctt aga 1758 Leu Gin Val Asn He Asn Ser Pro He Thr Gin Arg Tyr Arg Leu Arg 510 515 520 ttc aga tac get tct tct aga gat get aga gtt ate gtt ctt act gga 1806 Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val He Val Leu Thr Gly 525 530 535 get get tct act gga gtt gga gga cag gtt tct gtt aac atg cct ctt 1854 Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu 540 545 550 555 cag aag act atg gag ate gga gag aac ctt act tct aga act ttc aga 1902 Gin Lys Thr Met Glu He Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg 560 565 570 tac act gat ttc tct aac cct ttc tct ttc aga get aac cct gat att 1950 Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp He 575 580 585 ate gga ate tct gag cag cct ctt ttc gga get gga tct ate tct tct 1998 He Gly He Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser He Ser Ser file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (13 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 590 595 600 gga gag ctt tac ate gat aaa ate gag ate ate ctt get gat get act 2046 Gly Glu Leu Tyr lie Asp Lys lie Glu He He Leu Ala Asp Ala Thr 605 610 615 ttc gag get gag tct gat tta gag aga tga 2076 Phe Glu Ala Glu Ser Asp Leu Glu Arg 620 625 <210> 4 <211> 2442 <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> CDS <222> (1)..(1038) <220> <221> Intron <222> (1039)..(1227) <220> <221> CDS <222> (1228)..(2439) <400> 4 atg get tct ate tct tct tct gtt get act gtt tct aga act get cct 48 Met Ala Ser He Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 get cag get aac atg gtt get cct ttc act gga ctt aag tct aac get 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 get ttc cct act act aag aag get aac gat ttc tct act ctt cct tct 144 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (14 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 35 40 45 aac gga gga aga gtt cag tgt atg cag gtt tgg cct get tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg get cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 act gtt atg atg get tct tct get act get gtt get cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 ctt aag tct act get tct ctt cct gtt get aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 ctt gga aac gtt tct aac gga gga aga ate aga tgt gag gag aac aac 384 Leu Gly Asn Val Ser Asn Gly Gly Arg lie Arg Cys Glu Glu Asn Asn 115 120 125 cag aac cag tgt ate cct tac aac tgt ctt teg aac cct gag gag gtt 432 Gin Asn Gin Cys He Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val 130 135 140 ctt ctt gat gga gag aga ate tct act gga aac tct tct ate gat att 480 Leu Leu Asp Gly Glu Arg He Ser Thr Gly Asn Ser Ser He Asp He 145 150 155 160 tct ctt tct ctt gtt cag ttc ctt gtt tct aac ttc gtt cct gga gga 528 Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly 165 170 175 gga ttc ctt gtt gga ctt ate gat ttc gtt tgg gga ate gtt gga cct 576 Gly Phe Leu Val Gly Leu He Asp Phe Val Trp Gly He Val Gly Pro 180 185 190 tct cag tgg gat get ttc ctt gtt cag ate gag cag ctt ate aac gag 624 Ser Gin Trp Asp Ala Phe Leu Val Gin He Glu Gin Leu He Asn Glu 195 200 205 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (15 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt aga ate get gag ttc get aga aac get get ate get aac ctt gag gga 672 Arg lie Ala Glu Phe Ala Arg Asn Ala Ala He Ala Asn Leu Glu Gly 210 215 220 ctt gga aac aac ttc aac ate tac gtt gag get ttc aag gag tgg gag 720 Leu Gly Asn Asn Phe Asn He Tyr Val Glu Ala Phe Lys Glu Trp Glu 225 230 235 240 gag gat cct aac aac cct gag act aga act aga gtt ate gat aga ttc 768 Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val He Asp Arg Phe 245 250 255 aga ate ctt gat gga ctt ctt gag aga gat att cct tct ttc aga ate 816 Arg He Leu Asp Gly Leu Leu Glu Arg Asp He Pro Ser Phe Arg He 260 265 270 tct gga ttc gag gtt cct ctt ctt tct gtt tac get cag get get aac 864 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn 275 280 285 ctt cat ctt get ate ctt aga gat tct gtt ate ttc gga gag aga tgg 912 Leu His Leu Ala He Leu Arg Asp Ser Val He Phe Gly Glu Arg Trp 290 295 300 gga ctt act act ate aac gtt aac gag aac tac aac aga ctt ate aga 960 Gly Leu Thr Thr He Asn Val Asn Glu Asn Tyr Asn Arg Leu He Arg 305 310 315 320 cat ate gat gag tac get gat cat tgt get aac act tac aac aga gga 1008 His He Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly 325 330 335 ctt aac aac ctt cct aag tct act tac cag gtaagtttct gcttctacct 1058 Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin 340 345 ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt 1118 tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat 1178 tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcag gac tgg ate 1236 Asp Trp He file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (16 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt act tac aac aga ctt aga aga gat ctt act ctt act gtt ctt gat att 1284 Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp lie 350 355 360 365 get get ttc ttc cct aac tac gat aac aga aga tac cct ate cag cct 1332 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro He Gin Pro 370 375 380 gtt gga cag ctt act aga gag gtt tac act gat cct ctt ate aac ttc 1380 Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu He Asn Phe 385 390 395 aac cct cag ctt cag tct gtt get cag ctt cct act ttc aac gtt atg 1428 Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met 400 405 410 gag tct tct get ate aga aac cct cat ctt ttc gat att ctt aac aac 1476 Glu Ser Ser Ala He Arg Asn Pro His Leu Phe Asp He Leu Asn Asn 415 420 425 ctt act ate ttc act gac tgg ttc tct gtt gga aga aac ttc tac tgg 1524 Leu Thr He Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp 430 435 440 445 gga gga cat aga gtt ate tct tct ctt ate gga gga gga aac ate act 1572 Gly Gly His Arg Val He Ser Ser Leu He Gly Gly Gly Asn He Thr 450 455 460 tct cct ate tac gga aga gag get aac cag gag cct cct aga tct ttc 1620 Ser Pro He Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe 465 470 475 act ttc aac gga cct gtt ttc aga act ctt tct aac cct act ctt aga 1668 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg 480 485 490 ctt ctt cag cag cct tgg cct get cct cct ttc aac ctt aga gga gtt 1716 Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val 495 500 505 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (17 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt gag gga gtt gag ttc tct act cct act aac tct ttc act tac aga gga 1764 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly 510 515 520 525 aga gga act gtt gat tct ctt act gag ctt cct cct gag gat aac tct 1812 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser 530 535 540 gtt cct cct aga gag gga tac tct cat aga ctt tgt cat get act ttc 1860 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 545 550 555 gtt cag aga tct gga act cct ttc ctt act act gga gtt gtt ttc tct 1908 Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser 560 565 570 tgg act cat aga tct get act ctt act aac act ate gat cct gag agg 1956 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr lie Asp Pro Glu Arg 575 580 585 ate aac cag ate cct ctt gtt aag gga ttc aga gtt tgg gga gga act 2004 He Asn Gin He Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr 590 595 600 605 tct gtt ate act gga cct gga ttc act gga gga gat att ctt aga aga 2052 Ser Val He Thr Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg 610 615 620 aac act ttc gga gat ttc gtt tct ctt cag gtt aac ate aac tct cct 2100 Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn He Asn Ser Pro 625 630 635 ate act cag aga tac aga ctt aga ttc aga tac get tct tct aga gat 2148 He Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp 640 645 650 get aga gtt ate gtt ctt act gga get get tct act gga gtt gga gga 2196 Ala Arg Val He Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly 655 660 665 cag gtt tct gtt aac atg cct ctt cag aag act atg gag ate gga gag 2244 Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu He Gly Glu file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (18 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 670 675 680 685 aac ctt act tct aga act ttc aga tac act gat ttc tct aac cct ttc 2292 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe 690 695 700 tct ttc aga get aac cct gat att ate gga ate tct gag cag cct ctt 2340 Ser Phe Arg Ala Asn Pro Asp lie He Gly He Ser Glu Gin Pro Leu 705 710 715 ttc gga get gga tct ate tct tct gga gag ctt tac ate gat aaa ate 2388 Phe Gly Ala Gly Ser He Ser Ser Gly Glu Leu Tyr He Asp Lys He 720 725 730 gag ate ate ctt get gat get act ttc gag get gag tct gat tta gag 2436 Glu He He Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu 735 740 745 aga tga 2442 Arg 750 <210> 5 <211> 750 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 5 Met Ala Ser He Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (19 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 Leu Gly Asn Val Ser Asn Gly Gly Arg lie Arg Cys Glu Glu Asn Asn 115 120 125 Gin Asn Gin Cys He Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val 130 135 140 Leu Leu Asp Gly Glu Arg He Ser Thr Gly Asn Ser Ser He Asp He 145 150 155 160 Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly 165 170 175 Gly Phe Leu Val Gly Leu He Asp Phe Val Trp Gly He Val Gly Pro 180 185 190 Ser Gin Trp Asp Ala Phe Leu Val Gin He Glu Gin Leu He Asn Glu 195 200 205 Arg He Ala Glu Phe Ala Arg Asn Ala Ala He Ala Asn Leu Glu Gly file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (20 of 73) [3/06/2011 12:22:38 p.m.J file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 210 215 220 Leu Gly Asn Asn Phe Asn lie Tyr Val Glu Ala Phe Lys Glu Trp Glu 225 230 235 240 Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val He Asp Arg Phe 245 250 255 Arg He Leu Asp Gly Leu Leu Glu Arg Asp He Pro Ser Phe Arg He 260 265 270 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn 275 280 285 Leu His Leu Ala He Leu Arg Asp Ser Val He Phe Gly Glu Arg Trp 290 295 300 Gly Leu Thr Thr He Asn Val Asn Glu Asn Tyr Asn Arg Leu He Arg 305 310 315 320 His He Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly 325 330 335 Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp He Thr Tyr Asn 340 345 350 Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp He Ala Ala Phe 355 360 365 Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro He Gin Pro Val Gly Gin 370 375 380 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (21 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu lie Asn Phe Asn Pro Gin 385 390 395 400 Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser 405 410 415 Ala He Arg Asn Pro His Leu Phe Asp He Leu Asn Asn Leu Thr He 420 425 430 Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His 435 440 445 Arg Val He Ser Ser Leu He Gly Gly Gly Asn He Thr Ser Pro He 450 455 460 Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn 465 470 475 480 Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin 485 490 495 Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val 500 505 510 Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr 515 520 525 Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro 530 535 540 Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg 545 550 555 560 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (22 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His 565 570 575 Arg Ser Ala Thr Leu Thr Asn Thr lie Asp Pro Glu Arg He Asn Gin 580 585 590 He Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val He 595 600 605 Thr Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Asn Thr Phe 610 615 620 Gly Asp Phe Val Ser Leu Gin Val Asn He Asn Ser Pro He Thr Gin 625 630 635 640 Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val 645 650 655 He Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser 660 665 670 Val Asn Met Pro Leu Gin Lys Thr Met Glu He Gly Glu Asn Leu Thr 675 680 685 Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg 690 695 700 Ala Asn Pro Asp He He Gly He Ser Glu Gin Pro Leu Phe Gly Ala 705 710 715 720 Gly Ser He Ser Ser Gly Glu Leu Tyr He Asp Lys He Glu He He 725 730 735 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (23 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg 740 745 750 <210> 6 <211> 2442 <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> CDS <222> (1)..(855) <220> <221> Intron <222> (856)..(1044) <220> <221> CDS <222> (1045)..(2439) <400> 6 atg get tct ate tct tct tct gtt get act gtt tct aga act get cct 48 Met Ala Ser lie Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 get cag get aac atg gtt get cct ttc act gga ctt aag tct aac get 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 get ttc cct act act aag aag get aac gat ttc tct act ctt cct tct 144 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 aac gga gga aga gtt cag tgt atg cag gtt tgg cct get tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (24 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg get cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 act gtt atg atg get tct tct get act get gtt get cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 ctt aag tct act get tct ctt cct gtt get aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 ctt gga aac gtt tct aac gga gga aga ate aga tgt gag gag aac aac 384 Leu Gly Asn Val Ser Asn Gly Gly Arg lie Arg Cys Glu Glu Asn Asn 115 120 125 cag aac cag tgt ate cct tac aac tgt ctt teg aac cct gag gag gtt 432 Gin Asn Gin Cys He Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val 130 135 140 ctt ctt gat gga gag aga ate tct act gga aac tct tct ate gat att 480 Leu Leu Asp Gly Glu Arg He Ser Thr Gly Asn Ser Ser He Asp He 145 150 155 160 tct ctt tct ctt gtt cag ttc ctt gtt tct aac ttc gtt cct gga gga 528 Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly 165 170 175 gga ttc ctt gtt gga ctt ate gat ttc gtt tgg gga ate gtt gga cct 576 Gly Phe Leu Val Gly Leu He Asp Phe Val Trp Gly He Val Gly Pro 180 185 190 tct cag tgg gat get ttc ctt gtt cag ate gag cag ctt ate aac gag 624 Ser Gin Trp Asp Ala Phe Leu Val Gin He Glu Gin Leu He Asn Glu 195 200 205 aga ate get gag ttc get aga aac get get ate get aac ctt gag gga 672 Arg He Ala Glu Phe Ala Arg Asn Ala Ala He Ala Asn Leu Glu Gly 210 215 220 ctt gga aac aac ttc aac ate tac gtt gag get ttc aag gag tgg gag 720 Leu Gly Asn Asn Phe Asn He Tyr Val Glu Ala Phe Lys Glu Trp Glu file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (25 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 225 230 235 240 gag gat cct aac aac cct gag act aga act aga gtt ate gat aga ttc 768 Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val lie Asp Arg Phe 245 250 255 aga ate ctt gat gga ctt ctt gag aga gat att cct tct ttc aga ate 816 Arg He Leu Asp Gly Leu Leu Glu Arg Asp He Pro Ser Phe Arg He 260 265 270 tct gga ttc gaa gtt cct ctt ctt tct gtt tac get cag gtaagtttct 865 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin 275 280 285 gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca 925 aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa 985 gtgtgtatat tttaatttat aacttttcta atatatgacc aaaacatggt gatgtgcag 1044 get get aac ctt cat ctt get ate ctt aga gat tct gtt ate ttc gga 1092 Ala Ala Asn Leu His Leu Ala He Leu Arg Asp Ser Val He Phe Gly 290 295 300 gag aga tgg gga ctt act act ate aac gtt aac gag aac tac aac aga 1140 Glu Arg Trp Gly Leu Thr Thr He Asn Val Asn Glu Asn Tyr Asn Arg 305 310 315 ctt ate aga cat ate gat gag tac get gat cat tgt get aac act tac 1188 Leu He Arg His He Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr 320 325 330 aac aga gga ctt aac aac ctt cct aag tct act tac cag gac tgg ate 1236 Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp He 335 340 345 act tac aac aga ctt aga aga gat ctt act ctt act gtt ctt gat att 1284 Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp He 350 355 360 365 get get ttc ttc cct aac tac gat aac aga aga tac cct ate cag cct 1332 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (26 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro lie Gin Pro 370 375 380 gtt gga cag ctt act aga gag gtt tac act gat cct ctt ate aac ttc 1380 Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu He Asn Phe 385 390 395 aac cct cag ctt cag tct gtt get cag ctt cct act ttc aac gtt atg 1428 Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met 400 405 410 gag tct tct get ate aga aac cct cat ctt ttc gat att ctt aac aac 1476 Glu Ser Ser Ala He Arg Asn Pro His Leu Phe Asp He Leu Asn Asn 415 420 425 ctt act ate ttc act gac tgg ttc tct gtt gga aga aac ttc tac tgg 1524 Leu Thr He Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp 430 435 440 445 gga gga cat aga gtt ate tct tct ctt ate gga gga gga aac ate act 1572 Gly Gly His Arg Val He Ser Ser Leu He Gly Gly Gly Asn He Thr 450 455 460 tct cct ate tac gga aga gag get aac cag gag cct cct aga tct ttc 1620 Ser Pro He Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe 465 470 475 act ttc aac gga cct gtt ttc aga act ctt tct aac cct act ctt aga 1668 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg 480 485 490 ctt ctt cag cag cct tgg cct get cct cct ttc aac ctt aga gga gtt 1716 Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val 495 500 505 gag gga gtt gag ttc tct act cct act aac tct ttc act tac aga gga 1764 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly 510 515 520 525 aga gga act gtt gat tct ctt act gag ctt cct cct gag gat aac tct 1812 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser 530 535 540 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (27 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt gtt cct cct aga gag gga tac tct cat aga ctt tgt cat get act ttc 1860 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 545 550 555 gtt cag aga tct gga act cct ttc ctt act act gga gtt gtt ttc tct 1908 Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser 560 565 570 tgg act cat aga tct get act ctt act aac act ate gat cct gag agg 1956 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr lie Asp Pro Glu Arg 575 580 585 ate aac cag ate cct ctt gtt aag gga ttc aga gtt tgg gga gga act 2004 He Asn Gin He Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr 590 595 600 605 tct gtt ate act gga cct gga ttc act gga gga gat att ctt aga aga 2052 Ser Val He Thr Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg 610 615 620 aac act ttc gga gat ttc gtt tct ctt cag gtt aac ate aac tct cct 2100 Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn He Asn Ser Pro 625 630 635 ate act cag aga tac aga ctt aga ttc aga tac get tct tct aga gat 2148 He Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp 640 645 650 get aga gtt ate gtt ctt act gga get get tct act gga gtt gga gga 2196 Ala Arg Val He Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly 655 660 665 cag gtt tct gtt aac atg cct ctt cag aag act atg gag ate gga gag 2244 Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu He Gly Glu 670 675 680 685 aac ctt act tct aga act ttc aga tac act gat ttc tct aac cct ttc 2292 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe 690 695 700 tct ttc aga get aac cct gat att ate gga ate tct gag cag cct ctt 2340 Ser Phe Arg Ala Asn Pro Asp He He Gly He Ser Glu Gin Pro Leu file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (28 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 705 710 715 ttc gga get gga tct ate tct tct gga gag ctt tac ate gat aaa ate 2388 Phe Gly Ala Gly Ser lie Ser Ser Gly Glu Leu Tyr He Asp Lys He 720 725 730 gag ate ate ctt get gat get act ttc gag get gag tct gat tta gag 2436 Glu He He Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu 735 740 745 aga tga 2442 Arg 750 <210> 7 <211> 750 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 7 Met Ala Ser He Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (29 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 Leu Gly Asn Val Ser Asn Gly Gly Arg lie Arg Cys Glu Glu Asn Asn 115 120 125 Gin Asn Gin Cys He Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val 130 135 140 Leu Leu Asp Gly Glu Arg He Ser Thr Gly Asn Ser Ser He Asp He 145 150 155 160 Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly 165 170 175 Gly Phe Leu Val Gly Leu He Asp Phe Val Trp Gly He Val Gly Pro 180 185 190 Ser Gin Trp Asp Ala Phe Leu Val Gin He Glu Gin Leu He Asn Glu 195 200 205 Arg He Ala Glu Phe Ala Arg Asn Ala Ala He Ala Asn Leu Glu Gly 210 215 220 Leu Gly Asn Asn Phe Asn He Tyr Val Glu Ala Phe Lys Glu Trp Glu 225 230 235 240 Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val He Asp Arg Phe file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (30 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 245 250 255 Arg lie Leu Asp Gly Leu Leu Glu Arg Asp He Pro Ser Phe Arg He 260 265 270 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn 275 280 285 Leu His Leu Ala He Leu Arg Asp Ser Val He Phe Gly Glu Arg Trp 290 295 300 Gly Leu Thr Thr He Asn Val Asn Glu Asn Tyr Asn Arg Leu He Arg 305 310 315 320 His He Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly 325 330 335 Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp He Thr Tyr Asn 340 345 350 Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp He Ala Ala Phe 355 360 365 Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro He Gin Pro Val Gly Gin 370 375 380 Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu He Asn Phe Asn Pro Gin 385 390 395 400 Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser 405 410 415 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (31 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ala lie Arg Asn Pro His Leu Phe Asp He Leu Asn Asn Leu Thr He 420 425 430 Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His 435 440 445 Arg Val He Ser Ser Leu He Gly Gly Gly Asn He Thr Ser Pro He 450 455 460 Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn 465 470 475 480 Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin 485 490 495 Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val 500 505 510 Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr 515 520 525 Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro 530 535 540 Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg 545 550 555 560 Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His 565 570 575 Arg Ser Ala Thr Leu Thr Asn Thr He Asp Pro Glu Arg He Asn Gin 580 585 590 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (32 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt lie Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val He 595 600 605 Thr Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Asn Thr Phe 610 615 620 Gly Asp Phe Val Ser Leu Gin Val Asn He Asn Ser Pro He Thr Gin 625 630 635 640 Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val 645 650 655 He Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser 660 665 670 Val Asn Met Pro Leu Gin Lys Thr Met Glu He Gly Glu Asn Leu Thr 675 680 685 Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg 690 695 700 Ala Asn Pro Asp He He Gly He Ser Glu Gin Pro Leu Phe Gly Ala 705 710 715 720 Gly Ser He Ser Ser Gly Glu Leu Tyr He Asp Lys He Glu He He 725 730 735 Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg 740 745 750 <210> 8 <211> 2313 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (33 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> CDS <222> (1)..(2310) <400> 8 atg get tct ate tct tct tct gtt get act gtt tct aga act get cct 48 Met Ala Ser lie Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 get cag get aac atg gtt get cct ttc act gga ctt aag tct aac get 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 get ttc cct act act aag aag get aac gat ttc tct act ctt cct tct 144 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 aac gga gga aga gtt cag tgt atg cag gtt tgg cct get tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg get cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 act gtt atg atg get tct tct get act get gtt get cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 ctt aag tct act get tct ctt cct gtt get aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 ctt gga aac gtt tct aac gga gga aga ate aga tgt act teg aac aga 384 Leu Gly Asn Val Ser Asn Gly Gly Arg He Arg Cys Thr Ser Asn Arg 115 120 125 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (34 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt aag aac gag aac gag ate ate aac get gtt tct aac cat tct get cag 432 Lys Asn Glu Asn Glu lie He Asn Ala Val Ser Asn His Ser Ala Gin 130 135 140 atg gat ctt ctt cct gat get aga ate gag gat tct ctt tgt ate get 480 Met Asp Leu Leu Pro Asp Ala Arg He Glu Asp Ser Leu Cys He Ala 145 150 155 160 gag gga aac aac ate gat cct ttc gtt tct get tct act gtt cag act 528 Glu Gly Asn Asn He Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr 165 170 175 ggt ate aac ate get gga aga att ctt gga gtt ctt gga gtt cct ttc 576 Gly He Asn He Ala Gly Arg He Leu Gly Val Leu Gly Val Pro Phe 180 185 190 get gga cag ctt get tct ttc tac tct ttc ctt gtt gga gag ctt tgg 624 Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp 195 200 205 cct aga gga aga gat cag tgg gag ate ttc ctt gag cat gtt gag cag 672 Pro Arg Gly Arg Asp Gin Trp Glu lie Phe Leu Glu His Val Glu Gin 210 215 220 ctt ate aac cag cag ate act gag aac get aga aac act get ctt get 720 Leu He Asn Gin Gin He Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala 225 230 235 240 aga ctt cag gga ctt gga gat tct ttc aga get tac cag cag tct ctt 768 Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu 245 250 255 gag gac tgg ctt gag aac aga gat gat get aga act aga tct gtt ctt 816 Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu 260 265 270 cat act cag tac ate get ctt gag ctt gat ttc ctt aac get atg cct 864 His Thr Gin Tyr He Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro 275 280 285 ctt ttc get ate aga aac cag gag gtt cct ctt ctt atg gtt tac get 912 Leu Phe Ala He Arg Asn Gin Glu Val Pro Leu Leu Met Val Tyr Ala file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (35 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 290 295 300 cag get get aac ctt cat ctt ctt ctt ctt aga gat get tct ctt ttc 960 Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe 305 310 315 320 gga tct gag ttc gga ctt act tct cag gag ate cag aga tat tac gag 1008 Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu lie Gin Arg Tyr Tyr Glu 325 330 335 aga cag gtt gag aga act aga gat tac tct gat tac tgt gtt gag tgg 1056 Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp 340 345 350 tac aac act gga ctt aac tct ctt aga gga act aac get get tct tgg 1104 Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp 355 360 365 gtt aga tac aac cag ttc aga aga gat ctt act ctt gga gtt ctt gat 1152 Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp 370 375 380 ctt gtt get ctt ttc cct tct tac gac act aga act tac cct ate aac 1200 Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro He Asn 385 390 395 400 act tct get cag ctt act aga gag gtt tac act gat get ate gga get 1248 Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala He Gly Ala 405 410 415 act gga gtt aac atg get tct atg aac tgg tac aac aac aac get cct 1296 Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro 420 425 430 tct ttc tct get ate gag get get get ate aga tct cct cat ctt ctt 1344 Ser Phe Ser Ala He Glu Ala Ala Ala He Arg Ser Pro His Leu Leu 435 440 445 gat ttc ctt gag cag ctt act ate ttc tct get tct tct aga tgg tct 1392 Asp Phe Leu Glu Gin Leu Thr He Phe Ser Ala Ser Ser Arg Trp Ser 450 455 460 aac act aga cac atg act tac tgg aga gga cat ace ate cag tct aga 1440 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (36 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr lie Gin Ser Arg 465 470 475 480 cct ate gga gga gga ctt aac act tct act cat gga get act aac act 1488 Pro He Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr 485 490 495 tct ate aac cct gtt act ctt aga ttc get tct aga gat gtt tac aga 1536 Ser He Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg 500 505 510 act gag tct tac get gga gtt ctt ctt tgg gga ate tac ctt gag cct 1584 Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly He Tyr Leu Glu Pro 515 520 525 ate cac gga gtt cct act gtt aga ttc aac ttc act aac cct cag aac 1632 He His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn 530 535 540 ate tct gat aga gga act get aac tac tct cag cct tac gag tct cct 1680 He Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro 545 550 555 560 gga ctt cag ctt aag gat tct gag act gag ctt cct cct gag act act 1728 Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr 565 570 575 gag aga cct aac tac gag tct tac tct cat aga ctt tct cat ate gga 1776 Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His He Gly 580 585 590 ate ate ctt cag tct aga gtt aac gtt cct gtt tac tct tgg act cat 1824 He He Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His 595 600 605 aga tct get gat aga act aac act ate gga cct aac aga ate act cag 1872 Arg Ser Ala Asp Arg Thr Asn Thr He Gly Pro Asn Arg He Thr Gin 610 615 620 ate cct atg gtt aag get tct gag ctt cct cag gga act act gtt gtt 1920 He Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val 625 630 635 640 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (37 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt aga gga cct gga ttc act gga gga gat ate ctt aga aga act aac act 1968 Arg Gly Pro Gly Phe Thr Gly Gly Asp lie Leu Arg Arg Thr Asn Thr 645 650 655 gga gga ttc gga cct ate aga gtt act gtt aac gga cct ctt act cag 2016 Gly Gly Phe Gly Pro He Arg Val Thr Val Asn Gly Pro Leu Thr Gin 660 665 670 aga tac aga ate gga ttc aga tac get tct act gtt gat ttc gat ttc 2064 Arg Tyr Arg He Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe 675 680 685 ttc gtt tct aga gga gga act act gtt aac aac ttc aga ttc ctt aga 2112 Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg 690 695 700 act atg aac tct gga gat gag ctt aag tac gga aac ttc gtt aga aga 2160 Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg 705 710 715 720 get ttc act act cct ttc act ttc act cag ate cag gat ate ate aga 2208 Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin lie Gin Asp He He Arg 725 730 735 act tct ate cag gga ctt tct gga aac gga gag gtt tac ate gat aaa 2256 Thr Ser He Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr He Asp Lys 740 745 750 ate gag ate ate cct gtt act get act ttc gag get gag tac gat tta 2304 He Glu He He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu 755 760 765 gag aga tga 2313 Glu Arg 770 <210> 9 <211> 770 <212> PRT <213> Artificial file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (38 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <220> <223> Synthetic Construct <400> 9 Met Ala Ser lie Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 Leu Gly Asn Val Ser Asn Gly Gly Arg He Arg Cys Thr Ser Asn Arg 115 120 125 Lys Asn Glu Asn Glu He He Asn Ala Val Ser Asn His Ser Ala Gin 130 135 140 Met Asp Leu Leu Pro Asp Ala Arg He Glu Asp Ser Leu Cys He Ala 145 150 155 160 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (39 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Glu Gly Asn Asn lie Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr 165 170 175 Gly He Asn He Ala Gly Arg He Leu Gly Val Leu Gly Val Pro Phe 180 185 190 Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp 195 200 205 Pro Arg Gly Arg Asp Gin Trp Glu He Phe Leu Glu His Val Glu Gin 210 215 220 Leu He Asn Gin Gin He Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala 225 230 235 240 Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu 245 250 255 Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu 260 265 270 His Thr Gin Tyr He Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro 275 280 285 Leu Phe Ala He Arg Asn Gin Glu Val Pro Leu Leu Met Val Tyr Ala 290 295 300 Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe 305 310 315 320 Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu He Gin Arg Tyr Tyr Glu file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (40 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 325 330 335 Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp 340 345 350 Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp 355 360 365 Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp 370 375 380 Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro lie Asn 385 390 395 400 Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala He Gly Ala 405 410 415 Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro 420 425 430 Ser Phe Ser Ala He Glu Ala Ala Ala He Arg Ser Pro His Leu Leu 435 440 445 Asp Phe Leu Glu Gin Leu Thr He Phe Ser Ala Ser Ser Arg Trp Ser 450 455 460 Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr He Gin Ser Arg 465 470 475 480 Pro He Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr 485 490 495 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (41 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ser lie Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg 500 505 510 Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly He Tyr Leu Glu Pro 515 520 525 He His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn 530 535 540 He Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro 545 550 555 560 Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr 565 570 575 Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His He Gly 580 585 590 He He Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His 595 600 605 Arg Ser Ala Asp Arg Thr Asn Thr He Gly Pro Asn Arg He Thr Gin 610 615 620 He Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val 625 630 635 640 Arg Gly Pro Gly Phe Thr Gly Gly Asp He Leu Arg Arg Thr Asn Thr 645 650 655 Gly Gly Phe Gly Pro He Arg Val Thr Val Asn Gly Pro Leu Thr Gin 660 665 670 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (42 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Arg Tyr Arg lie Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe 675 680 685 Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg 690 695 700 Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg 705 710 715 720 Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin He Gin Asp He He Arg 725 730 735 Thr Ser He Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr He Asp Lys 740 745 750 He Glu He He Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu 755 760 765 Glu Arg 770 <210> 10 <211> 1947 <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> CDS <222> (1)..(1944) <400> 10 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (43 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt atg get act teg aac aga aag aac gag aac gag ate ate aac get gtt 48 Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu lie He Asn Ala Val 15 10 15 tct aac cat tct get cag atg gat ctt ctt cct gat get aga ate gag 96 Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg He Glu 20 25 30 gat tct ctt tgt ate get gag gga aac aac ate gat cct ttc gtt tct 144 Asp Ser Leu Cys He Ala Glu Gly Asn Asn He Asp Pro Phe Val Ser 35 40 45 get tct act gtt cag act ggt ate aac ate get gga aga att ctt gga 192 Ala Ser Thr Val Gin Thr Gly He Asn He Ala Gly Arg He Leu Gly 50 55 60 gtt ctt gga gtt cct ttc get gga cag ctt get tct ttc tac tct ttc 240 Val Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe 65 70 75 80 ctt gtt gga gag ctt tgg cct aga gga aga gat cag tgg gag ate ttc 288 Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu He Phe 85 90 95 ctt gag cat gtt gag cag ctt ate aac cag cag ate act gag aac get 336 Leu Glu His Val Glu Gin Leu He Asn Gin Gin He Thr Glu Asn Ala 100 105 110 aga aac act get ctt get aga ctt cag gga ctt gga gat tct ttc aga 384 Arg Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg 115 120 125 get tac cag cag tct ctt gag gac tgg ctt gag aac aga gat gat get 432 Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala 130 135 140 aga act aga tct gtt ctt cat act cag tac ate get ctt gag ctt gat 480 Arg Thr Arg Ser Val Leu His Thr Gin Tyr He Ala Leu Glu Leu Asp 145 150 155 160 ttc ctt aac get atg cct ctt ttc get ate aga aac cag gag gtt cct 528 Phe Leu Asn Ala Met Pro Leu Phe Ala He Arg Asn Gin Glu Val Pro 165 170 175 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (44 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt ctt ctt atg gtt tac get cag get get aac ctt cat ctt ctt ctt ctt 576 Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu 180 185 190 aga gat get tct ctt ttc gga tct gag ttc gga ctt act tct cag gag 624 Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu 195 200 205 ate cag aga tat tac gag aga cag gtt gag aga act aga gat tac tct 672 lie Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser 210 215 220 gat tac tgt gtt gag tgg tac aac act gga ctt aac tct ctt aga gga 720 Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly 225 230 235 240 act aac get get tct tgg gtt aga tac aac cag ttc aga aga gat ctt 768 Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu 245 250 255 act ctt gga gtt ctt gat ctt gtt get ctt ttc cct tct tac gac act 816 Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr 260 265 270 aga act tac cct ate aac act tct get cag ctt act aga gag gtt tac 864 Arg Thr Tyr Pro He Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr 275 280 285 act gat get ate gga get act gga gtt aac atg get tct atg aac tgg 912 Thr Asp Ala He Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp 290 295 300 tac aac aac aac get cct tct ttc tct get ate gag get get get ate 960 Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala He Glu Ala Ala Ala He 305 310 315 320 aga tct cct cat ctt ctt gat ttc ctt gag cag ctt act ate ttc tct 1008 Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr He Phe Ser 325 330 335 get tct tct aga tgg tct aac act aga cac atg act tac tgg aga gga 1056 Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (45 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 340 345 350 cat acc ate cag tct aga cct ate gga gga gga ctt aac act tct act 1104 His Thr lie Gin Ser Arg Pro lie Gly Gly Gly Leu Asn Thr Ser Thr 355 360 365 cat gga get act aac act tct ate aac cct gtt act ctt aga ttc get 1152 His Gly Ala Thr Asn Thr Ser He Asn Pro Val Thr Leu Arg Phe Ala 370 375 380 tct aga gat gtt tac aga act gag tct tac get gga gtt ctt ctt tgg 1200 Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp 385 390 395 400 gga ate tac ctt gag cct ate cac gga gtt cct act gtt aga ttc aac 1248 Gly He Tyr Leu Glu Pro He His Gly Val Pro Thr Val Arg Phe Asn 405 410 415 ttc act aac cct cag aac ate tct gat aga gga act get aac tac tct 1296 Phe Thr Asn Pro Gin Asn He Ser Asp Arg Gly Thr Ala Asn Tyr Ser 420 425 430 cag cct tac gag tct cct gga ctt cag ctt aag gat tct gag act gag 1344 Gin Pro Tyr Glu Ser Pro Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu 435 440 445 ctt cct cct gag act act gag aga cct aac tac gag tct tac tct cat 1392 Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His 450 455 460 aga ctt tct cat ate gga ate ate ctt cag tct aga gtt aac gtt cct 1440 Arg Leu Ser His He Gly He He Leu Gin Ser Arg Val Asn Val Pro 465 470 475 480 gtt tac tct tgg act cat aga tct get gat aga act aac act ate gga 1488 Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr He Gly 485 490 495 cct aac aga ate act cag ate cct atg gtt aag get tct gag ctt cct 1536 Pro Asn Arg He Thr Gin He Pro Met Val Lys Ala Ser Glu Leu Pro 500 505 510 cag gga act act gtt gtt aga gga cct gga ttc act gga gga gat ate 1584 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (46 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp lie 515 520 525 ctt aga aga act aac act gga gga ttc gga cct ate aga gtt act gtt 1632 Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro He Arg Val Thr Val 530 535 540 aac gga cct ctt act cag aga tac aga ate gga ttc aga tac get tct 1680 Asn Gly Pro Leu Thr Gin Arg Tyr Arg He Gly Phe Arg Tyr Ala Ser 545 550 555 560 act gtt gat ttc gat ttc ttc gtt tct aga gga gga act act gtt aac 1728 Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn 565 570 575 aac ttc aga ttc ctt aga act atg aac tct gga gat gag ctt aag tac 1776 Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr 580 585 590 gga aac ttc gtt aga aga get ttc act act cct ttc act ttc act cag 1824 Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin 595 600 605 ate cag gat ate ate aga act tct ate cag gga ctt tct gga aac gga 1872 He Gin Asp He He Arg Thr Ser He Gin Gly Leu Ser Gly Asn Gly 610 615 620 gag gtt tac ate gat aaa ate gag ate ate cct gtt act get act ttc 1920 Glu Val Tyr He Asp Lys He Glu He He Pro Val Thr Ala Thr Phe 625 630 635 640 gag get gag tac gat tta gag aga tga 1947 Glu Ala Glu Tyr Asp Leu Glu Arg 645 <210> 11 <211> 648 <212> PRT <213> Artificial <220> <223> Synthetic Construct file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (47 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <400> 11 Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu lie He Asn Ala Val 15 10 15 Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg He Glu 20 25 30 Asp Ser Leu Cys He Ala Glu Gly Asn Asn He Asp Pro Phe Val Ser 35 40 45 Ala Ser Thr Val Gin Thr Gly He Asn He Ala Gly Arg He Leu Gly 50 55 60 Val Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe 65 70 75 80 Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu He Phe 85 90 95 Leu Glu His Val Glu Gin Leu He Asn Gin Gin He Thr Glu Asn Ala 100 105 110 Arg Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg 115 120 125 Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala 130 135 140 Arg Thr Arg Ser Val Leu His Thr Gin Tyr He Ala Leu Glu Leu Asp 145 150 155 160 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (48 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Phe Leu Asn Ala Met Pro Leu Phe Ala lie Arg Asn Gin Glu Val Pro 165 170 175 Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu 180 185 190 Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu 195 200 205 He Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser 210 215 220 Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly 225 230 235 240 Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu 245 250 255 Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr 260 265 270 Arg Thr Tyr Pro He Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr 275 280 285 Thr Asp Ala He Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp 290 295 300 Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala He Glu Ala Ala Ala He 305 310 315 320 Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr He Phe Ser 325 330 335 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (49 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly 340 345 350 His Thr lie Gin Ser Arg Pro He Gly Gly Gly Leu Asn Thr Ser Thr 355 360 365 His Gly Ala Thr Asn Thr Ser He Asn Pro Val Thr Leu Arg Phe Ala 370 375 380 Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp 385 390 395 400 Gly He Tyr Leu Glu Pro He His Gly Val Pro Thr Val Arg Phe Asn 405 410 415 Phe Thr Asn Pro Gin Asn He Ser Asp Arg Gly Thr Ala Asn Tyr Ser 420 425 430 Gin Pro Tyr Glu Ser Pro Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu 435 440 445 Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His 450 455 460 Arg Leu Ser His He Gly He He Leu Gin Ser Arg Val Asn Val Pro 465 470 475 480 Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr He Gly 485 490 495 Pro Asn Arg He Thr Gin He Pro Met Val Lys Ala Ser Glu Leu Pro 500 505 510 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (50 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp lie 515 520 525 Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro He Arg Val Thr Val 530 535 540 Asn Gly Pro Leu Thr Gin Arg Tyr Arg He Gly Phe Arg Tyr Ala Ser 545 550 555 560 Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn 565 570 575 Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr 580 585 590 Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin 595 600 605 He Gin Asp He He Arg Thr Ser He Gin Gly Leu Ser Gly Asn Gly 610 615 620 Glu Val Tyr He Asp Lys He Glu He He Pro Val Thr Ala Thr Phe 625 630 635 640 Glu Ala Glu Tyr Asp Leu Glu Arg 645 <210> 12 <211> 2181 <212> DNA <213> Artificial file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (51 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <220> <223> modified sequence <220> <221> CDS <222> (1)..(2178) <400> 12 atg get tct ate tct tct tct gtt get act gtt tct aga act get cct 48 Met Ala Ser lie Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 get cag get aac atg gtt get cct ttc act gga ctt aag tct aac get 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 get ttc cct act act aag aag get aac gat ttc tct act ctt cct tct 144 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 aac gga gga aga gtt cag tgt atg cag gtt tgg cct get tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg get cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 act gtt atg atg get tct tct get act get gtt get cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 ctt aag tct act get tct ctt cct gtt get aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 ctt gga aac gtt tct aac gga gga aga ate aga tgt gag ate aac aac 384 Leu Gly Asn Val Ser Asn Gly Gly Arg He Arg Cys Glu He Asn Asn 115 120 125 cag aac cag tgt gtt cct tac aac tgt ctt tct aac cct aag gag ate 432 Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu He file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (52 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 130 135 140 ate ctt gga gag gag aga ctt gag act gga aac act gtt get gat ate 480 lie Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp lie 145 150 155 160 tct ctt gga ctt ate aac ttc ctt tac tct aac ttc gtt cct gga ggt 528 Ser Leu Gly Leu He Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly 165 170 175 gga ttc ate gtt gga ctt ctt gag ctt ate tgg gga ttc ate gga cct 576 Gly Phe He Val Gly Leu Leu Glu Leu He Trp Gly Phe He Gly Pro 180 185 190 tct cag tgg gat ate ttc ctt get cag ate gag cag ctt ate tct cag 624 Ser Gin Trp Asp He Phe Leu Ala Gin He Glu Gin Leu He Ser Gin 195 200 205 aga ate gag gag ttc get aga aac cag get ate tct aga ctt gag gga 672 Arg He Glu Glu Phe Ala Arg Asn Gin Ala He Ser Arg Leu Glu Gly 210 215 220 ctt tct aat ctt tac aaa gtt tac gtt aga get ttc tct gac tgg gag 720 Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu 225 230 235 240 aag gat cct act aac cct get ctt aga gag gag atg aga ate cag ttc 768 Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg He Gin Phe 245 250 255 aac gat atg aac tct get ctt ate act get ate cct ctt ttc aga gtt 816 Asn Asp Met Asn Ser Ala Leu He Thr Ala He Pro Leu Phe Arg Val 260 265 270 cag aac tac gag gtt get ctt ctt tct gtt tac gtt cag get get aac 864 Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn 275 280 285 ctt cat ctt tct ate ctt aga gat gtt tct gtt ttc gga gag aga tgg 912 Leu His Leu Ser He Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp 290 295 300 gga tac gat act get act ate aac aac aga tac tct gat ctt act tct 960 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (53 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Gly Tyr Asp Thr Ala Thr lie Asn Asn Arg Tyr Ser Asp Leu Thr Ser 305 310 315 320 ctt ate cat gtt tac act aac cat tgt gtt gat act tac aac cag gga 1008 Leu He His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly 325 330 335 ctt aga aga ctt gag gga aga ttc ctt tct gac tgg ate gtt tac aac 1056 Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp He Val Tyr Asn 340 345 350 aga ttc aga aga cag ctt act ate tct gtt ctt gat ate gtt get ttc 1104 Arg Phe Arg Arg Gin Leu Thr He Ser Val Leu Asp He Val Ala Phe 355 360 365 ttc cct aac tac gat ate aga act tac cct ate cag act get act cag 1152 Phe Pro Asn Tyr Asp He Arg Thr Tyr Pro He Gin Thr Ala Thr Gin 370 375 380 ctt act aga gag gtt tac ctt gat ctt cct ttc ate aac gag aac ctt 1200 Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe He Asn Glu Asn Leu 385 390 395 400 tct cct get get tct tac cct act ttc tct get get gag tct get ate 1248 Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala He 405 410 415 ate aga tct cct cat ctt gtt gat ttc ctt aac tct ttc act ate tac 1296 He Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr He Tyr 420 425 430 act gat tct ctt get aga tac get tac tgg gga gga cat ctt gtt aac 1344 Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn 435 440 445 tct ttc aga act gga act aca act aac ctt ate aga tct cct ctt tac 1392 Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu He Arg Ser Pro Leu Tyr 450 455 460 gga aga gag gga aac act gag aga cct gtt act ate act get tct cct 1440 Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr He Thr Ala Ser Pro 465 470 475 480 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (54 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt tct gtt cct ate ttc aga act ctt tct tac ate act gga ctt gat aac 1488 Ser Val Pro lie Phe Arg Thr Leu Ser Tyr He Thr Gly Leu Asp Asn 485 490 495 tct aac cct gtt get gga ate gag gga gtt gag ttc cag aac act ate 1536 Ser Asn Pro Val Ala Gly He Glu Gly Val Glu Phe Gin Asn Thr He 500 505 510 tct aga tct ate tac aga aag tct gga cct ate gat tct ttc tct gag 1584 Ser Arg Ser He Tyr Arg Lys Ser Gly Pro He Asp Ser Phe Ser Glu 515 520 525 ctt cct cct cag gat get tct gtt tct cct get ate gga tac tct cat 1632 Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala He Gly Tyr Ser His 530 535 540 aga ctt tgt cat get act ttc ctt gag aga ate tct gga cct aga ate 1680 Arg Leu Cys His Ala Thr Phe Leu Glu Arg He Ser Gly Pro Arg He 545 550 555 560 get gga act gtt ttc tct tgg act cat aga tct get tct cct act aac 1728 Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn 565 570 575 gag gtt tct cct tct aga ate act cag ate cct tgg gtt aag get cat 1776 Glu Val Ser Pro Ser Arg He Thr Gin He Pro Trp Val Lys Ala His 580 585 590 act ctt get tct gga get tct gtt ate aag gga cct gga ttc act gga 1824 Thr Leu Ala Ser Gly Ala Ser Val He Lys Gly Pro Gly Phe Thr Gly 595 600 605 gga gat ate ctt act aga aac tct atg gga gag ctt gga act ctt aga 1872 Gly Asp He Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg 610 615 620 gtt act ttc act gga aga ctt cct cag tct tac tac ate aga ttc aga 1920 Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr He Arg Phe Arg 625 630 635 640 tac get tct gtt get aac aga tct gga act ttc aga tac tct cag cct 1968 Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (55 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 645 650 655 cct tct tac gga ate tct ttc cct aag act atg gat get gga gag cct 2016 Pro Ser Tyr Gly lie Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro 660 665 670 ctt act tct aga tct ttc get cat aca act ctt ttc act cct ate act 2064 Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro He Thr 675 680 685 ttc tct aga get cag gag gag ttc gat eta tac ate cag tct gga gtt 2112 Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr He Gin Ser Gly Val 690 695 700 tac ate gat aga ate gag ttc ate cct gtt act get act ttc gag get 2160 Tyr He Asp Arg He Glu Phe He Pro Val Thr Ala Thr Phe Glu Ala 705 710 715 720 gag tac gat tta gag aga tga 2181 Glu Tyr Asp Leu Glu Arg 725 <210> 13 <211> 726 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 13 Met Ala Ser He Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (56 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 Leu Gly Asn Val Ser Asn Gly Gly Arg lie Arg Cys Glu He Asn Asn 115 120 125 Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu He 130 135 140 He Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp He 145 150 155 160 Ser Leu Gly Leu He Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly 165 170 175 Gly Phe He Val Gly Leu Leu Glu Leu He Trp Gly Phe He Gly Pro 180 185 190 Ser Gin Trp Asp He Phe Leu Ala Gin He Glu Gin Leu He Ser Gin 195 200 205 Arg He Glu Glu Phe Ala Arg Asn Gin Ala He Ser Arg Leu Glu Gly file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (57 of 73) [3/06/2011 12:22:38 p.m.J file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 210 215 220 Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu 225 230 235 240 Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg lie Gin Phe 245 250 255 Asn Asp Met Asn Ser Ala Leu He Thr Ala He Pro Leu Phe Arg Val 260 265 270 Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn 275 280 285 Leu His Leu Ser He Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp 290 295 300 Gly Tyr Asp Thr Ala Thr He Asn Asn Arg Tyr Ser Asp Leu Thr Ser 305 310 315 320 Leu He His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly 325 330 335 Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp He Val Tyr Asn 340 345 350 Arg Phe Arg Arg Gin Leu Thr He Ser Val Leu Asp He Val Ala Phe 355 360 365 Phe Pro Asn Tyr Asp He Arg Thr Tyr Pro He Gin Thr Ala Thr Gin 370 375 380 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (58 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe lie Asn Glu Asn Leu 385 390 395 400 Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala lie 405 410 415 He Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr He Tyr 420 425 430 Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn 435 440 445 Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu He Arg Ser Pro Leu Tyr 450 455 460 Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr He Thr Ala Ser Pro 465 470 475 480 Ser Val Pro He Phe Arg Thr Leu Ser Tyr He Thr Gly Leu Asp Asn 485 490 495 Ser Asn Pro Val Ala Gly He Glu Gly Val Glu Phe Gin Asn Thr He 500 505 510 Ser Arg Ser He Tyr Arg Lys Ser Gly Pro He Asp Ser Phe Ser Glu 515 520 525 Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala He Gly Tyr Ser His 530 535 540 Arg Leu Cys His Ala Thr Phe Leu Glu Arg He Ser Gly Pro Arg He 545 550 555 560 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (59 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn 565 570 575 Glu Val Ser Pro Ser Arg lie Thr Gin He Pro Trp Val Lys Ala His 580 585 590 Thr Leu Ala Ser Gly Ala Ser Val He Lys Gly Pro Gly Phe Thr Gly 595 600 605 Gly Asp He Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg 610 615 620 Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr He Arg Phe Arg 625 630 635 640 Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro 645 650 655 Pro Ser Tyr Gly He Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro 660 665 670 Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro He Thr 675 680 685 Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr He Gin Ser Gly Val 690 695 700 Tyr He Asp Arg He Glu Phe He Pro Val Thr Ala Thr Phe Glu Ala 705 710 715 720 Glu Tyr Asp Leu Glu Arg 725 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (60 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <210> 14 <211> 1815 <212> DNA <213> Artificial <220> <223> modified sequence <220> <221> CDS <222> (1)..( 1812) <400> 14 atg get gag ate aac aac cag aac cag tgt gtt cct tac aac tgt ctt 48 Met Ala Glu lie Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu 15 10 15 tct aac cct aag gag ate ate ctt gga gag gag aga ctt gag act gga 96 Ser Asn Pro Lys Glu He He Leu Gly Glu Glu Arg Leu Glu Thr Gly 20 25 30 aac act gtt get gat ate tct ctt gga ctt ate aac ttc ctt tac tct 144 Asn Thr Val Ala Asp He Ser Leu Gly Leu He Asn Phe Leu Tyr Ser 35 40 45 aac ttc gtt cct gga ggt gga ttc ate gtt gga ctt ctt gag ctt ate 192 Asn Phe Val Pro Gly Gly Gly Phe He Val Gly Leu Leu Glu Leu He 50 55 60 tgg gga ttc ate gga cct tct cag tgg gat ate ttc ctt get cag ate 240 Trp Gly Phe He Gly Pro Ser Gin Trp Asp He Phe Leu Ala Gin He 65 70 75 80 gag cag ctt ate tct cag aga ate gag gag ttc get aga aac cag get 288 Glu Gin Leu He Ser Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95 ate tct aga ctt gag gga ctt tct aat ctt tac aaa gtt tac gtt aga 336 He Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg 100 105 110 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (61 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt get ttc tct gac tgg gag aag gat cct act aac cct get ctt aga gag 384 Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 gag atg aga ate cag ttc aac gat atg aac tct get ctt ate act get 432 Glu Met Arg lie Gin Phe Asn Asp Met Asn Ser Ala Leu He Thr Ala 130 135 140 ate cct ctt ttc aga gtt cag aac tac gag gtt get ctt ctt tct gtt 480 He Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Leu Leu Ser Val 145 150 155 160 tac gtt cag get get aac ctt cat ctt tct ate ctt aga gat gtt tct 528 Tyr Val Gin Ala Ala Asn Leu His Leu Ser He Leu Arg Asp Val Ser 165 170 175 gtt ttc gga gag aga tgg gga tac gat act get act ate aac aac aga 576 Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr He Asn Asn Arg 180 185 190 tac tct gat ctt act tct ctt ate cat gtt tac act aac cat tgt gtt 624 Tyr Ser Asp Leu Thr Ser Leu He His Val Tyr Thr Asn His Cys Val 195 200 205 gat act tac aac cag gga ctt aga aga ctt gag gga aga ttc ctt tct 672 Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser 210 215 220 gac tgg ate gtt tac aac aga ttc aga aga cag ctt act ate tct gtt 720 Asp Trp He Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr He Ser Val 225 230 235 240 ctt gat ate gtt get ttc ttc cct aac tac gat ate aga act tac cct 768 Leu Asp He Val Ala Phe Phe Pro Asn Tyr Asp He Arg Thr Tyr Pro 245 250 255 ate cag act get act cag ctt act aga gag gtt tac ctt gat ctt cct 816 He Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro 260 265 270 ttc ate aac gag aac ctt tct cct get get tct tac cct act ttc tct 864 Phe He Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (62 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt 275 280 285 get get gag tct get ate ate aga tct cct cat ctt gtt gat ttc ctt 912 Ala Ala Glu Ser Ala lie He Arg Ser Pro His Leu Val Asp Phe Leu 290 295 300 aac tct ttc act ate tac act gat tct ctt get aga tac get tac tgg 960 Asn Ser Phe Thr He Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp 305 310 315 320 gga gga cat ctt gtt aac tct ttc aga act gga act aca act aac ctt 1008 Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu 325 330 335 ate aga tct cct ctt tac gga aga gag gga aac act gag aga cct gtt 1056 He Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val 340 345 350 act ate act get tct cct tct gtt cct ate ttc aga act ctt tct tac 1104 Thr He Thr Ala Ser Pro Ser Val Pro He Phe Arg Thr Leu Ser Tyr 355 360 365 ate act gga ctt gat aac tct aac cct gtt get gga ate gag gga gtt 1152 He Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly He Glu Gly Val 370 375 380 gag ttc cag aac act ate tct aga tct ate tac aga aag tct gga cct 1200 Glu Phe Gin Asn Thr He Ser Arg Ser He Tyr Arg Lys Ser Gly Pro 385 390 395 400 ate gat tct ttc tct gag ctt cct cct cag gat get tct gtt tct cct 1248 He Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro 405 410 415 get ate gga tac tct cat aga ctt tgt cat get act ttc ctt gag aga 1296 Ala He Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg 420 425 430 ate tct gga cct aga ate get gga act gtt ttc tct tgg act cat aga 1344 He Ser Gly Pro Arg He Ala Gly Thr Val Phe Ser Trp Thr His Arg 435 440 445 tct get tct cct act aac gag gtt tct cct tct aga ate act cag ate 1392 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (63 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg lie Thr Gin He 450 455 460 cct tgg gtt aag get cat act ctt get tct gga get tct gtt ate aag 1440 Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val He Lys 465 470 475 480 gga cct gga ttc act gga gga gat ate ctt act aga aac tct atg gga 1488 Gly Pro Gly Phe Thr Gly Gly Asp He Leu Thr Arg Asn Ser Met Gly 485 490 495 gag ctt gga act ctt aga gtt act ttc act gga aga ctt cct cag tct 1536 Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gin Ser 500 505 510 tac tac ate aga ttc aga tac get tct gtt get aac aga tct gga act 1584 Tyr Tyr He Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr 515 520 525 ttc aga tac tct cag cct cct tct tac gga ate tct ttc cct aag act 1632 Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly He Ser Phe Pro Lys Thr 530 535 540 atg gat get gga gag cct ctt act tct aga tct ttc get cat aca act 1680 Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr 545 550 555 560 ctt ttc act cct ate act ttc tct aga get cag gag gag ttc gat eta 1728 Leu Phe Thr Pro He Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu 565 570 575 tac ate cag tct gga gtt tac ate gat aga ate gag ttc ate cct gtt 1776 Tyr He Gin Ser Gly Val Tyr He Asp Arg He Glu Phe He Pro Val 580 585 590 act get act ttc gag get gag tac gat tta gag aga tga 1815 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 595 600 <210> 15 <211> 604 <212> PRT file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (64 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <213> Artificial <220> <223> Synthetic Construct <400> 15 Met Ala Glu lie Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu 15 10 15 Ser Asn Pro Lys Glu He He Leu Gly Glu Glu Arg Leu Glu Thr Gly 20 25 30 Asn Thr Val Ala Asp He Ser Leu Gly Leu He Asn Phe Leu Tyr Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe He Val Gly Leu Leu Glu Leu He 50 55 60 Trp Gly Phe He Gly Pro Ser Gin Trp Asp He Phe Leu Ala Gin He 65 70 75 80 Glu Gin Leu He Ser Gin Arg He Glu Glu Phe Ala Arg Asn Gin Ala 85 90 95 He Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg 100 105 110 Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu Met Arg He Gin Phe Asn Asp Met Asn Ser Ala Leu He Thr Ala 130 135 140 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (65 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt lie Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Leu Leu Ser Val 145 150 155 160 Tyr Val Gin Ala Ala Asn Leu His Leu Ser He Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr He Asn Asn Arg 180 185 190 Tyr Ser Asp Leu Thr Ser Leu He His Val Tyr Thr Asn His Cys Val 195 200 205 Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser 210 215 220 Asp Trp He Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr He Ser Val 225 230 235 240 Leu Asp He Val Ala Phe Phe Pro Asn Tyr Asp He Arg Thr Tyr Pro 245 250 255 He Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro 260 265 270 Phe He Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser 275 280 285 Ala Ala Glu Ser Ala He He Arg Ser Pro His Leu Val Asp Phe Leu 290 295 300 Asn Ser Phe Thr He Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp 305 310 315 320 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (66 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu 325 330 335 lie Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val 340 345 350 Thr He Thr Ala Ser Pro Ser Val Pro He Phe Arg Thr Leu Ser Tyr 355 360 365 He Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly He Glu Gly Val 370 375 380 Glu Phe Gin Asn Thr He Ser Arg Ser He Tyr Arg Lys Ser Gly Pro 385 390 395 400 He Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro 405 410 415 Ala He Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg 420 425 430 He Ser Gly Pro Arg He Ala Gly Thr Val Phe Ser Trp Thr His Arg 435 440 445 Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg He Thr Gin He 450 455 460 Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val He Lys 465 470 475 480 Gly Pro Gly Phe Thr Gly Gly Asp He Leu Thr Arg Asn Ser Met Gly 485 490 495 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (67 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gin Ser 500 505 510 Tyr Tyr lie Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr 515 520 525 Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly He Ser Phe Pro Lys Thr 530 535 540 Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr 545 550 555 560 Leu Phe Thr Pro He Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu 565 570 575 Tyr lie Gin Ser Gly Val Tyr He Asp Arg He Glu Phe He Pro Val 580 585 590 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg 595 600 <210> 16 <211> 372 <212> DNA <213> Artificial <220> <223> modified transit peptide <220> <221> CDS <222> (1)..(372) file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (68 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <400> 16 atg get tct ate tct tct tct gtt get act gtt tct aga act get cct 48 Met Ala Ser lie Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 get cag get aac atg gtt get cct ttc act gga ctt aag tct aac get 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 get ttc cct act act aag aag get aac gat ttc tct act ctt cct tct 144 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 aac gga gga aga gtt cag tgt atg cag gtt tgg cct get tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg get cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 act gtt atg atg get tct tct get act get gtt get cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 ctt aag tct act get tct ctt cct gtt get aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 ctt gga aac gtt tct aac gga gga aga ate aga tgt 372 Leu Gly Asn Val Ser Asn Gly Gly Arg He Arg Cys 115 120 <210> 17 <211> 124 <212> PRT <213> Artificial <220> <223> Synthetic Construct <400> 17 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (69 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt Met Ala Ser lie Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro 15 10 15 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala 20 25 30 Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser 35 40 45 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn 50 55 60 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro 65 70 75 80 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly 85 90 95 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser 100 105 110 Leu Gly Asn Val Ser Asn Gly Gly Arg He Arg Cys 115 120 <210> 18 <211> 988 <212> DNA <213> Subterranean clover stunt virus <400> 18 ctagataatt gttattatca ataaaagaat ttttattgtt attgtgttat ttggtaattt 60 atgcttataa gtaattctat gattaattgt gaattaataa gactaatgag gataataatt 120 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (70 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt gaatttgatt aaattaactc tgcgaagcca tatgtctttc acgtgagagt cacgtgatgt 180 ctccgcgaca ggctggcacg gggcttagta ttacccccgt gccgggatca gagacatttg 240 actaaatgtt gacttggaat aatagccctt ggattagatg acacgtggac gctcaggatc 300 tgtgatgcta gtgaagcgct taagctgaac gaatctgacg gaagagcgga caaacgcaca 360 tggactatgg cccactgctt tattaaagaa gtgaatgaca gctgtctttg cttcaagacg 420 aagtaaagaa tagtggaaaa cgcgttaatt gttattatca ataaaagaat ttttattgtt 480 attgtgttat ttggtaattt atgcttataa gtaattctat gattaattgt gaattaataa 540 gactaatgag gataataatt gaatttgatt aaattaactc tgcgaagcta tatgtctttc 600 acgtgagagt cacgtgatgt ctccgcgaca ggctggcacg gggcttagta ttacccccgt 660 gccgggatca gagacatttg actaaatgtt gacttggaat aatagccctt ggattagatg 720 acacgtggac gctcaggatc tgtgatgcta gtgaagcgct taagctgaac gaatctgacg 780 gaagagcgga caaacgcaca tggactatgg cccactgctt tattaaagaa gtgaatgaca 840 gctgtctttg cttcaagacg aagtaaagaa tagtggaaaa cgcgtaaaga ataagcgtac 900 tcagtacgct tcgtggcttt ataaatagtg cttcgtctta ttcttcgttg tatcatcaac 960 gaagaagtta agctttgttc tgcgtttc 988 <210> 19 <211> 1042 <212> DNA <213> Subterranean clover stunt virus <400> 19 taattaatag taattatgat taattatgag ataagagttg ttattaatgc ttatgaggaa 60 taaagaatga ttaatattgt ttaattttat tccgcgaagc ggtgtgttat gtttttgttg 120 gagacatcac gtgactctca cgtgatgtct ccgcgacagg ctggcacggg gcttagtatt 180 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (71 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt acccccgtgc cgggatcaga gacatttgac taaatattga cttggaataa tagcccttgg 240 attagatgac acgtggacgc tcaggatctg tgatgctagt gaagcgctta agctgaacga 300 atctgacgga agagcggaca tacgcacatg gattatggcc cacatgtcta aagtgtatct 360 ctttacagct atatcgatgt gacgtaagat gctttacttc gcttcgaagt aaagtaggaa 420 attgctcgct aagttattct tttctgaaag aaattaattt aattctaatt aaattaaatg 480 agtggcctgc agtaattaat agtaattatg attaattatg agataagagt tgttattaat 540 gcttatgagg aataaagaat gattaatatt gtttaatttt attccgcgaa gcggtgtgtt 600 atgtttttgt tggagacatc acgtgactct cacgtgatgt ctccgcgaca ggctggcacg 660 gggcttagta ttacccccgt gccgggatca gagacatttg actaaatatt gacttggaat 720 aatagccctt ggattagatg acacgtggac gctcaggatc tgtgatgcta gtgaagcgct 780 taagctgaac gaatctgacg gaagagcgga catacgcaca tggattatgg cccacatgtc 840 taaagtgtat ctctttacag ctatatcgat gtgacgtaag atgctttact tcgcttcgaa 900 gtaaagtagg aaattgctcg ctaagttatt cttttctgaa agaaattaat ttaattctaa 960 attaaattaa atgagtggct ataaatagtg tcgatgctac ctcacatcgt attcttcttc 1020 gcatcgtctg ttctggtttt aa 1042 <210> 20 <211> 28 <212> DNA <213> Artificial <220> <223> primer <400> 20 tacttcgaac agaaagaacg agaacgag 28 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (72 of 73) [3/06/2011 12:22:38 p.m.] file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt <210> 21 <211> 24 <212> DNA <213> Artificial <220> <223> primer <400> 21 gtccagcgaa aggaactcca agaa 24 <210> 22 <211> 21 <212> DNA <213> Artificial <220> <223> primer <400> 22 aaccttgagg gacttggaaa c 21 <210> 23 <211> 23 <212> DNA <213> Artificial <220> <223> primer <400> 23 aagatgaggg tttctgatag cag 23 55 file:////wd.govt.nz/dfs/personal/homedrive/wne/templep/571952.txt (73 of 73) [3/06/2011 12:22:38 p.m.]