CA2924415A1 - Novel genes encoding insecticidal proteins - Google Patents

Abstract

The present invention relates to novel gene sequences encoding insecticidal proteins produced by Bacillus thuringiensis strains. Particularly, new chimeric genes encoding a Cry1C, Cry1B or Cry1D 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 one of such chimeric gene and at least one other of such chimeric genes.

Description

v75749-47D1 Novel genes encoding insecticidal proteins.
This application is a division of Canadian Application Serial No. 2,646,471 filed March 16, 2007 (parent application).
It should be understood that the expression "the present invention" or the like used in this specification encompasses not only the subject matter of this divisional application, but that of the parent application also.
INTRODUCTION
The present invention relates to new gene sequences encoding insecticidal proteins produced by Bacillus thuringiensis strains. Particularly, new chimeric genes encoding a Cry1C 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 cry1C chimeric gene and at least one other gene encoding an insecticidal protein, such as new gene sequences encoding a Cry1B or Cry1D 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 centiiry 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 Cry1Ac or a Cry1C Bt toxin, as well as crosses between these plants so that both the Cry1Ac and Cry1C toxins are expressed in the same plants, but these plants have not been commercialized. NewLeaf T"
potatoes comprising a Cry3A Coleopteran-active gene were briefly commercialized in Northern America, but have been withdrawn from the market in 2001.

7 5 -74 9 ¨ 4 7 , =

2 The current invention provides new genes encoding proteins of the Cry1C type of Bt proteins, which ideally are combiried with genes encoding proteins Of the Cry18 or Cry1D type Bt prOteins.
The DNA seqUericeb=Of the cry1C, cry1B or cry1 D 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.
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
plantorops, 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. Bressice species plants are protected from insects by-the new genes of the current Invention: B.
carinata, B.
elongate, B. fruticulosa, B. juncea, B. napus, B. narinosai B: nigra, B.
oleracea, B.
perviridis,B, raps, B. rUpestris, B. septieeps, 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_ottechnologles 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 ' WO 2007/107302

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 cry1C and a cry1B gene, a cry1C and a cry1D gene, or a combination of a cry1C, a cry1B and a cry1D 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 cry1B and cry1D genes, particularly cry1B or cryl D 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 Cry1C 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 y

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 Cryl C 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 El 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 Cry 1 D 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, WO 2007/107302 =PCT/EP2007/002342 the second chimeric gene in these DNAs further comprises the leader sequence of the tapetum specific El 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 Cryl D 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 n ' ' 75749-47D1 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 tube rosum.
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.
The current invention as claimed relates to:
- a chimeric nucleic acid comprising the following operably-linked sequences: a) a first fragment of a Bacillus thuringiensis coding sequence encoding an insecticidal protein, b) a plant intron sequence, c) a second fragment of said Bacillus thuringiensis coding sequence, d) a promoter region capable of directing expression in plant cells, wherein no insecticidal protein can be produced from such gene in a given host cell wherein the intron is not spliced, and wherein said intron is effectively spliced in plant cells, wherein said effective splicing can be measured using the detection of a functional protein produced in plant cells and wherein the plant intron sequence is positioned between the first and second fragment;
- a transformed plant cell selected from the group consisting of: corn, cotton, watercress, horseradish, wasabi, arugula, cress, radish, canola, soybean, vegetable plants, Cruciferae plant species, cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale, broccoli, Brussels sprouts, and mustard spinach, comprising the chimeric nucleic acid as described herein; and - a transgenic plant cell, comprising the chimeric nucleic acid as described herein stably incorporated in its genome.

, 75749-47D1 6a 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, co/1BI, cry1B2, cry1D1, and cry1D2, and their encoded proteins are designated herein as Cry1C (e.g., Cry1C1, Cry1C3, and Cry1C4), Cry1B (e.g., Cry1B1 and Cry1B2) and Cry1D (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 "Cry1C 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 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.
(also named Cry1C3 protein herein) or SEQ ID No. 7 (also named Cry1C4 protein herein).
A Cry1C 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 Cry1C
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 Cry1C-based protein variants, hybrids or mutants retaining substantially the same insecticidal activity as that of the Cry1C protein of the invention defined above.

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, "Cry1D 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 Cryl D
protein , = 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, "Cry1B 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

Cry1B 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 /0, 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 Cry1B 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 Cry1C, Cry1B or Cry1D protein, respectively, as defined above.
This includes naturally occurring, artificial or synthetic DNA sequences encoding the Cry1C, Cry1B or Cry1D 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 ,vhich are similar enough to any one of the DNA sequences of SEQ ID No. 1, 3, 4, 6, 8, 10, 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 radio-labeled 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 %

= CA 02924415 2016-03-21 s 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 cry1C, cry1B or ryylD DNA of this invention are n DNA encoding the insecticidal Cryl C, Cry1B
or Cry1D
= protein variants described above.
Also included herein as a Cry1C 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, or f) a DNA
.=
comprising the nucleotide sequence of SEQ ID No. 6 from nucleotide position 7 to nucleotide position 2439.
Also included herein as a Cry1D 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 Cry1B 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 cryl C, 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-, .
==
:.1 . =
e WO 2007/107302 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 Cry1D DNA, a fragment of the prior art sequence (of equal length) was selected to secure optimal alignment, but even then only 72.5 `)/0 sequence identity was the closest sequence identity with any known DNA
sequence listed in the available databases.
Hence, also included herein as cry1C, cry1B or cry1D genes are DNA sequences encoding an insecticidal protein with at least 80 %, 90 %, preferably at least 93 to 97 %, pnrticulnrly f Q8 % rnt Ic.st 99 hi irmcvi irientity trI
nny nnP thP r-nriing 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 Cry1C, Cry1B or Cry1D
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., ' WO 2007/107302 PCT/EP2007/002342 the exemplified transgenic Brassica cauliflower or cabbage plant cells or plants of the invention comprising the cry1C and/or cryl E3 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 = WO 2007/107302 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 "Cry1 protein/DNA" or "Cry protein/DNA of this invention", as used herein, refer to any one of the Cry1C, Cry1B, or Cry1D proteins or any one of the cry1C, cry1B
or cry1D DNA sequences as defined herein. A Cry or Cry1 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 cry1C, cry1B or cry1D genes of the invention.
The DNA sequences encoding the Cry1 proteins of the invention can be chemically synthesized using routine techniques, and can be inserted in expression vectors to produce high amounts of Cry1 proteins. The Cryl proteins can be used to prepare specific monoclonal or polyclonal antibodies in a conventional manner (1-16fte 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 cry1 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) cry1 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 cry1 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 cry1 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 cry1 gene of the invention, or both recognizing specifically the cry1 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 cry1 chimeric gene of the invention, so that a specific fragment ("integration fragment" or discriminating amplicon) is amplified from a nucleic acid sample comprising a cry1 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 cry1C, =

cry1B or cry1D 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 cry1 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 cry1 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 cry1 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 cry1 gene of the invention and the associated transit peptide sequence or the regulatory elements in the cry1 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.

= CA 02924415 2016-03-21 =

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 cry1 = 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 cry1 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 oryl 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 cry1 genes therein. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer, label) for identification of the cry1 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 cry1 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.

$ = WO 2007/107302 Examples of suitable primer combinations in accordance with the invention are (sequence 5' - 3') for the cry1B gene of the invention: P1B227 (TAC TTC GAA
CAG AM
GM CGA GM CGA G, SEQ ID No. 20) and P1B228 (GTC CAG CGA AAG GM CTC
CAA GAA, SEQ ID No. 21), and for the cryl C gene of the invention: P1C247 (MC
CTT
GAG GGA CU GGA MC, SEQ ID No. 22) and P1C252 (MG ATG AGG GTT TCT
GAT AGC AG, SEQ ID No. 23). Hence, any gene encoding an insecticidal Cry1B or Cry1C 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 cry1C, cry1B or cry1D 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 cry1 DNA sequence of the invention, particularly any such markers, primers or labeled probes do not detect or recognize any plant expressing a Cryl C, Cry1D or Cry1B
protein wherein such plant does not contain a DNA sequence of the invention (such as a cry1C, cry1D or cry1B 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 Cry1 protoxin can be removed or be replaced by another amino acid sequence without affecting the insecticidal activity of the Cry1 proteins of the invention).
Equivalents of the DNA sequences of the invention include DNA sequences with less f =

than 20, preferably 5-10, nucleotide differences compared to the cry1 genes of this invention as defined herein, but which encode an insecticidal Cry1 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 cry1 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, update on the Bt nomenclature website corresponding to the Crickmore et at, (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 cry1C DNA of SEQ ID No. 1 or 3, 73% for the cry1B DNA of SEQ
ID No. 10, and 72.5 % for the cry1D 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.

By an "insecticidally effective part (portion or fragment)" of DNA sequences encoding a Cry1 protein, also referred to herein as "truncated gene" or "truncated DNA", is meant a DNA sequence encoding a poiypeptide 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. colt, 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 cry1 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 cry1 genes of the = invention, preferably in the coding sequence of at least one of the cry1 genes of the invention. Any of the known plant introns (e.g., Brown, 1986, Brown and Simpson, 1998, = Brown et a)., 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 Solanum 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 = WO 2007/107302 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 oferaceae 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 at., 1986).

= ' . 7574947 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 Kletsgen et at. (1989), KlOsgen and Well (1991), Neuhaus & Rogers (1998), Bih et at. (1999), Morris et at. (1999), Hesse et al. (1989), Tavladoraki et al. (1998), Terashima et at. (1999), Park et al. (1997), Shcherban et at. (1995), 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 Cry1 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 cry1 genes herein, to prevent or delay the development of insect resistance to a plant expressing insecticidal toxins. Because of the characteristics of the new cry1 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 cry1C 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 Cry1C protein in a target insect.
Preferred , = = , WO 2007/107302 insect control proteins to combine with the Cry1C proteins of this invention, particularly for simultaneous expression in plants, preferably Brassica species plants, particularly cabbage and cauliflower, include the Cry1B protein of this invention or the Cry1D 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 cry1 gene of this invention, or by crossing plants transformed with the insect control protein and plants transformed with the cry1 gene of this invention. For Brassica species plants, preferably the cry1 gene is used as first gene and as second gene the Cry1B, Cry1D 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 cry IC
gene of the invention is located in one and the same locus as a second insect control gene, such as a Cry1B or Cry1D 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 or glyphosate.
The insecticidally effective cry1 gene, preferably the cry1 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-plastinid vectors each contain the insecticidally effective cry gene part between the border sequences, or CL least IGGCLGA to the Iaff of the right border OGMUGUnG, of the T_MAA of the Ti _ iC Jv 1%.
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

7.
=
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 cry1C 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 cry1 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 cell (herein named a "plant-expressible promoter"). This is preferably accomplished by inserting the cry1 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 promoter described by Odell et al. (1985), promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen etal., 1992, see also Cornejo et at., 1993), the gos2 promoter (de Pater et at., 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 at. (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 at.
(1995) or Schunmann et at. (2003), an alcohol dehydrogenase promoter, e.g., pAdh1S
(GenBank accession numbers X04049, X00581), and the TR1' promoter and the TR2' promoter (the "TR1' 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 at. (1994), or the Agrobacterium TR2' or mannopine synthase promoter (Velten et at., 1984) or a promoter inducible by chemical factors.

= CA 02924415 2016-03-21 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 cry1 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. elongate, B. fruticulosa, B. juncea, B.
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 cry1B and cry1C 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 Brassicaceee plants such as B. napus, B.
rape, B.
juncea or B. carinata, that contain the gene or genes of the invention, such as the cry1B
and cry1C genes of the invention, from crossings with a transformed B.
oleracea plant or , WO 2007/107302 the progeny thereof, or to B. oleracea plants that contain the gene or genes of the invention, such as the cry1B and cry1C 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 Cry1 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 (DuImage, 1981; Bernhard and Utz, 1993) to provide high yields of cells. Under appropriate conditions which are well understood (DuImage, 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 = CA 02924415 2016-03-21 =

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 cry1 genes or Cry1 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 bra ssicae, Manduca sexta, Choristoneura fumiferana, Choristoneura occidentalis, Choristoneura rosaceana, Pandemis pyrusana, Platynota stultana, Lymantria dispar, Orgyia leucostigma, Malacosoma disstria, Lambina fiscellaria, Chile suppressalis, Chile 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 Cry1C, Cryl B and Cryl D 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 cryi G genes of the invention in a field, or by securing the presence of a Cry1C 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 Cry1C
protein of this invention). The invention also relates to the use of the cry1 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 cry1B or cry1D
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 SEQ ID No.2: amino acid sequence of the Cry1C1 protein encoded by SEQ ID No. 1 . CA 02924415 2016-03-21 , = = , 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 S'EQ ID 40 .Q. optimized cry 1C4 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 cryl B1 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: Cryl B2 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: cry1B gene primer P1B227 SEQ IDr:Io. 21: cry1B gene primer P1 B228 SEQ ID No. 22: cry1C gene primer P1C247 SEQ ID No. 23: cry1C 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.

The cry1C1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 1. This DNA encodes the insecticidal Cryl Cl protein of the invention (SEQ ID No. 2). For transformation of plants, a first chimeric gene (the cryl CI 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 El gene (GE1) of Oryza sativa (Michiels et al., 1992), the cryl Cl 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 Haveria bidentis (3' Mel, Marshall et al., 1996).
A similar cryl C chimeric gene was made, wherein the ST-LS1 intron 2 is at position 489 of the cryl C DNA (i.e., the cryl C2 DNA), this is the cryl C2 chimeric gene, otherwise constructed exactly like the cryl Cl chimeric gene.
To secure targeting of the Cryl C protein to the plant cell chloroplast, variants of the cryl Cl and cryl C2 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 cryl C coding region so that a transit peptide fusion =
protein is expressed in plant cells. These are the cryl C3 and cryl C4 chimeric genes, comprising the cryl C3 and cryl C4 coding sequences, respectively, which each contain the sequence of the modified chloroplast transit peptide of SEQ ID No.16. The cryl C3 DNA sequence is shown in SEQ ID No. 4, it is a fusion of the cryl Cl sequence of SEQ
ID No. 1 with the transit peptide coding sequence of SEQ ID No. 16. The cryl sequence is shown in SEQ ID No. 6, it is a fusion of the cryl C2 sequence of SEQ ID No.
3 with the transit peptide coding sequence of SEQ ID No. 16.
The cryl B1 DNA which was designed for optimal expression in plant cells is represented in SEQ ID No. 8. This DNA encodes the insecticidal Cryl B1 protein of the invention (SEQ ID No. 9). For transformation of plants, a chimeric gene (the cryl 61 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 at., 1995, SEQ
ID No.

= 33 19), the leader sequence of the El gene (GE1) of Otyza sativa (Michiels et al., 1992), the cryl B1 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' Mel, Marshall et al., 1996).
A second form of the cry 1B chimeric gene was also made, using the cryl B2 DNA
(SEQ
ID No. 10), wherein no sequence encoding an optimized transit peptide is contained, so that cytoplasmic accumulation of the Cryl B protein occurs in plant cells.
This is the Cryl B2 chimeric gene.
The cryl D1 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 cryl D1 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 El gene (GE1) of Oryza sat iva (Michiels et al., 1992), the cryl D1 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' Mel, Marshall et al., 1996).
A second form of the cryl D chimeric gene was also made, using the cryl D2 DNA, wherein no sequence encoding an optimized transit peptide is contained, so that cytoplasmic accumulation of the C.ryl n protein occurs in plant cells. This is the rryi n9 chimeric gene.
= A DNA transformation vector (pT1C4B1) is made comprising between the T-DNA
borders the cry1C4 chimeric gene and the cryl B1 chimeric gene in a head-to-tail orientation (3'Mel-cry1C4-GE1 leader-S7S7 ¨ 5454-GE1 leader-cry1B1-31viel), as well as a transfer vector (pT1C2B2) comprising between the T-DNA borders the cryl = chimeric gene and the cryl B2 chimeric gene in a head-to-tail orientation (3'Mel-cryl C2-GE1 leader-S7S7 ¨ S4S4-GE1 leader-cry1B2-3'Mel). In such manner, with both T-DNA
vectors, the cryl C and cryl B genes of the invention will be co-transferred to the plant cell and will be located at one locus after successful transformation.

. WO 2007/107302 PCT/EP2007/002342 Similar T-DNA vectors are constructed which contain the above cry1C chimeric genes but which contain as second chimeric gene the cry1D1 or cry1D2 chimeric genes instead of the above cry1B chimeric genes. Also a triple cry gene transformation vector is constructed, comprising both the cryl C, crylD and cry1B 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 coil 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 coil 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 cry1C genes but having the ST-LS1 intron at a position 489). Also these vectors contain the cry1B1 or cry1B2 chimeric genes, or the cry1D1 or cry1D2 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 transf"rm,tirµn regenerntiNI.
The above transformation vectors pT1C4B1 and pT1C2B2 containing the cry1C 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/I
silver = nitrate and 250 mg/I of both Triacillin and Carbenicillin and 10 mg/I
phosphinothricin for selection of transformation events.
= Every two weeks explants are transferred to fresh medium. Every week explants are checked for callus formation. Celli 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 cry1C and cry1B 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).

= WO 2007/107302 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 Cry1C-and Cry1B-specific ELISA assays or Western blots are used to select those transformed plants showing optimal expression levels of the Cry1C and Cry1B proteins.
RT-PCR experiments on RNA collected from cauliflower plants shown to be transformed with the cry1C 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 Cry1C
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 Cry1C and Cry1B 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 Cry1C or Cry1B 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 cry1 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) cry1B and cry1C genes of the invention in different cabbage, cauliflower or oilseed rape genetic backgrounds, optimally adapted to different areas or climatic conditions.

= CA 02924415 2016-03-21 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 thuringinsi.õAn 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).
Bih et al. (1999) J. Biol. Chem. 274, 22884-22894.
Boevink et al. (1995) Virology, 207, 354-361.
Bolivar et al. (1977) Gene, 2: 95-113.
Bradford et al. (1976) Anal. Biochem. 72, 248-254.
Brown (1986) Nucleic Acids Res. 1986 14, 9549-9559.
Brown and Simpson (1998) Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 77-95.
Brown et at (1996) Plant Mol Biol. 32, 531-535.
Christensen et al. (1992) Plant Mol. Biol. 18, 675-689.
Christou et al. (1990). Trends Biotechnology 8, 145.
Cordera et al. (1994) The Plant Journal 6, 141.
=
Cornejo et al. (1993) Plant Mol. Biol. 23, 567-581.
Cornelissen & Vandewiele (1989) Nucleic Acids Research, 17: 19-25.
Cornelissen et al. (1986) EMBO J. 5, 37-40.
Crickmore et al. (1998) Microbiol. Mol. Biol Rev. 62(3), 807-13.
natta et al. (1990) r,iofTechnology 8, 736-740.
De Block et al. (1989) Plant Physiol., 91: 694.
De Pater et al., 1992, Plant J. 2, 834-844.
Depicker et al. (1982) Journal of Molecular and Applied Genetics, 1: 561-573.
DuImage (1981), "Production of Bacteria for Biological Control of insects" in Biological Control in Crop Production, Ed. Papanzas, D.C., Osmun Publishers, Totowa, N.J., USA, pp. 129-141 (1981).
Eckes et al. (1986) Molecular and General Genetics, 205: 14-22.
Estruch et al., (1996), Proc Natl Acad Sci USA 93, 5389-94.
Ffrench-Constant and Bowen (2000) Cell Mol Life Sci 57, 828-33.
Franck et al. (1980) Cell 21, 285-294.
Fromm et al. (1990) BiofTechnology 8, 833-839.

Gardner et al. (1981) Nucleic Acids Research 9, 2871-2887.
Gielen et al. (1984) EMBO J 3, 835-845.
Gordon-Kamm et al. (1990) The Plant Cell 2, 603-618.
Hesse et al. (1989), EMBO J. 8 2453-2461.
Hinchee et al. (1988) BiofTechnology 6, 915.
Ho et al. (1989). Gene 77, 51-59.
Hate et al. (1988) Appl. and Environm. Microbiol. 54, 2010-2017.
Hate and Whiteley (1989) Microbiological Review 53, 242-255.
Hull and Howell (1987) Virology 86, 482-493.
ltoh et al. (1984) Plasmid, 11: 206-220.
Jansens et al. (1997) Crop Science 37, 1616-1624.
Keil et al. (1986), Nucl. Acids Res. 14, 5641-5650.
KlOsgen et al. (1989), Mol. Gen. Genet. 217, 155-161.
Klosgen and Weil (1991), Mol. Gen. Genet. 225, 297-304.
Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1840-1845.
Last et al. (1990) Theor. Appl. Genet. 81, 581-588.
Lebrun et at. (1996) US Patent 5,510,471.
Lin et al. (2003) Bot. Bull. Acad. Sin. 44:199-210.
Mahillon et al, FEMS Microbiol. Letters 60, 205-210 (1989).
Marshall et al. (1996) Plant Physiology, 111: 1251-1261.
Michiels et al. (1992) published PCT application W092/13956.
Morris et al. (1999), Biochem. Biophys. Res. Commun. 255, 328-333.
Needleman and Wunsch (1970) J. Mol. Biol., 48: 443-53.
Neuhaus & Rogers (1998), Plant Mol. Biol. 38, 127-144.
Odell et al. (1985) Nature, 313: 810-812.
Oelmuller et al., Mol. Gen. Genet. 237, 261-272 (1993).
Oka et al. (1981) Journal of Molecular Biology, 147: 217-226.
Park et al. (1997), J. Biol. Chem. 272, 6876-6881.
Rice et al. (2000) Trends in Genetics, 16: 276-277.
SchOnmann et al. (2003) Functional Plant Biology 30, 453 ¨ 460.
Shcherban et at. (1995) Proc. Natl. Acad. Sci USA 92, 9245-9249.
Shimamoto et al. (1989) Nature 338, 274-276.
Stanssens et al. (1989), Nucleic Acids Research 12, 4441-4454.
Sutliff et al. (1991) Plant Molec. Biol. 16, 579-591.

Tavladoraki et at. (1998), FEBS Lett. 426, 62-66.
Terashima et at. (1999), Appl. Microbial. Biotechnol. 52, 516-523.
Thompson et at. (1987) The EMBO Journal, 6: 2519-2523.
Van Den Broeck at at., 1985, Nature 313, 358.
Van Rie et at. (1990) Science 247, 72.
Velten et at. (1984) J., EMBO J 3,2723-2730.
Velten and Schell (1985) Nucleic Acids Research 13, 6981-6998.
Verdaguer at at. (1998) Plant Mal. Biol. 37, 1055-1067.
VVaterfield et at. (2001) Trends Microbiol 9, 185-91.
White at at. (1989)Trends in Genet. 5, 185-189.
Wong et at. (1992) Plant Malec. Bio1.20, 81-93.
Zambryski (1988) Annual Review of Genetics, 22: .1-30.
Zhang et at. (1991) The Plant Cell 3,1155-1165.
Zhao at at. (2003) Nature Biotechnology, 21: 1493¨ 1497.
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.

SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII text format (file: 75749-47D1 Seq 16-MAR-16 v1.txt).
A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in the following table.
SEQUENCE TABLE
<110> Bayer CropScience NV
<120> Novel genes encoding insecticidal proteins <130> 75749-47D1 <140> Division of CA 2,646,471 <141> 2007-03-16 <160> 23 <170> PatentIn version 3.3 <210> 1 <211> 2076 <212> DNA
<213> Artificial Sequence <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 gct gag gag aac aac cag aac cag tgt atc cct tac aac tgt ctt 48 Met Ala Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu tog aac cct gag gag gtt ctt ctt gat gga gag aga atc tot act gga 96 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly aac tot tot atc gat att tot ctt tot ctt gtt cag ttc ctt gtt tot 144 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt atc gat ttc gtt 192 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val tgg gga atc gtt gga cct tot cag tgg gat got ttc ctt gtt cag atc 240 Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile gag cag ctt atc aac gag aga atc got gag ttc got aga aac got got 288 Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala atc got aac ctt gag gga ctt gga aac aac ttc aac atc tac gtt gag 336 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu got 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 aga gtt atc gat aga ttc aga atc ctt gat gga ctt ctt gag aga gat 432 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp att cct tot ttc aga atc tot gga ttc gag gtt cct ctt ctt tot gtt 480 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val tac got cag got got aac ctt cat ctt got atc ctt aga gat tot gtt 528 Tyr Ala Gin Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val atc ttc gga gag aga tgg gga ctt act act atc aac gtt aac gag aac 576 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn tac aac aga ctt atc aga cat atc gat gag tac got gat cat tgt got 624 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala , , 42 aac act tac aac aga gga ctt aac aac ctt cct aag tot act tac cag Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin gtaagtttct gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcag gac tgg atc act tac aac aga ctt aga aga gat ctt act ctt Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu act gtt ctt gat att got got ttc ttc cot aac tac gat aac aga aga Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg tac cot atc cag cot gtt gga cag ctt act aga gag gtt tac act gat Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp cot ctt atc aac ttc aac cot cag ctt cag tot gtt got cag ctt cot Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro act ttc aac gtt atg gag tot tot got atc aga aac cot cat ctt ttc Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe gat att Ott aac aac ctt act atc ttc act gac tgg ttc tot gtt gga Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly aga aac ttc tac tgg gga gga cat aga gtt atc tot tot ctt atc gga Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly gga gga aac atc act tot cot atc tac gga aga gag got aac cag gag Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu cot cot aga tot ttc act ttc aac gga cot gtt ttc aga act ctt tot Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser aac cot act ctt aga ctt ctt cag cag cot tgg cot got cct cct ttc Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe aac ctt aga gga gtt gag gga gtt gag ttc tot act cot act aac tot Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser ttc act tac aga gga aga gga act gtt gat tot ctt act gag ctt cot Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro cot gag gat aac tct gtt cot cot 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 tgt cat got 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 gga gtt gtt ttc tct tgg act cat aga tct got act ctt act aac act 1575 Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr atc gat cot gag agg atc aac cag atc cot ctt gtt aag gga ttc aga 1623 Ile Asp Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg gtt tgg gga gga act tct gtt atc act gga cct gga ttc act gga gga 1671 Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly gat att ctt aga aga aac act ttc gga gat ttc gtt tct ctt cag gtt 1719 Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val aac atc aac tct cot atc act cag aga tac aga ctt aga ttc aga tac 1767 Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr got tct tct aga gat got aga gtt atc gtt ctt act gga got got tct 1815 Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser act gga gtt gga gga cag gtt tct gtt aac atg cot ctt cag aag act 1863 Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr atg gag atc gga gag aac ctt act tct aga act ttc aga tac act gat 1911 Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp ttc tct aac cot ttc tct ttc aga got aac cot gat att atc gga atc 1959 Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile tct gag cag cct ctt ttc gga gct gga tct atc tct tct gga gag ctt 2007 Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu tac atc gat aaa atc gag atc atc ctt got gat got act ttc gag got 2055 Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala gag tct gat tta gag aga tga 2076 Glu Ser Asp Leu Glu Arg <210> 2 <211> 628 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 2 Met Ala Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg <210> 3 <211> 2076 <212> DNA
<213> Artificial Sequence <220>
<223> modified sequence <220>
<221> exon <222> (1)..(489) <220>
<221> Intron <222> (490)..(678) <220>
<221> exon <222> (679)..(2073) <400> 3 atg got gag gag aac aac cag aac cag tgt atc cct tac aac tgt ctt 48 Met Ala Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu tcg aac cot gag gag gtt ctt ctt gat gga gag aga atc tot act gga 96 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly aac tot tot atc gat att tot ctt tot ctt gtt cag ttc ctt gtt tot 144 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser aac ttc gtt cct gga gga gga ttc ctt gtt gga ctt atc gat ttc gtt 192 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Lou Ile Asp Phe Val tgg gga atc gtt gga cct tot cag tgg gat got ttc ctt gtt cag atc 240 Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile gag cag ctt atc aac gag aga atc got gag ttc got aga aac got got 288 Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala atc got aac ctt gag gga ctt gga aac aac ttc aac atc tac gtt gag 336 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu got 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 aga gtt atc gat aga ttc aga atc ctt gat gga ctt ctt gag aga gat 432 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp att cct tot ttc aga atc tot gga ttc gaa gtt cct ctt ctt tot gtt 480 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Lou Leu Ser Val tac got 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 got got aac ctt cat ctt got atc 702 Ala Ala Asn Lou His Leu Ala Ile ctt aga gat tot gtt atc ttc gga gag aga tgg gga ctt act act atc 750 Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile . .
, aac gtt aac gag aac tac aac aga ctt atc aga cat atc gat gag tac Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr gct gat cat tgt gct aac act tac aac aga gga ctt aac aac ctt cot Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro aag tct act tac cag gac tgg atc act tac aac aga ctt aga aga gat Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp ctt act ctt act gtt ctt gat att gct gct ttc ttc cot aac tac gat Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp aac aga aga tac cct atc cag cot gtt gga cag ctt act aga gag gtt Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val tac act gat cot ctt atc aac ttc aac cot cag ctt cag tct gtt gct Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala cag ctt cot act ttc aac gtt atg gag tct tct gct atc aga aac cot Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro cat ctt ttc gat att ctt aac aac ctt act atc ttc act gac tgg ttc His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe tct gtt gga aga aac ttc tac tgg gga gga cat aga gtt atc tct tct Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser ctt atc gga gga gga aac atc act tct cot atc tac gga aga gag gct Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala aac cag gag cot cot aga tct ttc act ttc aac gga cot gtt ttc aga Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg act ctt tct aac cot act ctt aga ctt ctt cag cag cot tgg cot gct Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala cot cot ttc aac ctt aga gga gtt gag gga gtt gag ttc tct act cot Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro act aac tct ttc act tac aga gga aga gga act gtt gat tct ctt act Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr gag ctt cot cot gag gat aac tot gtt cot cot aga gag gga tac tot 1470 Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser cat aga ctt tgt cat got act ttc gtt cag aga tot gga act cot ttc 1518 His Arg Leu Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe ctt act act gga gtt gtt ttc tot tgg act cat aga tot got act ctt 1566 Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu act aac act atc gat cot gag agg atc aac cag atc cot ctt gtt aag 1614 Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys gga ttc aga gtt tgg gga gga act tot gtt atc act gga cot gga ttc 1662 Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe act gga gga gat att ctt aga aga aac act ttc gga gat ttc gtt tot 1710 Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser ctt cag gtt aac atc aac tot cot atc act cag aga tac aga ctt aga 1758 Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg ttc aga tac got tot tot aga gat got aga gtt atc gtt ctt act gga 1806 Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly got got tot act gga gtt gga gga cag gtt tot gtt aac atg cot ctt 1854 Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu cag aag act atg gag atc gga gag aac ctt act tot aga act ttc aga 1902 Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg tac act gat ttc tot aac cot ttc tot ttc aga got aac cot gat att 1950 Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile atc gga atc tot gag cag cot ctt ttc gga got gga tot atc tot tot 1998 Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser gga gag ctt tac etc gat aaa atc gag atc atc ctt got gat got act 2046 Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr ttc gag got gag tot gat tta gag aga tga 2076 Phe Glu Ala Glu Ser Asp Leu Glu Arg , . .
, <210> 4 <211> 2442 <212> DNA
<213> Artificial Sequence <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 gct tot atc tot tct tot gtt gct act gtt tot aga act gct cct Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro gct cag gct aac atg gtt gct cot ttc act gga ctt aag tot aac gct Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala gct ttc cot act act aag aag gct aac gat ttc tot act ctt cot tot Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser aac gga gga aga gtt cag tgt atg cag gtt tgg cot gct tac gga aac Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn aag aag ttc gag act ctt tot tac ctt cot cot ctt tot atg gct cot Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro act gtt atg atg gct tot tot gct act gct gtt gct cot ttc cag gga Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly ctt aag tot act gct tot ctt cot gtt gct aga aga tot tot aga tot Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser ctt gga aac gtt tot aac gga gga aga atc aga tgt gag gag aac aac Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn cag aac cag tgt atc cot tac aac tgt ctt tog aac cot gag gag gtt Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val ctt ctt gat gga gag aga atc tct act gga aac tct tct atc gat att 480 Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile 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 gga ttc ctt gtt gga ctt atc gat ttc gtt tgg gga atc gtt gga cot 576 Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro tct cag tgg gat gct ttc ctt gtt cag atc gag cag ctt atc aac gag 624 Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu aga atc gct gag ttc gct aga aac gct gct atc gct aac ctt gag gga 672 Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly ctt gga aac aac ttc aac atc tac gtt gag gct ttc aag gag tgg gag 720 Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu gag gat cot aac aac cot gag act aga act aga gtt atc gat aga ttc 768 Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe aga atc ctt gat gga ctt ctt gag aga gat att cct tct ttc aga atc 816 Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile tct gga ttc gag gtt cot ctt ctt tct gtt tac gct cag gct gct aac 864 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn ctt cat ctt gct atc ctt aga gat tct gtt atc ttc gga gag aga tgg 912 Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp gga ctt act act atc aac gtt aac gag aac tac aac aga ctt atc aga 960 Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg cat atc gat gag tac gct gat cat tgt gct aac act tac aac aga gga 1008 His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly ctt aac aac ctt cot aag tct act tac cag gtaagtttct gcttctacct 1058 Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin ttgatatata tataataatt atcattaatt agtagtaata taatatttca aatatttttt 1118 tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa gtgtgtatat 1178 tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcag gac tgg atc 1236 Asp Trp Ile 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 Ile gct gct ttc ttc cct aac tac gat aac aga aga tac cct atc cag cct 1332 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro gtt gga cag ctt act aga gag gtt tac act gat cct ctt atc aac ttc 1380 Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe aac cct cag ctt cag tot gtt gct 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 gag tot tot gct atc aga aac cct cat ctt ttc gat att ctt aac aac 1476 Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn ctt act atc ttc act gac tgg ttc tot gtt gga aga aac ttc tac tgg 1524 Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp gga gga cat aga gtt atc tot tot ctt atc gga gga gga aac atc act 1572 Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr tot cct atc tac gga aga gag gct aac cag gag cct cct aga tot ttc 1620 Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe act ttc aac gga cct gtt ttc aga act ctt tot aac cct act ctt aga 1668 Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg ctt ctt cag cag cct tgg cct gct 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 gag gga gtt gag ttc tot act cct act aac tot ttc act tac aga gga 1764 Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly aga gga act gtt gat tot ctt act gag ctt cct cct gag gat aac tot 1812 Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser gtt cct cct aga gag gga tac tot cat aga ctt tgt cat gct act ttc 1860 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe gtt cag aga tct gga act cot 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 tgg act cat aga tct got act ctt act aac act atc gat cot gag agg 1956 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg atc aac cag atc cot ctt gtt aag gga ttc aga gtt tgg gga gga act 2004 Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr tct gtt atc act gga cot gga ttc act gga gga gat att ctt aga aga 2052 Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg aac act ttc gga gat ttc gtt tct ctt cag gtt aac atc aac tct cct 2100 Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro atc act cag aga tac aga ctt aga ttc aga tac got tct tct aga gat 2148 Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp got aga gtt atc gtt ctt act gga got got tct act gga gtt gga gga 2196 Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly cag gtt tct gtt aac atg cot ctt cag aag act atg gag atc gga gag 2244 Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu aac ctt act tct aga act ttc aga tac act gat ttc tct aac cot ttc 2292 Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe tct ttc aga gct aac cot gat att atc gga atc tct gag cag cot ctt 2340 Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu ttc gga got gga tct atc tct tct gga gag ctt tac atc gat aaa atc 2388 Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile gag atc atc ctt got gat got act ttc gag got gag tct gat tta gag 2436 Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu aga tga 2442 Arg <210> 5 <211> 750 =

<212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 5 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Tie Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg <210> 6 <211> 2442 <212> DNA
<213> Artificial Sequence <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 gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala gct ttc cct act act aag aag gct 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 aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser ctt gga aac gtt tct aac gga gga aga atc aga tgt gag gag aac aac 384 Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn cag aac cag tgt atc cct tac aac tgt ctt tog aac cct gag gag gtt 432 Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val ctt ctt gat gga gag aga atc tct act gga aac tct tct atc gat att 480 Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile 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 gga ttc ctt gtt gga ctt atc gat ttc gtt tgg gga atc gtt gga cot 576 Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro tct cag tgg gat got ttc ctt gtt cag atc gag cag ctt atc aac gag 624 Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu aga atc got gag ttc got aga aac got got atc gct aac ctt gag gga 672 Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly ctt gga aac aac ttc aac atc tac gtt gag got ttc aag gag tgg gag 720 Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu gag gat cct aac aac cct gag act aga act aga gtt atc gat aga ttc 768 Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe aga atc ctt gat gga ctt ctt gag aga gat att cct tot ttc aga atc 816 Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile tot gga ttc gaa gtt cct ctt ctt tot gtt tac got cag gtaagtttct 865 Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca 925 aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa 985 gtgtgtatat tttaatttat aacttttcta atatatgacc aaaacatggt gatgtgcag 1044 got got aac ctt cat ctt got atc ctt aga gat tot gtt atc ttc gga 1092 Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly gag aga tgg gga ctt act act atc aac gtt aac gag aac tac aac aga 1140 Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg ctt atc aga cat atc gat gag tac got gat cat tgt got aac act tac 1188 Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr aac aga gga ctt aac aac ctt cct aag tot act tac cag gac tgg atc 1236 Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile 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 Ile got got ttc ttc cct aac tac gat aac aga aga tac cct atc cag cct 1332 Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro gtt gga cag ctt act aga gag gtt tac act gat cct ctt atc aac ttc 1380 Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe aac cct cag ctt cag tct gtt gct 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 gag tct tct gct atc aga aac cct cat ctt ttc gat att ctt aac aac 1476 Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn ctt act atc ttc act gac tgg ttc tct gtt gga aga aac ttc tac tgg 1524 Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp gga gga cat aga gtt atc tct tct ctt atc gga gga gga aac atc act 1572 Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr tct cct atc tac gga aga gag gct aac cag gag cct cct aga tct ttc 1620 Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe 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 ctt ctt cag cag cct tgg cct gct 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 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 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 gtt cct cct aga gag gga tac tct cat aga ctt tgt cat gct act ttc 1860 Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe 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 tgg act cat aga tct gct act ctt act aac act atc gat cct gag agg 1956 Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg atc aac cag atc cct ctt gtt aag gga ttc aga gtt tgg gga gga act 2004 Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr , , 58 tot gtt atc act gga cot gga ttc act gga gga gat att ctt aga aga Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg aac act ttc gga gat ttc gtt tot ctt cag gtt aac atc aac tot cot Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro atc act cag aga tac aga ctt aga ttc aga tac got tot tot aga gat Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp got aga gtt atc gtt ctt act gga got got tot act gga gtt gga gga Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly cag gtt tot gtt aac atg cct ctt cag aag act atg gag atc gga gag Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu aac ctt act tot aga act ttc aga tac act gat ttc tot aac cot ttc Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe tot ttc aga got aac cot gat att atc gga atc tot gag cag cot ctt Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu ttc gga got gga tot atc tot tot gga gag ctt tac atc gat aaa atc Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile gag atc atc ctt got gat got act ttc gag got gag tot gat tta gag Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu aga tga Arg <210> 7 <211> 750 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 7 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Glu Asn Asn Gin Asn Gin Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gin Phe Leu Val Ser Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val Trp Gly Ile Val Gly Pro Ser Gin Trp Asp Ala Phe Leu Val Gin Ile Glu Gin Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu Ala Phe Lys Glu Trp Glu Glu Asp Pro Asn Asn Pro Glu Thr Arg Thr Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr Ala Gin Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro Glu Arg Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg <210> 8 <211> 2313 <212> DNA
<213> Artificial Sequence <220>
<223> modified sequence <220>
<221> CDS
<222> (1)..(2310) <400> 8 atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96 Ala Gin Ala Asn Met Val Ala Pro She Thr Gly Leu Lys Ser Asn Ala , . .
, gct ttc cot act act aag aag gct aac gat ttc tct act ctt cct tct Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser aac gga gga aga gtt cag tgt atg cag gtt tgg cot gct tac gga aac Asn Gly Gly Arg Val Gln Cys Met Gin Val Trp Pro Ala Tyr Gly Asn aag aag ttc gag act ctt tct tac ctt cot cct ctt tct atg gct cct Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro act gtt atg atg gct tct tct gct act gct gtt gct cot ttc cag gga Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly ctt aag tct act gct tct ctt cot gtt gct aga aga tct tct aga tct Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser ctt gga aac gtt tct aac gga gga aga atc aga tgt act tog aac aga Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Thr Ser Asn Arg aag aac gag aac gag atc atc aac gct gtt tct aac cat tct gct cag Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gin atg gat ctt ctt cot gat gct aga atc gag gat tct ctt tgt atc gct Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala gag gga aac aac atc gat cot ttc gtt tct gct tct act gtt cag act Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr ggt atc aac atc gct gga aga att ctt gga gtt ctt gga gtt cot ttc Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe gct gga cag ctt gct tct ttc tac tct ttc ctt gtt gga gag ctt tgg Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp cot aga gga aga gat cag tgg gag atc ttc ctt gag cat gtt gag cag Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe Leu Glu His Val Glu Gin ctt atc aac cag cag atc act gag aac gct aga aac act gct ctt gct Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala aga ctt cag gga ctt gga gat tct ttc aga gct tac cag cag tct ctt Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu gag gac tgg ctt gag aac aga gat gat gct aga act aga tot gtt ctt 816 Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu cat act cag tac atc gct ctt gag ctt gat ttc ctt aac gct atg cct 864 His Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro ctt ttc gct atc aga aac cag gag gtt cot ctt ctt atg gtt tac gct 912 Leu Phe Ala Ile Arg Asn Gln Glu Val Pro Leu Leu Met Val Tyr Ala cag gct gct aac ctt cat ctt ctt ctt ctt aga gat gct tot ctt ttc 960 Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe gga tot gag ttc gga ctt act tot cag gag atc cag aga tat tac gag 1008 Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu aga cag gtt gag aga act aga gat tac tot gat tac tgt gtt gag tgg 1056 Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp tac aac act gga ctt aac tot ctt aga gga act aac gct gct tot tgg 1104 Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp gtt aga tac aac cag ttc aga aga gat ctt act ctt gga gtt ctt gat 1152 Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp ctt gtt gct ctt ttc cot tot tac gac act aga act tac cct atc aac 1200 Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn act tct gct cag ctt act aga gag gtt tac act gat gct atc gga gct 1248 Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala act gga gtt aac atg gct tot atg aac tgg tac aac aac aac gct cot 1296 Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro tot ttc tot gct atc gag gct gct gct atc aga tot cot cat ctt ctt 1344 Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu gat ttc ctt gag cag ctt act atc ttc tot gct tot tot aga tgg tot 1392 Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser aac act aga cac atg act tac tgg aga gga cat acc atc cag tot aga 1440 Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gln Ser Arg cct atc gga gga gga ctt aac act tot act cat gga gct act aac act 1488 Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr tot atc aac cct gtt act ctt aga ttc got tot aga gat gtt tac aga 1536 Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg act gag tot tac got gga gtt ctt ctt tgg gga atc tac ctt gag cot 1584 Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro atc cac gga gtt cot act gtt aga ttc aac ttc act aac cot cag aac 1632 Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn atc tot gat aga gga act got aac tac tot cag cot tac gag tot cct 1680 Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro gga ctt cag ctt aag gat tot gag act gag ctt cot cot gag act act 1728 Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr gag aga cot aac tac gag tot tac tot cat aga ctt tot cat atc gga 1776 Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly atc atc ctt cag tct aga gtt aac gtt cot gtt tac tot tgg act cat 1824 Ile Ile Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His aga tot got gat aga act aac act atc gga cot aac aga atc act cag 1872 Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gin atc cot atg gtt aag got tot gag ctt cct cag gga act act gtt gtt 1920 Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val aga gga cot gga ttc act gga gga gat atc ctt aga aga act aac act 1968 Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr gga gga ttc gga cot atc aga gtt act gtt aac gga cot ctt act cag 2016 Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gin aga tac aga atc gga ttc aga tac got tot act gtt gat ttc gat ttc 2064 Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe ttc gtt tot 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 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 gct ttc act act cct ttc act ttc act cag atc cag gat atc atc aga 2208 Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin Ile Gin Asp Ile Ile Arg act tct atc cag gga ctt tct gga aac gga gag gtt tac atc gat aaa 2256 Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys atc gag atc atc cot gtt act got act ttc gag got gag tac gat tta 2304 Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu gag aga tga 2313 Glu Arg <210> 9 <211> 770 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 9 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser Asn Gly Gly Arg Val Gin Cys Met Gln Val Trp Pro Ala Tyr Gly Asn Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly Lou Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Lou Trp . .
. .
Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe Leu Glu His Val Glu Gin Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu His Thr Gin Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gin Glu Val Pro Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu Ile Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gin Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Ile Ile Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gin Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr , . .
, Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gin Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin Ile Gin Asp Ile Ile Arg Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg <210> 10 <211> 1947 <212> DNA
<213> Artificial Sequence <220>
<223> modified sequence <220>
<221> CDS
<222> (1)..(1944) <400> 10 atg gct act tcg aac aga aag aac gag aac gag atc atc aac gct gtt Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val tct aac cat tct gct cag atg gat ctt ctt cct gat gct aga atc gag Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg Ile Glu gat tct ctt tgt atc gct gag gga aac aac atc gat cct ttc gtt tct Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser gct tct act gtt cag act ggt atc aac atc gct gga aga att ctt gga Ala Ser Thr Val Gin Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly gtt ctt gga gtt cct ttc gct gga cag ctt gct tct ttc tac tct ttc Val Leu Gly Val Pro Phe Ala Gly Gin Leu Ala Ser Phe Tyr Ser Phe ctt gtt gga gag ctt tgg cct aga gga aga gat cag tgg gag atc ttc Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe ctt gag cat gtt gag cag ctt atc aac cag cag atc act gag aac gct 336 Leu Glu His Val Glu Gin Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala aga aac act gct ctt gct 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 gct tac cag cag tct ctt gag gac tgg ctt gag aac aga gat gat gct 432 Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala aga act aga tct gtt ctt cat act cag tac atc gct ctt gag ctt gat 480 Arg Thr Arg Ser Val Leu His Thr Gin Tyr Ile Ala Leu Glu Leu Asp ttc ctt aac gct atg cot ctt ttc gct atc aga aac cag gag gtt cct 528 Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gin Glu Val Pro ctt ctt atg gtt tac gct cag gct gct aac ctt cat ctt ctt ctt ctt 576 Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu aga gat gct 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 atc cag aga tat tac gag aga cag gtt gag aga act aga gat tac tct 672 Ile Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser 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 act aac gct gct 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 act ctt gga gtt ctt gat ctt gtt gct ctt ttc cot tct tac gac act 816 Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr aga act tac cot atc aac act tot gct cag ctt act aga gag gtt tac 864 Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr act gat gct atc gga gct act gga gtt aac atg gct tct atg aac tgg 912 Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp tac aac aac aac gct cot tct ttc tct gct atc gag gct gct gct atc 960 Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile aga tot cot cat ctt ctt gat ttc ctt gag cag ctt act atc ttc tot 1008 Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr Ile Phe Ser got tot tot aga tgg tot 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 cat acc atc cag tot aga cot atc gga gga gga ctt aac act tot act 1104 His Thr Ile Gin Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr cat gga got act aac act tot atc aac cot gtt act ctt aga ttc got 1152 His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala tot aga gat gtt tac aga act gag tot tac got gga gtt ctt ctt tgg 1200 Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp gga atc tac ctt gag cot atc cac gga gtt cot act gtt aga ttc aac 1248 Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn ttc act aac cot cag aac atc tot gat aga gga act got aac tac tot 1296 Phe Thr Asn Pro Gin Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser cag cot tac gag tot cot gga ctt cag ctt aag gat tot gag act gag 1344 Gin Pro Tyr Glu Ser Pro Gly Leu Gin Leu Lys Asp Ser Glu Thr Glu ctt cot cot gag act act gag aga cot aac tac gag tot tac tot cat 1392 Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His aga ctt tot cat atc gga atc atc ctt cag tot aga gtt aac gtt cct 1440 Arg Leu Ser His Ile Gly Ile Ile Leu Gin Ser Arg Val Asn Val Pro gtt tac tot tgg act cat aga tot got gat aga act aac act atc gga 1488 Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly cot aac aga atc act cag atc cot atg gtt aag got tot gag ctt cot 1536 Pro Asn Arg Ile Thr Gin Ile Pro Met Val Lys Ala Ser Glu Leu Pro cag gga act act gtt gtt aga gga cot gga ttc act gga gga gat atc 1584 Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile ctt aga aga act aac act gga gga ttc gga cot atc aga gtt act gtt 1632 Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val aac gga cct ctt act cag aga tac aga atc gga ttc aga tac gct tot 1680 Asn Gly Pro Leu Thr Gin Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser act gtt gat ttc gat ttc ttc gtt tot aga gga gga act act gtt aac 1728 Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn aac ttc aga ttc ctt aga act atg aac tot gga gat gag ctt aag tac 1776 Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr gga aac ttc gtt aga aga got ttc act act cot ttc act ttc act cag 1824 Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gin atc cag gat atc atc aga act tot atc cag gga ctt tot gga aac gga 1872 Ile Gin Asp Ile Ile Arg Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly gag gtt tac atc gat aaa atc gag atc atc cot gtt act got act ttc 1920 Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe gag got gag tac gat tta gag aga tga 1947 Glu Ala Glu Tyr Asp Leu Glu Arg <210> 11 <211> 648 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 11 Met Ala Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser Asn His Ser Ala Gin Met Asp Leu Leu Pro Asp Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp Pro Phe Val Ser Ala Ser Thr Val Gin Thr Gly Ile Asn Ile Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Gin Trp Glu Ile Phe Leu Glu His Val Glu Gin Leu Ile Asn Gin Gin Ile Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala Arg Leu Gin Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gin Gin Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg Thr Arg Ser Val Leu His Thr Gin Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn Gin Glu Val Pro Leu Leu Met Val Tyr Ala Gin Ala Ala Asn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu Thr Ser Gin Glu Ile Gin Arg Tyr Tyr Glu Arg Gin Val Glu Arg Thr Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr Asn Ala Ala Ser Trp Val Arg Tyr Asn Gin Phe Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gin Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ala Ile Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gin Leu Thr Ile Phe Ser Ala Ser Ser Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gin Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Thr Asn Pro Gin Asn Ile Ser Asp Arg Gly Thr Ala Asn Tyr Ser Gin Pro Tyr Glu Ser Pro Gly Lou Gin Leu Lys Asp Ser Glu Thr Glu Lou Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Ile Ile Leu Gin Ser Arg Val Asn Val Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr Gin Ile Pro Met Val Lys Ala Ser Glu Leu Pro Gin Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro Ile Arg Val Thr Val Asn Gly Pro Leu Thr Gin Arg Tyr Arg Ile Gly Phe Arg Tyr Ala Ser Thr Val Asp Phe Asp Phe Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn Phe Arg Phe Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr . , . 71 Gly Asn Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Ile Gin Asp Ile Ile Arg Thr Ser Ile Gin Gly Leu Ser Gly Asn Gly Glu Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg <210> 12 <211> 2181 <212> DNA
<213> Artificial Sequence <220>
<223> modified sequence <220>
<221> CDS
<222> (1)..(2178) <400> 12 atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala gct ttc cct act act aag aag gct aac gat ttc tct act ctt cct tct Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser ctt gga aac gtt tct aac gga gga aga atc aga tgt gag atc aac aac Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Ile Asn Asn =

cag aac cag tgt gtt Oct tac aac tgt ctt tot aac cct aag gag atc Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile atc ctt gga gag gag aga ctt gag act gga aac act gtt got gat atc Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile tot ctt gga ctt atc aac ttc ctt tac tot aac ttc gtt cot gga ggt Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly gga ttc atc gtt gga ctt ctt gag ctt atc tgg gga ttc atc gga cot Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro tot cag tgg gat atc ttc ctt got cag atc gag cag ctt atc tot cag Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile Glu Gin Leu Ile Ser Gin aga atc gag gag ttc got aga aac cag got atc tot aga ctt gag gga Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu Glu Gly ctt tot aat ctt tac aaa gtt tac gtt aga got ttc tot gac tgg gag Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu aag gat cot act aac cct got ctt aga gag gag atg aga atc cag ttc Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gin Phe aac gat atg aac tot got ctt atc act got atc cot ctt ttc aga gtt Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe Arg Val cag aac tac gag gtt got ctt ctt tot gtt tac gtt cag got gat aac Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn ctt cat ctt tot atc ctt aga gat gtt tot gtt ttc gga gag aga tgg Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp gga tac gat act got act atc aac aac aga tac tot gat ctt act tot Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser ctt atc cat gtt tac act aac cat tgt gtt gat act tac aac cag gga Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly ctt aga aga ctt gag gga aga ttc ctt tot gac tgg atc gtt tac aac Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn aga ttc aga aga cag ctt act atc tot gtt ctt gat atc gtt got ttc 1104 Arg Phe Arg Arg Gin Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe ttc cot aac tac gat atc aga act tac cct atc cag act got act cag 1152 Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gin Thr Ala Thr Gin ctt act aga gag gtt tac ctt gat ctt cot ttc atc aac gag aac ctt 1200 Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu tot cot got got tot tac cot act ttc tot got got gag tot got atc 1248 Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile atc aga tot cot cat ctt gtt gat ttc ctt aac tot ttc act atc tac 1296 Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr act gat tot ctt got aga tac got 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 tot ttc aga act gga act aca act aac ctt atc aga tot cot ctt tac 1392 Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr gga aga gag gga aac act gag aga cot gtt act atc act got tot cot 1440 Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro tot gtt cot atc ttc aga act ctt tot tac atc act gga ctt gat aac 1488 Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn tot aac cot gtt got gga atc gag gga gtt gag ttc cag aac act atc 1536 Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gin Asn Thr Ile tot aga tot atc tac aga aag tot gga cot atc gat tot ttc tot gag 1584 Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu ctt cct cct cag gat got tot gtt tot cot got atc gga tac tot cat 1632 Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His aga ctt tgt cat got act ttc ctt gag aga atc tot gga cot aga atc 1680 Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile got gga act gtt ttc tot tgg act cat aga tot got tot cot act aac 1728 Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn . .
, 74 gag gtt tct cct tot aga atc act cag atc cct tgg gtt aag got cat Glu Val Ser Pro Ser Arg Ile Thr Gin Ile Pro Trp Val Lys Ala His act ctt got tot gga got tot gtt atc aag gga cct gga ttc act gga Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly gga gat atc ctt act aga aac tot atg gga gag ctt gga act ctt aga Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg gtt act ttc act gga aga ctt cct cag tot tac tac atc aga ttc aga Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr Ile Arg Phe Arg tac got tot gtt got aac aga tot gga act ttc aga tac tot cag cct Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro cct tot tac gga atc tot ttc cct aag act atg gat got gga gag cct Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro ctt act tot aga tot ttc got cat aca act ctt ttc act cct atc act Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr ttc tot aga got cag gag gag ttc gat cta tac atc cag tot gga gtt Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr Ile Gin Ser Gly Val tac atc gat aga atc gag ttc atc cct gtt act got act ttc gag got Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala gag tac gat tta gag aga tga Glu Tyr Asp Leu Glu Arg <210> 13 <211> 726 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 13 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser . 75 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys Glu Ile Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile Glu Gin Leu Ile Ser Gin Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gin Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala Ile Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn , = 76 Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gin Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gin Ile Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr Ile Gin Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg <210> 14 <211> 1815 <212> DNA
<213> Artificial Sequence <220>
<223> modified sequence <220>
<221> CDS
<222> (1)..(1812) <400> 14 atg gct gag atc aac aac cag aac cag tgt gtt cct tac aac tgt ctt Met Ala Glu Ile Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu tct aac cct aag gag atc atc ctt gga gag gag aga ctt gag act gga Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly aac act gtt gct gat atc tct ctt gga ctt atc aac ttc ctt tac tct Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser aac ttc gtt cot gga ggt gga ttc atc gtt gga ctt ctt gag ctt atc 192 Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile tgg gga ttc atc gga cot tot cag tgg gat atc ttc ctt got cag atc 240 Trp Gly Phe Ile Gly Pro Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile gag cag ctt atc tot cag aga atc gag gag ttc got aga aac cag got 288 Glu Gin Leu Ile Ser Gin Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala atc tot aga ctt gag gga ctt tot aat ctt tac aaa gtt tac gtt aga 336 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg got ttc tot gac tgg gag aag gat cot act aac cot got ctt aga gag 384 Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu gag atg aga atc cag ttc aac gat atg aac tot got ctt atc act got 432 Glu Met Arg Ile Gin Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala atc cot ctt ttc aga gtt cag aac tac gag gtt got ctt ctt tot gtt 480 Ile Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Lou Leu Ser Val tac gtt cag got got aac ctt cat ctt tot atc ctt aga gat gtt tot 528 Tyr Val Gin Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser gtt ttc gga gag aga tgg gga tac gat act got act atc aac aac aga 576 Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg tac tot gat ctt act tot ctt atc cat gtt tac act aac cat tgt gtt 624 Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val gat act tac aac cag gga ctt aga aga ctt gag gga aga ttc ctt tot 672 Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser gac tgg atc gtt tac aac aga ttc aga aga cag ctt act atc tot gtt 720 Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr Ile Ser Val ctt gat atc gtt got ttc ttc cot aac tac gat atc aga act tac cot 768 Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro atc cag act got act cag ctt act aga gag gtt tac ctt gat ctt cct 816 Ile Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro . ' .
, 78 ttc atc aac gag aac ctt tct cot gct gct tct tac cct act ttc tct Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser gct gct gag tct gct atc atc aga tct cct cat ctt gtt gat ttc ctt Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu aac tct ttc act atc tac act gat tct ctt gct aga tac gct tac tgg Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp gga gga cat ctt gtt aac tct ttc aga act gga act aca act aac ctt Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu atc aga tct cot ctt tac gga aga gag gga aac act gag aga cct gtt Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val act atc act gct tct cot tct gtt cot atc ttc aga act ctt tct tac Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr atc act gga ctt gat aac tct aac cot gtt gct gga atc gag gga gtt Ile Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val gag ttc cag aac act atc tct aga tct atc tac aga aag tct gga cot Glu Phe Gin Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro atc gat tct ttc tct gag ctt cot cot cag gat gct tct gtt tct cct Ile Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro gct atc gga tac tct cat aga ctt tgt cat gct act ttc ctt gag aga Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg atc tct gga cot aga atc gct gga act gtt ttc tct tgg act cat aga Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg tct gct tct cot act aac gag gtt tct cot tct aga atc act cag atc Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gin Ile cot tgg gtt aag gct cat act ctt gct tct gga gct tct gtt atc aag Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys gga cot gga ttc act gga gga gat atc ctt act aga aac tct atg gga Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly 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 tac tac atc aga ttc aga tac gct tct gtt gct aac aga tct gga act 1584 Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr ttc aga tac tct cag cct cot tct tac gga atc tct ttc cct aag act 1632 Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr atg gat gct gga gag cot ctt act tct aga tct ttc gct cat aca act 1680 Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr ctt ttc act cct atc act ttc tct aga gct cag gag gag ttc gat cta 1728 Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu tac atc cag tct gga gtt tac atc gat aga atc gag ttc atc cot gtt 1776 Tyr Ile Gin Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val act gct act ttc gag gct gag tac gat tta gag aga tga 1815 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg <210> 15 <211> 604 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 15 Met Ala Glu Ile Asn Asn Gin Asn Gin Cys Val Pro Tyr Asn Cys Leu Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile Trp Gly Phe Ile Gly Pro Ser Gin Trp Asp Ile Phe Leu Ala Gin Ile Glu Gin Leu Ile Ser Gin Arg Ile Glu Glu Phe Ala Arg Asn Gin Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala , == 80 Ile Pro Leu Phe Arg Val Gin Asn Tyr Glu Val Ala Leu Leu Ser Val Tyr Val Gin Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val Asp Thr Tyr Asn Gin Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gin Leu Thr Ile Ser Val Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro Ile Gin Thr Ala Thr Gin Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Ser Tyr Pro Thr Phe Ser Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Tyr Ala Tyr Trp Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr Ile Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val Glu Phe Gin Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro Ile Asp Ser Phe Ser Glu Leu Pro Pro Gin Asp Ala Ser Val Ser Pro Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gin Ile Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gin Ser Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr Phe Arg Tyr Ser Gin Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gin Glu Glu Phe Asp Leu Tyr Ile Gin Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg <210> 16 <211> 372 <212> DNA
<213> Artificial Sequence <220>
<223> modified transit peptide <220>
<221> CDS
<222> (1)..(372) <400> 16 atg gct tct atc tct tct tct gtt gct act gtt tct aga act gct cct 48 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro gct cag gct aac atg gtt gct cct ttc act gga ctt aag tct aac gct 96 Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala gct ttc cct act act aag aag gct 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 aac gga gga aga gtt cag tgt atg cag gtt tgg cct gct tac gga aac 192 Asn Gly Gly Arg Val Gin Cys Met Gin Val Trp Pro Ala Tyr Gly Asn aag aag ttc gag act ctt tct tac ctt cct cct ctt tct atg gct cct 240 Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro act gtt atg atg gct tct tct gct act gct gtt gct cct ttc cag gga 288 Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gin Gly ctt aag tct act gct tct ctt cct gtt gct aga aga tct tct aga tct 336 Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser ctt gga aac gtt tct aac gga gga aga atc aga tgt 372 Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys <210> 17 <211> 124 <212> PRT
<213> Artificial Sequence <220>
<223> Synthetic Construct <400> 17 Met Ala Ser Ile Ser Ser Ser Val Ala Thr Val Ser Arg Thr Ala Pro Ala Gln Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys Ser Asn Ala Ala Phe Pro Thr Thr Lys Lys Ala Asn Asp Phe Ser Thr Leu Pro Ser Asn Gly Gly Arg Val Gln Cys Met Gln Val Trp Pro Ala Tyr Gly Asn Lys Lys Phe Glu Thr Leu Ser Tyr Leu Pro Pro Leu Ser Met Ala Pro Thr Val Met Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln Gly Leu Lys Ser Thr Ala Ser Leu Pro Val Ala Arg Arg Ser Ser Arg Ser Leu Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys <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 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 = , 83 acccccgtgc cgggatcaga gacatttgac taaatattga cttggaataa tagcccttgg attagatgac acgtggacgc tcaggatctg tgatgctagt gaagcgctta agctgaacga atctgacgga agagcggaca tacgcacatg gattatggcc cacatgtcta aagtgtatct ctttacagct atatcgatgt gacgtaagat gctttacttc gcttcgaagt aaagtaggaa attgctcgct aagttattct tttctgaaag aaattaattt aattctaatt aaattaaatg agtggcctgc agtaattaat agtaattatg attaattatg agataagagt tgttattaat gcttatgagg aataaagaat gattaatatt gtttaatttt attccgcgaa gcggtgtgtt atgtttttgt tggagacatc acgtgactct cacgtgatgt ctccgcgaca ggctggcacg gggcttagta ttacccccgt gccgggatca gagacatttg actaaatatt gacttggaat aatagccctt ggattagatg acacgtggac gctcaggatc tgtgatgcta gtgaagcgct taagctgaac gaatctgacg gaagagcgga catacgcaca tggattatgg cccacatgtc taaagtgtat ctctttacag ctatatcgat gtgacgtaag atgctttact tcgcttcgaa gtaaagtagg aaattgctcg ctaagttatt cttttctgaa agaaattaat ttaattctaa attaaattaa atgagtggct ataaatagtg tcgatgctac ctcacatcgt attcttcttc gcatcgtctg ttctggtttt aa <210> 20 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 20 tacttcgaac agaaagaacg agaacgag <210> 21 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 21 gtccagcgaa aggaactcca agaa <210> 22 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 22 aaccttgagg gacttggaaa c <210> 23 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> primer <400> 23 aagatgaggg tttctgatag cag 23

Claims (14)

CLAIMS:

1. A chimeric nucleic acid comprising the following operably-linked sequences:
a) a first fragment of a Bacillus thuringiensis coding sequence encoding an insecticidal protein, b) a plant intron sequence, c) a second fragment of said Bacillus thuringiensis coding sequence, d) a promoter region capable of directing expression in plant cells, wherein no insecticidal protein can be produced from such gene in a given host cell wherein the intron is not spliced, and wherein said intron is effectively spliced in plant cells, wherein said effective splicing can be measured using the detection of a functional protein produced in plant cells and wherein the plant intron sequence is positioned between the first and second fragment.

2. The chimeric nucleic acid of claim 1, wherein the plant intron is the second intron of the ST-LS1 gene of Solanum tuberosum.

3. The chimeric nucleic acid of claim 1 or 2, wherein said intron is of a dicot plant gene when the gene is to be expressed in dicot plant cells, and wherein said intron is of a monocot plant gene when the gene is to be expressed in monocot plant cells.

4. The chimeric nucleic acid of any one of claims 1 to 3, wherein said promoter comprises the sequence of SEQ ID No. 18 or 19.

5. The chimeric nucleic acid of any one of claims 1 to 4, further comprising a 3' polyadenylation and transcript termination region.

6. The chimeric nucleic acid of claim 5, wherein said 3' polyadenylation and transcript termination region is of the NADP-malic enzyme gene from Flaveriabidentis.

7. The chimeric nucleic acid of any one of claims 1 to 6, further comprising the leader sequence of the tapetum specific E1 gene of Oryza sativa between the promoter and the coding sequence.

8. The chimeric gene of any one of claims 1 to 7, wherein the Bacillus thuringiensis coding sequence comprises:
(a) a DNA sequence encoding an insecticidal Cry1 C protein, comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, comprising the amino acid sequence of any one of SEQ ID No. 2, 5 or 7, or comprising an amino acid sequence with a sequence identity of at least 95 %
to the amino acid sequence of SEQ ID No. 2 from amino acid position 29 to the amino acid at position 627, (b) a DNA sequence encoding an insecticidal Cry1 B protein, comprising the amino acid sequence of SEQ ID No. 11 from amino acid position 31 to amino acid position 648, comprising the amino acid sequence of SEQ ID No. 11, or comprising an amino acid sequence with a sequence identity of at least 95 % to the amino acid sequence of SEQ ID No. 15 from amino acid position 31 to amino acid position 648, or (c) a DNA sequence encoding an insecticidal Cry1D protein, comprising the amino acid sequence of SEQ ID No. 15 from amino acid position 29 to amino acid position 604, or comprising the amino acid sequence of SEQ ID No. 13 or 15, or comprising an amino acid sequence with a sequence identity of at least 95 % to the amino acid sequence from amino acid position 29 to amino acid position 604 in SEQ
ID No. 15.

9. The chimeric nucleic acid of any one of claims 1 to 7, wherein the Bacillus thuringiensis coding sequence encodes an insecticidal Cry1C protein comprising the amino acid sequence of SEQ ID No. 2 from amino acid position 29 to amino acid position 627, comprising the amino acid sequence of any one of SEQ
ID No. 2, 5 or 7, or 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 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.

10. The chimeric nucleic acid of any one of claims 1 to 7, wherein the Bacillus thuringiensis coding sequence encodes an insecticidal Cry1B protein comprising the amino acid sequence of SEQ ID No. 11 from amino acid position 31 to amino acid position 648, comprising the amino acid sequence of SEQ ID No. 11, or comprising an amino acid sequence with a sequence identity of at least 95 % to the amino acid sequence of SEQ ID No. 15 from amino acid position 31 to amino acid position 648, or 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.

11. The chimeric nucleic acid of any one of claims 1 to 7, wherein the Bacillus thuringiensis coding sequence encodes an insecticidal Cry1D protein comprising the amino acid sequence of SEQ ID No. 15 from amino acid position 29 to amino acid position 604, or comprising the amino acid sequence of SEQ ID No.
13 or 15, or comprising an amino acid sequence with a sequence identity of at least 95 %
to the amino acid sequence from amino acid position 29 to amino acid position 604 in SEQ ID No. 15.

12. The chimeric nucleic acid of any one of claims 1 to 11, wherein said Bacillus thuringiensis coding sequence is an artificial, synthetically-made DNA
sequence having a different nucleotide sequence compared to the naturally-occurring DNA sequence.

13. A transformed plant cell selected from the group consisting of: corn, cotton, watercress, horseradish, wasabi, arugula, cress, radish, canola, soybean, vegetable plants, Cruciferae plant species, cauliflower, cabbage, Chinese cabbage, turnip, mustard, oilseed rape, kale, broccoli, Brussels sprouts, and mustard spinach, comprising the chimeric nucleic acid of any one of claims 1 to 12.

14. A transgenic plant cell, comprising the chimeric nucleic acid of any one of claims 1 to 12 stably incorporated in its genome.