Patent application title: METHODS AND COMPOSITIONS FOR CONTROLLING PLANT PESTS
Inventors:
Kimberly S. Sampson (Durham, NC, US)
Athenix Corporation
Daniel J. Tomso (Bahama, NC, US)
Assignees:
ATHENIX CORPORATION
IPC8 Class: AC07K14325FI
USPC Class:
514 45
Class name: Designated organic active ingredient containing (doai) peptide (e.g., protein, etc.) containing doai insect destroying or inhibiting
Publication date: 2013-08-15
Patent application number: 20130210712
Abstract:
Compositions and methods for conferring pesticidal activity to bacteria,
plants, plant cells, tissues and seeds are provided. Compositions
comprising a coding sequence for a delta-endotoxin polypeptide are
provided. The coding sequences can be used in DNA constructs or
expression cassettes for transformation and expression in plants and
bacteria. Compositions also comprise transformed bacteria, plants, plant
cells, tissues, and seeds. In particular, isolated delta-endotoxin
nucleic acid molecules are provided. Additionally, amino acid sequences
corresponding to the polynucleotides are encompassed, and antibodies
specifically binding to those amino acid sequences. In particular, the
present invention provides for isolated nucleic acid molecules comprising
nucleotide sequences encoding the amino acid sequence shown in SEQ ID
NO:13-24 or the nucleotide sequence set forth in SEQ ID NO:1-12 and
25-44, as well as variants and fragments thereof.Claims:
1. A recombinant polypeptide with pesticidal activity, selected from the
group consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:14-20, 22, and 24; b) a polypeptide comprising an amino acid
sequence having at least 95% sequence identity to the amino acid sequence
of SEQ ID NO:14-20, 22, and 24; and c) a polypeptide that is encoded by
SEQ ID NO:2-8, 10, and 11.
2. The polypeptide of claim 1 further comprising heterologous amino acid sequences.
3. A composition comprising the recombinant polypeptide of claim 1.
4. The composition of claim 3, wherein said composition is selected from the group consisting of a powder, dust, pellets, granules, a spray, an emulsion, a colloid, and a solution.
5. The composition of claim 4, wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of bacterial cells.
6. The composition of claim 3, wherein said composition is selected from the group consisting of a powder, dust, pellets and granules, and the composition comprises from about 1% to about 99% by weight of said recombinant polypeptide.
7. A method for controlling a lepidopteran, coleopteran, nematode, or dipteran pest population comprising contacting said population with a pesticidally-effective amount of the polypeptide of claim 1.
8. A method for killing a lepidopteran, coleopteran, nematode, or dipteran pest, comprising contacting said pest with, or feeding to said pest, a pesticidally-effective amount of the polypeptide of claim 1.
9. A method for producing a polypeptide with pesticidal activity, comprising culturing a host cell comprising a nucleic acid molecule encoding the recombinant polypeptide of claim 1 under conditions in which the nucleic acid molecule encoding the polypeptide is expressed.
10. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2-8, 10, and 11; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:14-20, 22, and 24; and c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:14-20, 22, and 24.
11. The recombinant nucleic acid molecule of claim 10, wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.
12. The recombinant nucleic acid molecule of claim 11, wherein said sequence is selected from the group consisting of SEQ ID NO:27-36 and 39-41.
13. The recombinant nucleic acid molecule of claim 10, wherein said nucleotide sequence is operably linked to a promoter capable of directing expression of said nucleotide sequence in a plant cell.
14. A vector comprising the nucleic acid molecule of claim 10.
15. The vector of claim 14, further comprising a nucleic acid molecule encoding a heterologous polypeptide.
16. A host cell that contains the vector of claim 14.
17. The host cell of claim 16, wherein the host cell is a bacterial host cell.
18. The host cell of claim 16, wherein the host cell is a plant cell.
19. A transgenic plant comprising a plant cell as a host cell, wherein the plant cell contains a vector comprising the nucleic acid molecule of claim 10.
20. The transgenic plant of claim 19, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.
21. A transgenic seed comprising the nucleic acid molecule of claim 10.
22. A plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2-8, 10, and 11; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:14-20, 22, and 24; and c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:14-20, 22, and 24; wherein said nucleotide sequence is operably linked to a promoter that drives expression of a coding sequence in a plant cell.
23. The plant of claim 22, wherein said plant is a plant cell.
24. A method for protecting a plant from a pest, comprising expressing in a plant or cell thereof a nucleotide sequence that encodes a pesticidal polypeptide, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2-8, 10, and 11; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:14-20, 22, and 24; and c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:14-20, 22, and 24.
25. The method of claim 24, wherein said plant produces a pesticidal polypeptide having pesticidal activity against a lepidopteran, coleopteran, heteropteran, nematode, or dipteran pest.
26. A method for increasing yield in a plant comprising growing in a field a plant or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence set forth in SEQ ID NO:2-8, 10, and 11; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:14-20, 22, and 24; and c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:14-20, 22, and 24; wherein said field is infested with a pest against which said polypeptide has pesticidal activity.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a Continuation of U.S. patent application Ser. No. 13/301,190, filed Nov. 21, 2011, which is a Divisional application of U.S. patent application Ser. No. 13/027,816 filed Feb. 15, 2011, now U.S. Pat. No. 8,076,533, which is a Continuation of U.S. patent application Ser. No. 12/718,059 filed Mar. 5, 2010, now U.S. Pat. No. 7,919,272, and claims the benefit of U.S. Provisional Application Ser. No. 61/158,133, filed Mar. 6, 2009, the contents of which are herein incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "APA064USSEQLIST.txt", created on Apr. 16, 2013, and having a size of 143 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] This invention relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of transgenic pest-resistant plants.
BACKGROUND OF THE INVENTION
[0004] Bacillus thuringiensis is a Gram-positive spore forming soil bacterium characterized by its ability to produce crystalline inclusions that are specifically toxic to certain orders and species of insects, but are harmless to plants and other non-targeted organisms. For this reason, compositions including Bacillus thuringiensis strains or their insecticidal proteins can be used as environmentally-acceptable insecticides to control agricultural insect pests or insect vectors for a variety of human or animal diseases.
[0005] Crystal (Cry) proteins (delta-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against predominantly Lepidopteran, Dipteran, and Coleopteran larvae. These proteins also have shown activity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari pest orders, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson (1993), The Bacillus Thuringiensis family tree. Advanced Engineered Pesticides, Marcel Dekker, Inc., New York, N.Y.). These proteins were originally classified as CryI to CryV based primarily on their insecticidal activity. The major classes were Lepidoptera-specific (I), Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III), Diptera-specific (IV), and nematode-specific (V) and (VI). The proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as Cry1A, Cry1B, Cry1C, etc. Even more closely related proteins within each division were given names such as Cry1C1, Cry1C2, etc.
[0006] A new nomenclature was recently described for the Cry genes based upon amino acid sequence homology rather than insect target specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In the new classification, each toxin is assigned a unique name incorporating a primary rank (an Arabic number), a secondary rank (an uppercase letter), a tertiary rank (a lowercase letter), and a quaternary rank (another Arabic number). In the new classification, Roman numerals have been exchanged for Arabic numerals in the primary rank. Proteins with less than 45% sequence identity have different primary ranks, and the criteria for secondary and tertiary ranks are 78% and 95%, respectively.
[0007] The crystal protein does not exhibit insecticidal activity until it has been ingested and solubilized in the insect midgut. The ingested protoxin is hydrolyzed by proteases in the insect digestive tract to an active toxic molecule. (Hofie and Whiteley (1989) Microbiol. Rev. 53:242-255). This toxin binds to apical brush border receptors in the midgut of the target larvae and inserts into the apical membrane creating ion channels or pores, resulting in larval death.
[0008] Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II consists of three beta-sheets arranged in a Greek key configuration, and domain III consists of two antiparallel beta-sheets in "jelly-roll" formation (de Maagd et al., 2001, supra). Domains II and III are involved in receptor recognition and binding, and are therefore considered determinants of toxin specificity.
[0009] Aside from delta-endotoxins, there are several other known classes of pesticidal protein toxins. The VIP1/VIP2 toxins (see, for example, U.S. Pat. No. 5,770,696) are binary pesticidal toxins that exhibit strong activity on insects by a mechanism believed to involve receptor-mediated endocytosis followed by cellular toxification, similar to the mode of action of other binary ("A/B") toxins. A/B toxins such as VIP, C2, CDT, CST, or the B. anthracis edema and lethal toxins initially interact with target cells via a specific, receptor-mediated binding of "B" components as monomers. These monomers then form homoheptamers. The "B" heptamer-receptor complex then acts as a docking platform that subsequently binds and allows the translocation of an enzymatic "A" component(s) into the cytosol via receptor-mediated endocytosis. Once inside the cell's cytosol, "A" components inhibit normal cell function by, for example, ADP-ribosylation of G-actin, or increasing intracellular levels of cyclic AMP (cAMP). See Barth et al. (2004) Microbiol Mol Biol Rev 68:373-402.
[0010] The intensive use of B. thuringiensis-based insecticides has already given rise to resistance in field populations of the diamondback moth, Plutella xylostella (Ferre and Van Rie (2002) Annu. Rev. Entomol. 47:501-533). The most common mechanism of resistance is the reduction of binding of the toxin to its specific midgut receptor(s). This may also confer cross-resistance to other toxins that share the same receptor (Ferre and Van Rie (2002)).
SUMMARY OF THE INVENTION
[0011] Compositions and methods for conferring pest resistance to bacteria, plants, plant cells, tissues and seeds are provided. Compositions include nucleic acid molecules encoding sequences for delta-endotoxin polypeptides, vectors comprising those nucleic acid molecules, and host cells comprising the vectors. Compositions also include the polypeptide sequences of the endotoxin, and antibodies to those polypeptides. The nucleotide sequences can be used in DNA constructs or expression cassettes for transformation and expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be synthetic sequences that have been designed for expression in an organism including, but not limited to, a microorganism or a plant. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds.
[0012] In particular, isolated nucleic acid molecules corresponding to delta-endotoxin nucleic acid sequences are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence shown in any of SEQ ID NO:13-24, or a nucleotide sequence set forth in any of SEQ ID NO:1-12, as well as variants and fragments thereof. Nucleotide sequences that are complementary to a nucleotide sequence of the invention, or that hybridize to a sequence of the invention are also encompassed.
[0013] The compositions and methods of the invention are useful for the production of organisms with pesticide resistance, specifically bacteria and plants. These organisms and compositions derived from them are desirable for agricultural purposes. The compositions of the invention are also useful for generating altered or improved delta-endotoxin proteins that have pesticidal activity, or for detecting the presence of delta-endotoxin proteins or nucleic acids in products or organisms.
[0014] The following embodiments are encompassed by the present invention:
[0015] 1. A recombinant nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
[0016] a) the nucleotide sequence set forth in any of SEQ ID NO:1-12;
[0017] b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:13-24; and
[0018] c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:13-24.
[0019] 2. The recombinant nucleic acid molecule of embodiment 1, wherein said nucleotide sequence is a synthetic sequence that has been designed for expression in a plant.
[0020] 3. The recombinant nucleic acid molecule of embodiment 2, wherein said sequence is set forth in any of SEQ ID NO:25-44.
[0021] 4. The recombinant nucleic acid molecule of claim 1, wherein said nucleotide sequence is operably linked to a promoter capable of directing expression of said nucleotide sequence in a plant cell.
[0022] 5. A vector comprising the nucleic acid molecule of embodiment 1.
[0023] 6. The vector of embodiment 5, further comprising a nucleic acid molecule encoding a heterologous polypeptide.
[0024] 7. A host cell that contains the vector of embodiment 5.
[0025] 8. The host cell of embodiment 7 that is a bacterial host cell.
[0026] 9. The host cell of embodiment 7 that is a plant cell.
[0027] 10. A transgenic plant comprising the host cell of embodiment 9.
[0028] 11. The transgenic plant of embodiment 10, wherein said plant is selected from the group consisting of maize, sorghum, wheat, cabbage, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.
[0029] 12. A transgenic seed comprising the nucleic acid molecule of embodiment 1.
[0030] 13. A recombinant polypeptide with pesticidal activity, selected from the group consisting of:
[0031] a) a polypeptide comprising the amino acid sequence of any of SEQ ID NO:13-24;
[0032] b) a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:13-24; and
[0033] c) a polypeptide that is encoded by any of SEQ ID NO:1-12.
[0034] 14. The polypeptide of embodiment 13 further comprising heterologous amino acid sequences.
[0035] 15. A composition comprising the recombinant polypeptide of embodiment 13.
[0036] 16. The composition of embodiment 15, wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.
[0037] 17. The composition of embodiment 15, wherein said composition is prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of bacterial cells.
[0038] 18. The composition of embodiment 15, comprising from about 1% to about 99% by weight of said polypeptide.
[0039] 19. A method for controlling a lepidopteran, coleopteran, heteropteran, nematode, or dipteran pest population comprising contacting said population with a pesticidally-effective amount of the polypeptide of embodiment 13.
[0040] 20. A method for killing a lepidopteran, coleopteran, heteropteran, nematode, or dipteran pest, comprising contacting said pest with, or feeding to said pest, a pesticidally-effective amount of the polypeptide of embodiment 13.
[0041] 21. A method for producing a polypeptide with pesticidal activity, comprising culturing the host cell of embodiment 7 under conditions in which the nucleic acid molecule encoding the polypeptide is expressed.
[0042] 22. A plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
[0043] a) the nucleotide sequence set forth in any of SEQ ID NO:1-12;
[0044] b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:13-24; and
[0045] c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:13-24;
[0046] wherein said nucleotide sequence is operably linked to a promoter that drives expression of a coding sequence in a plant cell.
[0047] 23. The plant of embodiment 22, wherein said plant is a plant cell.
[0048] 24. A method for protecting a plant from a pest, comprising expressing in a plant or cell thereof a nucleotide sequence that encodes a pesticidal polypeptide, wherein said nucleotide sequence is selected from the group consisting of:
[0049] a) the nucleotide sequence set forth in any of SEQ ID NO:1-12;
[0050] b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:13-24; and
[0051] c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:13-24.
[0052] 25. The method of embodiment 24, wherein said plant produces a pesticidal polypeptide having pesticidal activity against a lepidopteran, coleopteran, heteropteran, nematode, or dipteran pest.
[0053] 26. A method for increasing yield in a plant comprising growing in a field a plant of or a seed thereof having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a protein having pesticidal activity, wherein said nucleotide sequence is selected from the group consisting of:
[0054] a) the nucleotide sequence set forth in any of SEQ ID NO:1-12;
[0055] b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:13-24; and
[0056] c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any of SEQ ID NO:13-24;
[0057] wherein said field is infested with a pest against which said polypeptide has pesticidal activity.
DETAILED DESCRIPTION
[0058] The present invention is drawn to compositions and methods for regulating pest resistance in organisms, particularly plants or plant cells. The methods involve transforming organisms with a nucleotide sequence encoding a delta-endotoxin protein of the invention. In particular, the nucleotide sequences of the invention are useful for preparing plants and microorganisms that possess pesticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are delta-endotoxin nucleic acids and proteins of Bacillus thuringiensis. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other delta-endotoxin genes, and for the generation of altered pesticidal proteins by methods known in the art, such as domain swapping or DNA shuffling. The proteins find use in controlling or killing lepidopteran, coleopteran, and nematode pest populations, and for producing compositions with pesticidal activity.
[0059] By "delta-endotoxin" is intended a toxin from Bacillus thuringiensis that has toxic activity against one or more pests, including, but not limited to, members of the Lepidoptera, Diptera, and Coleoptera orders or members of the Nematoda phylum, or a protein that has homology to such a protein. In some cases, delta-endotoxin proteins have been isolated from other organisms, including Clostridium bifermentans and Paenibacillus popilliae. Delta-endotoxin proteins include amino acid sequences deduced from the full-length nucleotide sequences disclosed herein, and amino acid sequences that are shorter than the full-length sequences, either due to the use of an alternate downstream start site, or due to processing that produces a shorter protein having pesticidal activity. Processing may occur in the organism the protein is expressed in, or in the pest after ingestion of the protein.
[0060] In various embodiments, the sequences disclosed herein have homology to delta-endotoxin proteins. Delta-endotoxins include proteins identified as cry1 through cry53, cyt1 and cyt2, and Cyt-like toxin. There are currently over 250 known species of delta-endotoxins with a wide range of specificities and toxicities. For an expansive list see Crickmore et al. (1998), Microbiol. Mol. Biol. Rev. 62:807-813, and for regular updates see Crickmore et al. (2003) "Bacillus thuringiensis toxin nomenclature," at www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.
[0061] In other embodiments, the sequences encompassed herein are MTX-like sequences. The term "MTX" is used in the art to delineate a set of pesticidal proteins that are produced by Bacillus sphaericus. The first of these, often referred to in the art as MTX1, is synthesized as a parasporal crystal which is toxic to mosquitoes. The major components of the crystal are two proteins of 51 and 42 kDa. Since the presence of both proteins are required for toxicity, MTX1 is considered a "binary" toxin (Baumann et al. (1991) Microbiol. Rev. 55:425-436).
[0062] By analysis of different Bacillus sphaericus strains with differing toxicities, two new classes of MTX toxins have been identified. MTX2 and MTX3 represent separate, related classes of pesticidal toxins that exhibit pesticidal activity. See, for example, Baumann et al. (1991) Microbiol. Rev. 55:425-436, herein incorporated by reference in its entirety. MTX2 is a 100-kDa toxin. More recently MTX3 has been identified as a separate toxin, though the amino acid sequence of MTX3 from B. sphaericus is 38% identical to the MTX2 toxin of B. sphaericus SSII-1 (Liu, et al. (1996) Appl. Environ. Microbiol. 62:2174-2176). Mtx toxins may be useful for both increasing the insecticidal activity of B. sphaericus strains and managing the evolution of resistance to the Bin toxins in mosquito populations (Wirth et al. (2007) Appl Environ Microbiol 73(19):6066-6071).
[0063] Provided herein are novel isolated nucleotide sequences that confer pesticidal activity. Also provided are the amino acid sequences of the delta-endotoxin proteins. The 10 protein resulting from translation of this gene allows cells to control or kill pests that ingest it.
Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof
[0064] One aspect of the invention pertains to isolated or recombinant nucleic acid molecules comprising nucleotide sequences encoding delta-endotoxin proteins and polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify delta-endotoxin encoding nucleic acids. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
[0065] An "isolated" nucleic acid sequence (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a recombinant bacterial or plant host cell. In some embodiments, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the invention, "isolated" when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the isolated delta-endotoxin encoding nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A delta-endotoxin protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-delta-endotoxin protein (also referred to herein as a "contaminating protein").
[0066] Nucleotide sequences encoding the proteins of the present invention include the sequence set forth in SEQ ID NO:1-12, and variants, fragments, and complements thereof. By "complement" is intended a nucleotide sequence that is sufficiently complementary to a given nucleotide sequence such that it can hybridize to the given nucleotide sequence to thereby form a stable duplex. The corresponding amino acid sequence for the delta-endotoxin protein encoded by this nucleotide sequence are set forth in SEQ ID NO:13-24.
[0067] Nucleic acid molecules that are fragments of these delta-endotoxin encoding nucleotide sequences are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence encoding a delta-endotoxin protein. A fragment of a nucleotide sequence may encode a biologically active portion of a delta-endotoxin protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Nucleic acid molecules that are fragments of a delta-endotoxin nucleotide sequence comprise at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350 contiguous nucleotides, or up to the number of nucleotides present in a full-length delta-endotoxin encoding nucleotide sequence disclosed herein depending upon the intended use. By "contiguous" nucleotides is intended nucleotide residues that are immediately adjacent to one another. Fragments of the nucleotide sequences of the present invention will encode protein fragments that retain the biological activity of the delta-endotoxin protein and, hence, retain pesticidal activity. By "retains activity" is intended that the fragment will have at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or higher of the pesticidal activity of the delta-endotoxin protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety.
[0068] A fragment of a delta-endotoxin encoding nucleotide sequence that encodes a biologically active portion of a protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100 contiguous amino acids, or up to the total number of amino acids present in a full-length delta-endotoxin protein of the invention. In some embodiments, the fragment is a proteolytic cleavage fragment. For example, the proteolytic cleavage fragment may have an N-terminal or a C-terminal truncation of at least about 100 amino acids, about 120, about 130, about 140, about 150, or about 160 amino acids relative to SEQ ID NO:13-24. In some embodiments, the fragments encompassed herein result from the removal of the C-terminal crystallization domain, e.g., by proteolysis or by insertion of a stop codon in the coding sequence.
[0069] Preferred delta-endotoxin proteins of the present invention are encoded by a nucleotide sequence sufficiently identical to the nucleotide sequence of SEQ ID NO:1-12. By "sufficiently identical" is intended an amino acid or nucleotide sequence that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity compared to a reference sequence using one of the alignment programs described herein using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.
[0070] To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length. In another embodiment, the comparison is across the entirety of the reference sequence (e.g., across the entirety of one of SEQ ID NO:1-12, or across the entirety of one of SEQ ID NO:13-24). The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
[0071] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to delta-endotoxin-like nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to delta-endotoxin protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection.
[0072] Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA/amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOCT®. GENEDOC® (Karl Nicholas) allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
[0073] Unless otherwise stated, GAP Version 10, which uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used to determine sequence identity or similarity using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity or % similarity for an amino acid sequence using GAP weight of 8 and length weight of 2, and the BLOSUM62 scoring program. Equivalent programs may also be used. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
[0074] The invention also encompasses variant nucleic acid molecules. "Variants" of the delta-endotoxin encoding nucleotide sequences include those sequences that encode the delta-endotoxin proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code as well as those that are sufficiently identical as discussed above. Naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the delta-endotoxin proteins disclosed in the present invention as discussed below. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining pesticidal activity. By "retains activity" is intended that the variant will have at least about 30%, at least about 50%, at least about 70%, or at least about 80% of the pesticidal activity of the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83: 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety.
[0075] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded delta-endotoxin proteins, without altering the biological activity of the proteins. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
[0076] For example, conservative amino acid substitutions may be made at one or more predicted, nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered from the wild-type sequence of a delta-endotoxin protein without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0077] Delta-endotoxins generally have five conserved sequence domains, and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II consists of three beta-sheets arranged in a Greek key configuration, and domain III consists of two antiparallel beta-sheets in "jelly-roll" formation (de Maagd et al., 2001, supra). Domains II and III are involved in receptor recognition and binding, and are therefore considered determinants of toxin specificity.
[0078] Amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of the amino acid sequences of the present invention and known delta-endotoxin sequences. Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of the amino acid sequences of the present invention and known delta-endotoxin sequences. However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues.
[0079] Alternatively, variant nucleotide sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer delta-endotoxin activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
[0080] Using methods such as PCR, hybridization, and the like corresponding delta-endotoxin sequences can be identified, such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY).
[0081] In a hybridization method, all or part of the delta-endotoxin nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sam brook and Russell, 2001, supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known delta-endotoxin-encoding nucleotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleotide sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of delta-endotoxin encoding nucleotide sequence of the invention or a fragment or variant thereof. Methods for the preparation of probes for hybridization are generally known in the art and are disclosed in Sambrook and Russell, 2001, supra herein incorporated by reference.
[0082] For example, an entire delta-endotoxin sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding delta-endotoxin-like sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique and are preferably at least about 10 nucleotides in length, or at least about 20 nucleotides in length. Such probes may be used to amplify corresponding delta-endotoxin sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0083] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
[0084] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
[0085] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Isolated Proteins and Variants and Fragments Thereof
[0086] Delta-endotoxin proteins are also encompassed within the present invention. By "delta-endotoxin protein" is intended a protein having the amino acid sequence set forth in SEQ ID NO:13-24. Fragments, biologically active portions, and variants thereof are also provided, and may be used to practice the methods of the present invention. An "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. An "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
[0087] "Fragments" or "biologically active portions" include polypeptide fragments comprising amino acid sequences sufficiently identical to the amino acid sequence set forth in any of SEQ ID NO:13-24 and that exhibit pesticidal activity. A biologically active portion of a delta-endotoxin protein can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for pesticidal activity. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety. As used here, a fragment comprises at least 8 contiguous amino acids of SEQ ID NO:13-24. The invention encompasses other fragments, however, such as any fragment in the protein greater than about 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids.
[0088] By "variants" is intended proteins or polypeptides having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any of SEQ ID NO:13-24. Variants also include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecule of SEQ ID NO:1-12, or a complement thereof, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining pesticidal activity. In some embodiments, the variants have improved activity relative to the native protein. Methods for measuring pesticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety.
[0089] Bacterial genes, such as the axmi genes of this invention, quite often possess multiple methionine initiation co dons in proximity to the start of the open reading frame. Often, translation initiation at one or more of these start codons will lead to generation of a functional protein. These start codons can include ATG codons. However, bacteria such as Bacillus sp. also recognize the codon GTG as a start codon, and proteins that initiate translation at GTG codons contain a methionine at the first amino acid. Furthermore, it is not often determined a priori which of these codons are used naturally in the bacterium. Thus, it is understood that use of one of the alternate methionine codons may also lead to generation of delta-endotoxin proteins that encode pesticidal activity. These delta-endotoxin proteins are encompassed in the present invention and may be used in the methods of the present invention.
[0090] Antibodies to the polypeptides of the present invention, or to variants or fragments thereof, are also encompassed. Methods for producing antibodies are well known in the art (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; and U.S. Pat. No. 4,196,265).
Altered or Improved Variants
[0091] It is recognized that DNA sequences of a delta-endotoxin may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by a delta-endotoxin of the present invention. This protein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of SEQ 10 NO:13-24, including up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 105, about 110, about 115, about 120, about 125, about 130 or more amino acid substitutions, deletions or insertions.
[0092] Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a delta-endotoxin protein can be prepared by mutations in the DNA. This may also be accomplished by one of several forms of mutagenesis and/or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess the desired pesticidal activity. However, it is understood that the ability of a delta-endotoxin to confer pesticidal activity may be improved by the use of such techniques upon the compositions of this invention. For example, one may express a delta-endotoxin in host cells that exhibit high rates of base misincorporation during DNA replication, such as XL-1 Red (Stratagene). After propagation in such strains, one can isolate the delta-endotoxin DNA (for example by preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a vector), culture the delta-endotoxin mutations in a non-mutagenic strain, and identify mutated delta-endotoxin genes with pesticidal activity, for example by performing an assay to test for pesticidal activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293. Such assays can include contacting plants with one or more pests and determining the plant's ability to survive and/or cause the death of the pests. Examples of mutations that result in increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.
[0093] Alternatively, alterations may be made to the protein sequence of many proteins at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.
[0094] Variant nucleotide and amino acid sequences of the present invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different delta-endotoxin protein coding regions can be used to create a new delta-endotoxin protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a delta-endotoxin gene of the invention and other known delta-endotoxin genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
[0095] Domain swapping or shuffling is another mechanism for generating altered delta-endotoxin proteins. Domains II and III may be swapped between delta-endotoxin proteins, resulting in hybrid or chimeric toxins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925).
Vectors
[0096] A delta-endotoxin sequence of the invention may be provided in an expression cassette for expression in a plant of interest. By "plant expression cassette" is intended a DNA construct that is capable of resulting in the expression of a protein from an open reading frame in a plant cell. Typically these contain a promoter and a coding sequence. Often, such constructs will also contain a 3' untranslated region. Such constructs may contain a "signal sequence" or "leader sequence" to facilitate co-translational or post-translational transport of the peptide to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus.
[0097] By "signal sequence" is intended a sequence that is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation. By "leader sequence" is intended any sequence that when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like.
[0098] By "plant transformation vector" is intended a DNA molecule that is necessary for efficient transformation of a plant cell. Such a molecule may consist of one or more plant expression cassettes, and may be organized into more than one "vector" DNA molecule. For example, binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). "Vector" refers to a nucleic acid construct designed for transfer between different host cells. "Expression vector" refers to a vector that has the ability to incorporate, integrate and express heterologous DNA sequences or fragments in a foreign cell. The cassette will include 5' and 3' regulatory sequences operably linked to a sequence of the invention. By "operably linked" is intended a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
[0099] "Promoter" refers to a nucleic acid sequence that functions to direct transcription of a downstream coding sequence. The promoter together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") are necessary for the expression of a DNA sequence of interest.
[0100] Such an expression cassette is provided with a plurality of restriction sites for insertion of the delta-endotoxin sequence to be under the transcriptional regulation of the regulatory regions.
[0101] The expression cassette will include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a DNA sequence of the invention, and a translational and transcriptional termination region (Le., termination region) functional in plants. The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "native" or "homologous" to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the DNA sequence of the invention, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
[0102] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the DNA sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0103] Where appropriate, the gene(s) may be optimized for increased expression in the transformed host cell. That is, the genes can be synthesized using host cell-preferred codons for improved expression, or may be synthesized using codons at a host-preferred codon usage frequency. Generally, the GC content of the gene will be increased. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
[0104] In one embodiment, the delta-endotoxin is targeted to the chloroplast for expression. In this manner, where the delta-endotoxin is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the delta-endotoxin to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.
[0105] The delta-endotoxin gene to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.
Plant Transformation
[0106] Methods of the invention involve introducing a nucleotide construct into a plant. By "introducing" is intended to present to the plant the nucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not require that a particular method for introducing a nucleotide construct to a plant is used, only that the nucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
[0107] By "plant" is intended whole plants, plant organs (e.g., leaves, sterns, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
[0108] "Transgenic plants" or "transformed plants" or "stably transformed" plants or cells or tissues refers to plants that have incorporated or integrated exogenous nucleic acid sequences or DNA fragments into the plant cell. These nucleic acid sequences include those that are exogenous, or not present in the untransformed plant cell, as well as those that may be endogenous, or present in the untransformed plant cell. "Heterologous" generally refers to the nucleic acid sequences that are not endogenous to the cell or part of the native genome in which they are present, and have been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like.
[0109] The transgenic plants of the invention express one or more of the pesticidal sequences disclosed herein. In various embodiments, the transgenic plant further comprises one or more additional genes for insect resistance, for example, one or more additional genes for controlling coleopteran, lepidopteran, heteropteran, or nematode pests. It will be understood by one of skill in the art that the transgenic plant may comprise any gene imparting an agronomic trait of interest.
[0110] Transformation of plant cells can be accomplished by one of several techniques known in the art. The delta-endotoxin gene of the invention may be modified to obtain or enhance expression in plant cells. Typically a construct that expresses such a protein would contain a promoter to drive transcription of the gene, as well as a 3' untranslated region to allow transcription termination and polyadenylation. The organization of such constructs is well known in the art. In some instances, it may be useful to engineer the gene such that the resulting peptide is secreted, or otherwise targeted within the plant cell. For example, the gene can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum. It may also be preferable to engineer the plant expression cassette to contain an intron, such that mRNA processing of the intron is required for expression.
[0111] Typically this "plant expression cassette" will be inserted into a "plant transformation vector". This plant transformation vector may be comprised of one or more DNA vectors needed for achieving plant transformation. For example, it is a common practice in the art to utilize plant transformation vectors that are comprised of more than one contiguous DNA segment. These vectors are often referred to in the art as "binary vectors". Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules. Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a "gene of interest" (a gene engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired). Also present on this plasmid vector are sequences required for bacterial replication. The cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein. For example, the selectable marker gene and the delta-endotoxin are located between the left and right borders. Often a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells. This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as is understood in the art (Hellens and Mullineaux (2000) Trends in Plant Science 5:446-451). Several types of Agrobacterium strains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used for plant transformation. The second plasmid vector is not necessary for transforming the plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
[0112] In general, plant transformation methods involve transferring heterologous DNA into target plant cells (e.g. immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass. Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent. The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grows into a mature plant and produces fertile seeds (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of the same medium and cultured routinely. A general description of the techniques and methods for generating transgenic plants are found in Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239 and Bommineni and Jauhar (1997) Maydica 42:107-120. Since the transformed material contains many cells; both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells. The ability to kill non-transformed cells and allow transformed cells to proliferate results in transformed plant cultures. Often, the ability to remove non-transformed cells is a limitation to rapid recovery of transformed plant cells and successful generation of transgenic plants.
[0113] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Generation of transgenic plants may be performed by one of several methods, including, but not limited to, microinjection, electroporation, direct gene transfer, introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, ballistic particle acceleration, aerosol beam transformation (U.S. Published Application No. 20010026941; U.S. Pat. No. 4,945,050; International Publication No. WO 91/00915; U.S. Published Application No. 2002015066), Lec1 transformation, and various other non-particle direct-mediated methods to transfer DNA.
[0114] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
[0115] Following integration of heterologous foreign DNA into plant cells, one then applies a maximum threshold level of appropriate selection in the medium to kill the untransformed cells and separate and proliferate the putatively transformed cells that survive from this selection treatment by transferring regularly to a fresh medium. By continuous passage and challenge with appropriate selection, one identifies and proliferates the cells that are transformed with the plasmid vector. Molecular and biochemical methods can then be used to confirm the presence of the integrated heterologous gene of interest into the genome of the transgenic plant.
[0116] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
Evaluation of Plant Transformation
[0117] Following introduction of heterologous foreign DNA into plant cells, the transformation or integration of heterologous gene in the plant genome is confirmed by various methods such as analysis of nucleic acids, proteins and metabolites associated with the integrated gene.
[0118] PCR analysis is a rapid method to screen transformed cells, tissue or shoots for the presence of incorporated gene at the earlier stage before transplanting into the soil (Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). PCR is carried out using oligonucleotide primers specific to the gene of interest or Agrobacterium vector background, etc.
[0119] Plant transformation may be confirmed by Southern blot analysis of genomic DNA (Sambrook and Russell, 2001, supra). In general, total DNA is extracted from the transformant, digested with appropriate restriction enzymes, fractionated in an agarose gel and transferred to a nitrocellulose or nylon membrane. The membrane or "blot" is then probed with, for example, radiolabeled 32P target DNA fragment to confirm the integration of introduced gene into the plant genome according to standard techniques (Sambrook and Russell, 2001, supra).
[0120] In Northern blot analysis, RNA is isolated from specific tissues of transformant, fractionated in a formaldehyde agarose gel, and blotted onto a nylon filter according to standard procedures that are routinely used in the art (Sambrook and Russell, 2001, supra). Expression of RNA encoded by the delta-endotoxin is then tested by hybridizing the filter to a radioactive probe derived from a delta-endotoxin, by methods known in the art (Sambrook and Russell, 2001, supra).
[0121] Western blot, biochemical assays and the like may be carried out on the transgenic plants to confirm the presence of protein encoded by the delta-endotoxin gene by standard procedures (Sambrook and Russell, 2001, supra) using antibodies that bind to one or more epitopes present on the delta-endotoxin protein.
Pesticidal Activity in Plants
[0122] In another aspect of the invention, one may generate transgenic plants expressing a delta-endotoxin that has pesticidal activity. Methods described above by way of example may be utilized to generate transgenic plants, but the manner in which the transgenic plant cells are generated is not critical to this invention. Methods known or described in the art such as Agrobacterium-mediated transformation, biolistic transformation, and non-particle-mediated methods may be used at the discretion of the experimenter. Plants expressing a delta-endotoxin may be isolated by common methods described in the art, for example by transformation of callus, selection of transformed callus, and regeneration of fertile plants from such transgenic callus. In such process, one may use any gene as a selectable marker so long as its expression in plant cells confers ability to identify or select for transformed cells.
[0123] A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like. Other genes that encode a product involved in chloroplast metabolism may also be used as selectable markers. For example, genes that provide resistance to plant herbicides such as glyphosate, bromoxynil, or imidazolinone may find particular use. Such genes have been reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314 (bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene). Additionally, the genes disclosed herein are useful as markers to assess transformation of bacterial or plant cells. Methods for detecting the presence of a transgene in a plant, plant organ (e.g., leaves, stems, roots, etc.), seed, plant cell, propagule, embryo or progeny of the same are well known in the art. In one embodiment, the presence of the transgene is detected by testing for pesticidal activity.
[0124] Fertile plants expressing a delta-endotoxin may be tested for pesticidal activity, and the plants showing optimal activity selected for further breeding. Methods are available in the art to assay for pest activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293.
[0125] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plants of interest include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
[0126] Vegetables include, but are not limited to, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. Preferably, plants of the present invention are crop plants (for example, maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape., etc.).
Use in Pest Control
[0127] General methods for employing strains comprising a nucleotide sequence of the present invention, or a variant thereof, in pesticide control or in engineering other organisms as pesticidal agents are known in the art. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.
[0128] The Bacillus strains containing a nucleotide sequence of the present invention, or a variant thereof, or the microorganisms that have been genetically altered to contain a pesticidal gene and protein may be used for protecting agricultural crops and products from pests. In one aspect of the invention, whole, i.e., unlysed, cells of a toxin (pesticide)-producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s).
[0129] Alternatively, the pesticide is produced by introducing a delta-endotoxin gene into a cellular host. Expression of the delta-endotoxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. In one aspect of this invention, these cells are then treated under conditions that prolong the activity of the toxin produced in the cell when the cell is applied to the environment of target pest(s). The resulting product retains the toxicity of the toxin. These naturally encapsulated pesticides may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest, e.g., soil, water, and foliage of plants. See, for example EPA 0192319, and the references cited therein. Alternatively, one may formulate the cells expressing a gene of this invention such as to allow application of the resulting material as a pesticide.
[0130] Pesticidal Compositions
[0131] The active ingredients of the present invention are normally applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other compounds. These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, pesticidal soaps, dormant oils, polymers, and/or time-release or biodegradable carrier formulations that permit long-term dosing of a target area following a single application of the formulation. They can also be selective herbicides, chemical insecticides, virucides, microbicides, amoebicides, pesticides, fungicides, bacteriocides, nematocides, molluscicides or mixtures of several of these preparations, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Likewise the formulations may be prepared into edible "baits" or fashioned into pest "traps" to permit feeding or ingestion by a target pest of the pesticidal formulation.
[0132] Methods of applying an active ingredient of the present invention or an agrochemical composition of the present invention that contains at least one of the pesticidal proteins produced by the bacterial strains of the present invention include leaf application, seed coating and soil application. The number of applications and the rate of application depend on the intensity of infestation by the corresponding pest.
[0133] The composition may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenation, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of cells comprising the polypeptide. In all such compositions that contain at least one such pesticidal polypeptide, the polypeptide may be present in a concentration of from about 1% to about 99% by weight.
[0134] Lepidopteran, coleopteran, or nematode pests may be killed or reduced in numbers in a given area by the methods of the invention, or may be prophylactically applied to an environmental area to prevent infestation by a susceptible pest. Preferably the pest ingests, or is contacted with, a pesticidally-effective amount of the polypeptide. By "pesticidally-effective amount" is intended an amount of the pesticide that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development. This amount will vary depending on such factors as, for example, the specific target pests to be controlled, the specific environment, location, plant, crop, or agricultural site to be treated, the environmental conditions, and the method, rate, concentration, stability, and quantity of application of the pesticidally-effective polypeptide composition. The formulations may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of pest infestation.
[0135] The pesticide compositions described may be made by formulating either the bacterial cell, crystal and/or spore suspension, or isolated protein component with the desired agriculturally-acceptable carrier. The compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline or other buffer. The formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. The term "agriculturally-acceptable carrier" covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in pesticide formulation technology; these are well known to those skilled in pesticide formulation. The formulations may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional formulation techniques. Suitable formulations and application methods are described in U.S. Pat. No. 6,468,523, herein incorporated by reference.
[0136] The plants can also be treated with one or more chemical compositions, including one or more herbicide, insecticides, or fungicides. Exemplary chemical compositions include: Fruits/Vegetables Herbicides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Halosulfuron Gowan, Paraquat, Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam; Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, Diazinon, Malathion, Abamectin, Cyfluthrinlbeta-cyfluthrin, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran, Fluacrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen, Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr, Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen, lmidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate, Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox, Methomyl, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on; Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs, Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam, Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid, Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin, Pyraclostrobin, Cytlufenamid, Boscalid; Cereals Herbicides: Isoproturon, Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, Fenoxaprop, Florasulam, Fluroxypyr, Metsulfuron, Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph, Epoxiconazole, Kresoximmethyl, Quinoxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides: Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, β-cytluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides Diazinon, Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Carbofuran, Benfuracarb; Rice Fungicides Thiophanate-methyl, Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos, Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole, Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; Cotton Herbicides: Diuron, Fluometuron, MSMA, Oxytluorfen, Prometryn, Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate, Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron, Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin, Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid, Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl, Flonicamid, Flubendiamide, Triflurnuron, Rynaxypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thiamethoxarn, Thiacloprid, Dinetofuran, Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides: Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor, Bentazone, Tritluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim, Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole, Deltamethrin, B-Cyfluthrin, gamma and lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb, beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides: Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides: Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid, Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin, 4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on, Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; Canola Herbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides: Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin, Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran, β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr, 4-[[(6-Chlorpyridin-3-yl)methyl] (2,2-difluorethyl)amino] furan-2(5H)-on.
[0137] "Pest" includes but is not limited to, insects, fungi, bacteria, nematodes, mites, ticks, and the like. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera, Lepidoptera, and Diptera.
[0138] The order Coleoptera includes the suborders Adephaga and Polyphaga. Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea, while suborder Polyphaga includes the superfamilies Hydrophiloidea, Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea, Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea, Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea, Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes the families Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoidea includes the family Gyrinidae. Superfamily Hydrophiloidea includes the family Hydrophilidae. Superfamily Staphylinoidea includes the families Silphidae and Staphylinidae. Superfamily Cantharoidea includes the families Cantharidae and Lampyridae. Superfamily Cleroidea includes the families Cleridae and Dermestidae. Superfamily Elateroidea includes the families Elateridae and Buprestidae. Superfamily Cucujoidea includes the family Coccinellidae. Superfamily Meloidea includes the family Meloidae. Superfamily Tenebrionoidea includes the family Tenebrionidae. Superfamily Scarabaeoidea includes the families Passalidae and Scarabaeidae. Superfamily Cerambycoidea includes the family Cerambycidae. Superfamily Chrysomeloidea includes the family Chrysomelidae. Superfamily Curculionoidea includes the families Curculionidae and Scolytidae.
[0139] The order Diptera includes the Suborders Nematocera, Brachycera, and Cyclorrhapha. Suborder Nematocera includes the families Tipulidae, Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae, Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the families Stratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae, and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschiza and Aschiza. Division Aschiza includes the families Phoridae, Syrphidae, and Conopidae. Division Aschiza includes the Sections Acalyptratae and Calyptratae. Section Acalyptratae includes the families Otitidae, Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptratae includes the families Hippoboscidae, Oestridae, Tachinidae, Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.
[0140] The order Lepidoptera includes the families Papilionidae, Pieridae, Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae, Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae, and Tineidae.
[0141] Nematodes include parasitic nematodes such as root-knot, cyst, and lesion nematodes, including Heterodera spp., Meloidogyne spp., and Globodera spp.; particularly members of the cyst nematodes, including, but not limited to, Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode); and Globodera rostochiensis and Globodera pailida (potato cyst nematodes). Lesion nematodes include Pratylenchus spp.
[0142] Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera/rugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephala borealis, northern masked chafer (white grub); Cyclocephala immaculata, southern masked chafer (white grub); Popillia japonica, Japanese beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis, corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta, thief ant; Tetranychus urticae, twospotted spider mite; Sorghum: Chilo partellus, sorghum borer; Spodoptera, frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, lesser cornstalk borer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissus leucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghum midge; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, army worm; Spodoptera rugiperda, fall armyworm; Elasmopalpus lignosellus, lesser cornstalk borer; Agrotis orthogonia, western cutworm; Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus, cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabrotica undecimpunctata howardi, southern corn rootworm; Russian wheat aphid; Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Melanoplus sanguinipes, migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosis mosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemya coarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephus cinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower: Suleima helianthana, sunflower bud moth; Homoeosoma electellum, sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrus gibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seed midge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophora gossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphis gossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper; Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris, tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Thrips tabaci, onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae, twospotted spider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodoptera frugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspis brunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil; Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Soybean: Pseudoplusia includens, soybean looper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, green cloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, green stink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplus differentialis, differential grasshopper; Hylemya platura, seedcorn maggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychus turkestani, strawberry spider mite; Tetranychus urticae, twospotted spider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum, greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternum hilare, green stink bug; Euschistus servus, brown stink bug; Delia platura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbage aphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Bertha armyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Root maggots.
Methods for Increasing Plant Yield
[0143] Methods for increasing plant yield are provided. The methods comprise providing a plant or plant cell expressing a polynucleotide encoding the pesticidal polypeptide sequence disclosed herein and growing the plant or a seed thereof in a field infested with a pest against which said polypeptide has pesticidal activity. In some embodiments, the polypeptide has pesticidal activity against a lepidopteran, coleopteran, dipteran, hemipteran, or nematode pest, and said field is infested with a lepidopteran, hemipteran, coleopteran, dipteran, or nematode pest.
[0144] As defined herein, the "yield" of the plant refers to the quality and/or quantity of biomass produced by the plant. By "biomass" is intended any measured plant product. An increase in biomass production is any improvement in the yield of the measured plant product. Increasing plant yield has several commercial applications. For example, increasing plant leaf biomass may increase the yield of leafy vegetables for human or animal consumption. Additionally, increasing leaf biomass can be used to increase production of plant-derived pharmaceutical or industrial products. An increase in yield can comprise any statistically significant increase including, but not limited to, at least a 1% increase, at least a 3% increase, at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater increase in yield compared to a plant not expressing the pesticidal sequence.
[0145] In specific methods, plant yield is increased as a result of improved pest resistance of a plant expressing a pesticidal protein disclosed herein. Expression of the pesticidal protein results in a reduced ability of a pest to infest or feed on the plant, thus improving plant yield.
[0146] The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Example 1
Discovery of Novel Toxin Genes from Bacillus thuringiensis Strain ATX15903
[0147] Novel pesticidal genes are identified from the bacterial strains listed in Table 1 using methods such as:
Method 1
[0148] Preparation of extrachromosomal DNA from the strain, which includes plasmids that typically harbor delta-endotoxin genes
[0149] Mechanical shearing of extrachromosomal DNA to generate size-distributed fragments
[0150] Cloning of ˜2 Kb to ˜10 Kb fragments of extrachromosomal DNA
[0151] Outgrowth of ˜1500 clones of the extrachromosomal DNA
[0152] Partial sequencing of the 1500 clones using primers specific to the cloning vector (end reads)
[0153] Identification of putative toxin genes via homology analysis via the MiDAS approach (as described in U.S. Patent Publication No. 20040014091, which is herein incorporated by reference in its entirety)
[0154] Sequence finishing (walking) of clones containing fragments of the putative toxin genes of interest
Method 2
[0154]
[0155] Preparation of extrachromosomal DNA from the strain (which contains a mixture of some or all of the following: plasmids of various size; phage chromosomes; genomic DNA fragments not separated by the purification protocol; other uncharacterized extrachromosomal molecules)
[0156] Mechanical or enzymatic shearing of the extrachromosomal DNA to generate size-distributed fragments
[0157] Sequencing of the fragmented DNA by high-throughput pyrosequencing methods
[0158] Identification of putative toxin genes via homology and/or other computational analyses
TABLE-US-00001
[0158] TABLE 1 Novel pesticidal genes from Strain ATX15903 Molecular Weight Percent (kDa) Amino Identity to and Length Nucleotide Acid Closest Closest (amino SEQ ID SEQ ID Sequence in Sequence Gene acids) NO: NO: Art in Art axmi077 141.6, 1 13 35.3% Cry43Ba1 1249 32.5% axmi087 axmi078 91.9, 2 14 28.7% Cry24Ba1 820 26% axmi014 axmi083 79.8, 3 15 75.2% Cry30Aa1 710 32% axmi007 axmi084 63.8, 4 16 84.3% Cry39Orf2 563 87% axmi086 axmi085 77.7, 5 17 31.7% Cry8Aa1 690 66% axmi009 axmi086 64.6, 6 18 85.2% Cry39Orf2 571 88% axmi090 axmi089 69.4, 7 19 36% Cry29Aa1 627 31% axmi085 axmi090 64.3, 8 20 85.9% Cry39orf2 566 89% axmi086 axmi094 33.0, 9 21 19.8% Mtx3 296 26% axmi095 axmi095 34.4 10 22 22.3% Mtx2 308 26% axmi094 Axmi105 141.2, 11 23 37% Cry43Aa2 1244 74% axmi077 axmi0106 58.0, 12 24 58% Cyt1Ca1 521
Example 2
Expression in Bacillus
[0159] The insecticidal gene disclosed herein is amplified by PCR from pAX980, and the PCR product is cloned into the Bacillus expression vector pAX916, or another suitable vector, by methods well known in the art. The resulting Bacillus strain, containing the vector with axmi gene is cultured on a conventional growth media, such as CYS media (10 g/l Bacto-casitone; 3 g/l yeast extract; 6 g/l KH2PO4; 14 g/l K2HPO4; 0.5 mM MgSO4; 0.05 mM MnCl2; 0.05 mM FeSO4), until sporulation is evident by microscopic examination. Samples are prepared and tested for activity in bioassays.
Example 3
Construction of Synthetic Sequences
[0160] In one aspect of the invention, synthetic axmi sequences were generated. These synthetic sequences have an altered DNA sequence relative to the parent axmi sequence, and encode a protein that is collinear with the parent AXMI protein to which it corresponds, but lacks the C-terminal "crystal domain" present in many delta-endotoxin proteins. Synthetic genes are presented in Table 2.
TABLE-US-00002 TABLE 2 Wildtype Gene Name Synthetic Gene Name SEQ ID NO: axmi077 axmi077bv01 25 axmi077bv02 26 axmi078 axmi078bv01 27 axmi078bv02 28 axmi083 axmi083_1bv01 29 axmi083_1bv02 30 axmi083_2bv01 31 axmi083_2bv02 32 axmi085 axmi085bv01 33 axmi085bv02 34 axmi089 axmi089bv01 35 axmi089bv02 36 axmi094 axmi094bv01 37 axmi094bv02 38 axmi095 axmi095bv01 39 axmi095bv02 40 asmi105 axmi105bv01 41 axmi106 axmi106bv01 43 axmi106bv02 44
[0161] In another aspect of the invention, modified versions of synthetic genes are designed such that the resulting peptide is targeted to a plant organelle, such as the endoplasmic reticulum or the apoplast. Peptide sequences known to result in targeting of fusion proteins to plant organelles are known in the art. For example, the N-terminal region of the acid phosphatase gene from the White Lupin Lupinus albus (Genebank ID GI:14276838; Miller et al. (2001). Plant Physiology 127: 594-606) is known in the art to result in endoplasmic reticulum targeting of heterologous proteins. If the resulting fusion protein also contains an endoplasmic retention sequence comprising the peptide N-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e. the "KDEL" motif (SEQ ID NO:45) at the C-terminus, the fusion protein will be targeted to the endoplasmic reticulum. If the fusion protein lacks an endoplasmic reticulum targeting sequence at the C-terminus, the protein will be targeted to the endoplasmic reticulum, but will ultimately be sequestered in the apoplast.
Example 4
Assays for Pesticidal Activity
[0162] The ability of a pesticidal protein to act as a pesticide upon a pest is often assessed in a number of ways. One way well known in the art is to perform a feeding assay. In such a feeding assay, one exposes the pest to a sample containing either compounds to be tested, or control samples. Often this is performed by placing the material to be tested, or a suitable dilution of such material, onto a material that the pest will ingest, such as an artificial diet. The material to be tested may be composed of a liquid, solid, or slurry. The material to be tested may be placed upon the surface and then allowed to dry. Alternatively, the material to be tested may be mixed with a molten artificial diet, then dispensed into the assay chamber. The assay chamber may be, for example, a cup, a dish, or a well of a microtiter plate.
[0163] Assays for sucking pests (for example aphids) may involve separating the test material from the insect by a partition, ideally a portion that can be pierced by the sucking mouth parts of the sucking insect, to allow ingestion of the test material. Often the test material is mixed with a feeding stimulant, such as sucrose, to promote ingestion of the test compound.
[0164] Other types of assays can include microinjection of the test material into the mouth, or gut of the pest, as well as development of transgenic plants, followed by test of the ability of the pest to feed upon the transgenic plant. Plant testing may involve isolation of the plant parts normally consumed, for example, small cages attached to a leaf, or isolation of entire plants in cages containing insects.
[0165] Other methods and approaches to assay pests are known in the art, and can be found, for example in Robertson, J. L. & H. K. Preisler. 1992. Pesticide bioassays, with arthropods. CRC, Boca Raton, Fla. Alternatively, assays are commonly described in the journals "Arthropod Management Tests" and "Journal of Economic Entomology" or by discussion with members of the Entomological Society of America (ESA).
Example 5
Vectoring of the Pesticidal Genes of the Invention for Plant Expression
[0166] Each of the coding regions of the genes of the invention are connected independently with appropriate promoter and terminator sequences for expression in plants. Such sequences are well known in the art and may include the rice actin promoter or maize ubiquitin promoter for expression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35S promoter for expression in dicots, and the nos or PinII terminators. Techniques for producing and confirming promoter--gene--terminator constructs also are well known in the art.
Example 6
Transformation of the Genes of the Invention into Plant Cells by Agrobacterium-Mediated Transformation
[0167] Ears are collected 8 to 12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are used for transformation. Embryos are plated scutellum side-up on a suitable incubation media, and incubated overnight at 25° C. in the dark. However, it is not necessary per se to incubate the embryos overnight. Embryos are contacted with an Agrobacterium strain containing the appropriate vectors for Ti plasmid mediated transfer for 5 to 10 minutes, and then plated onto co-cultivation media for 3 days (25° C. in the dark). After co-cultivation, explants are transferred to recovery period media for five days (at 25° C. in the dark). Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated as known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.
Example 7
Transformation of Maize Cells with the Pesticidal Genes of the Invention
[0168] Maize ears are collected 8 to 12 days after pollination. Embryos are isolated from the ears, and those embryos 0.8-1.5 mm in size are used for transformation. Embryos are plated scutellum side-up on a suitable incubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D), and incubated overnight at 25° C. in the dark.
[0169] The resulting explants are transferred to mesh squares (30-40 per plate), transferred onto osmotic media for 30-45 minutes, then transferred to a beaming plate (see, for example, PCT Publication No. WO/0138514 and U.S. Pat. No. 5,240,842).
[0170] DNA constructs designed to express the genes of the invention in plant cells are accelerated into plant tissue using an aerosol beam accelerator, using conditions essentially as described in PCT Publication No. WO/0138514. After beaming, embryos are incubated for 30 min on osmotic media, then placed onto incubation media overnight at 25° C. in the dark. To avoid unduly damaging beamed explants, they are incubated for at least 24 hours prior to transfer to recovery media. Embryos are then spread onto recovery period media, for 5 days, 25° C. in the dark, then transferred to a selection media. Explants are incubated in selection media for up to eight weeks, depending on the nature and characteristics of the particular selection utilized. After the selection period, the resulting callus is transferred to embryo maturation media, until the formation of mature somatic embryos is observed. The resulting mature somatic embryos are then placed under low light, and the process of regeneration is initiated by methods known in the art. The resulting shoots are allowed to root on rooting media, and the resulting plants are transferred to nursery pots and propagated as transgenic plants.
Materials
TABLE-US-00003
[0171] DN62A5S Media Component per liter Source Chu'S N6 Basal 3.98 g/L Phytotechnology Labs Salt Mixture (Prod. No. C 416) Chu's N6 Vitamin 1 mL/L Phytotechnology Labs Solution (Prod. (of 1000x Stock) No. C 149) L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L Sigma L-Proline 1.4 g/L Phytotechnology Labs Casaminoacids 100 mg/L Fisher Scientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L Sigma D-7299) (of 1 mg/mL Stock)
[0172] Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N KC1, add Gelrite (Sigma) to 3 g/L, and autoclave. After cooling to 50° C., add 2 ml/L of a 5 mg/ml stock solution of Silver Nitrate (Phytotechnology Labs). Recipe yields about 20 plates.
[0173] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0174] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
Sequence CWU
1
1
4513747DNABacillus thuringiensis 1atgaatggag gagagaatat gaatcaaaat
aatcaaaatg aaatgcacat aatagattct 60tcatccaatg attttagtca atcaaacaga
tatccaagat acccattagc taaagagtca 120aattataaag attggttagc tagttgtgat
gaatccaatc tagatagatt gtcaactcca 180agttctgtac aagatgcagt cgtgacaagt
ttgaatattt tttcttacat ctttggtttt 240ctagatgctg gagctacctc ggcaggtctt
ggtatactag gcgtgttatt tggtcaattt 300tggccatcaa ataataatgc ggtttgggag
acttttctac gtagtgttga agaactaatt 360gctcgagaaa tagatatcgt cgagagaaac
cgaatcatgg cccaatttga tggtttaaga 420aacgttatgt cgaattataa tggtgcactt
atagattggg atggaaatcg tgataatacc 480gcacttcaaa gtgaagtaag aagccgcttt
gataatgcag atgatgcttt tgcacttcgt 540attcctgaat ttagaataaa agattttgaa
atacaatcgc tagctgtata tgcacaggct 600gcaactcttc atttattatt attaagggat
gctgttgtta atgggcagct atggggagtc 660gatccagtaa cgactcaacg tcgttatgaa
aaactggtat gtctgagtgg tgcatatgca 720gatcattgta cattctttta tagacagggt
ttggaggagt taagaggaaa agggaattgg 780actgcattca ataattatcg tagaaacatg
aatattcaag tattggatgt tatttcttta 840ttttcaaatt atgaccctcg cttatatgga
aataatacaa acacacaact tacaagagaa 900atattcactg aaccacttgc tactcctgga
tggctggata ggtattctaa tccagatcag 960ttccaacaaa tagaatataa tcttaatcct
tcaccttcat tgtcttctac tctcttaaat 1020cttgcagcag ataccggtct tggatactat
ggtgccggtg ccgtgacacc taggccaata 1080atacaaagaa catcgatgcg tcgtttgaat
acaggggcta ctgttccttt cactactgct 1140tggcaaggcg cacctaatcc tttaatttca
caacaaaagc agttgtcatt tgaaggtttt 1200gatgttttta atatcaattc agtagtatct
agggaagttt ctagtcaaac tagctcatta 1260tttggagtcc aacaagctgt ttttcacact
gtgatcgctg gaggaaacat accacccact 1320atgaaaataa ttgatctaca accaagagga
aactattcca cttctgtaac gtctaatata 1380ccaggaaaaa gatcagcaac tgcaaccgct
tcagattata cccatagact atcttcgata 1440acttcaactt cagtaggaac ggcgtataga
gatagaacga atattatggc atatggatgg 1500acgcatgtta gttcagagaa aacgaataga
attctaccaa atagaattac acaaattcca 1560tttgtaaaag gaattattac ttcgtccggg
actcatgtaa gaagtggccc agatcatact 1620gggggaagcc tcgtatcaat gggaggggat
gctcaattcg gaatggtggt cacttcttca 1680gctaggcaaa ggtatcgtgt gcgcttacgt
tatgcagctt ctaattcagt ggattttcga 1740ttaagaattt cgccattagg ggtggattat
aattttacgt taccgggtgg tggaacttct 1800tttaatccag atttacgata tagctctttc
cgatatataa ctctaccaat agagtttgag 1860acgcctaatt ctctattaaa tttctctttt
gatctagaca ctttgactct tatgaatgga 1920acatgttttt ttgacagggt tgaattcctc
ccagttaatt ctatagcttt agaatatgaa 1980ggaaaacaaa agctagaaaa agcaaagcaa
gcggtggaca atttgtttac caatattggg 2040aaaaatgctt taaaggtaga tacgacggat
tatgatgtgg atcaagctgc aaatttagta 2100gaatgtgtgc caggggaact gtacacaaaa
gaaaaaatga tcctactgga tgaagtgaaa 2160catgccaaac aactcagtgc atctcgtaat
ctgattcaaa atgggaactt tgcattttat 2220acggatgaat ggacgacaag taataatgta
agtattcaaa cggataatca gatattcaaa 2280gggaactatc tcaaaatgcc aggggcgaga
gagacagagg gaggtacaac taggtttccg 2340acgtatgtac tccaaaaaat agatgaatcc
aaattaaaac cctatacacg ttataaagtc 2400agaggctttg tcggaagtag tcatgatgtg
aagttaattg tggaacgtta tggtaaagaa 2460gtggatgcaa tcctaaatgt gagaaatgat
ttagcccttg ataccgtatc ttcgtcctgt 2520gttgaagtta atcaatgtca gtcgcaaatg
tatcctatta tgcatgatgg atatctcgcg 2580aatgtaatag atacaaattc ttatgaagag
gctcagtcag atcatgctaa cttcaaaaaa 2640gaacagggga tgtgccatca atctcatcaa
tttgattttc acattgatac aggggaagta 2700cacctaaaca agaatccagg tatttgggtt
ctatttaaaa tttcttcgcc agaaggacac 2760gcaaccttag ataacattga gttaattgaa
gatggtccat tagtaggaga atcgctagcc 2820ctcgtgaaaa aacgagaaaa gaaatggaaa
catgagatga aaacaagatg gctccaaaca 2880aaagaagtgt acgaaaaggc aaaaggggca
atagatgcct tatttacaga tgcacaagat 2940cacgctataa aattcgacac aaacatttct
cacattattt cagcagagca tcttgtacaa 3000tctatgcctt atgtctataa caaatggtta
tcagatgtgc caggtatgaa ttatgacatc 3060tatacagaat tagaacgccg tattacgcag
gcgtactctt tatatgaacg tagaaatatc 3120attagaaatg gagattttaa ttacgattta
aatcattggt acgcgacacc tcatgccaaa 3180gtacaacaaa tagatagtac agctgtatta
gtacttccaa actggagttc caatgtgtct 3240caaaatctat gtgtagagca caaccgcggt
tatatattac gtgtaacagc aaaaaaagaa 3300gacatgggca aaggatatgt gactattagt
gactgcaatg gaaatcagga aacacttacg 3360ttcacttctt gtgataatta tgtatcaaac
gagatcacaa atgaccaatc ggagtatcat 3420ttcagtcaag agatgaatga acaacgtagt
tataatccaa atgaggccat aaacgagcaa 3480ttggattata gtctaggtca agtaagaaat
gaacaacgtt gttatactcg aaatgccatc 3540acaaatgacc agtcggaata tcattttagt
caagagatga atgaacaacg tagttataat 3600ccaaatgaaa ccataaatga gcaaaggaat
tatgtaacaa gaaccattga tttcttccca 3660gatacagatc aagtacgcat tgatattgga
gaaactgaag gtactttcaa agtagaaagt 3720atagaattga tttgtatgaa gagccaa
374722460DNABacillus thuringiensis
2gtggtatgcc taataacgat aggaggaata gttatgaatt catatcaaaa tagaaatgaa
60tacgaaatat tggatgcttc acccaatcat gggaacatat caaacagata tcctttcgca
120aagaatccaa acgctatgga aaatgtcaat tataaaaatt ggttgaatgt gcgtgaagac
180gttgcccctt ctttttttgg aggtgcactt ggaattatag ttaatctctt taaacaatat
240gtttctttta ttaaggcgcc ttccgtttct ggtggggtag gattcttacg aactattata
300ggtttaatga ctaatcgtaa tgtaataaat cttactatag atgatgtaca acgactaatt
360aatcaatcat tagataatat tacccgagat gcggcaaata ctaaatttac ttcaatacaa
420aataattata atcaatatct tctcaataga caaaactata gaaatagatc tttgcctaga
480aacatttttg tacaaagtct tcaaaatatt gagcgtgaat tgagaagtgc actagatagt
540acttttagtt tacagaatcg agagctacta ttgttaccaa actttacgca aattgcaatg
600ctacatttaa cagtattaag agatgctgta atatttcaag gtaacgatct aatagtgcct
660acgattagtg aaggacctat aaatccatta ttaactcgac ctcctagtaa tacatttgag
720gaggcacttt taactagcat aaggatatat tctaattatt gcgtaagaca atatgaagta
780ggtttaaatc tcttgagaaa cagggggaat actagtagaa actggttgga tttcaatgcc
840tatcgcttag aaatgacatt taaagtatta gattttgtta cattattttc attatttgat
900acagtaaaat atccagtatc gattgtgtct gaaactgatt ctgattctac atcacccgta
960gtttatcagt taagtagggt tatttatacg gatccagtag gtgctataag aagcgatggt
1020cgtggttggt ttgatccacc tgtaggaact gatagggtta cttttacatc aatagaaaat
1080gaaataccag cccctactac ttcccggcat ttatcagaat taacaatttc ttcaggcccg
1140cttggttttg gtgtaaatcc agctaggaca cattcgtggc aggggaatcg aaatgttaat
1200atttctgctc ctacggatgt ttctggagca atttctaatc gtacgcgaac tattcctgct
1260agaaatattt ttagagtgaa ttcacgtgtt tatactcttg attggaggct gtatggagtt
1320tatagagctg aattttttca gggtgctcac tcgcaagtat tttcagaaaa tcctccaaca
1380ggtattggtg cccaaagcgc aaataacttt agatttttac ctggagaaaa ttcagaaaca
1440ccaactccgc aagattatac tcatgtatta agcagagtag taaatgcaac tgtgggactt
1500acaccggcaa caggaaatca acgtaactct gtattaatat ttggttggac acataaaagt
1560ttaacctctg aaaatatata tagaatcaac gaaattacta aagtagctgc tgtgaataca
1620agaggtaact cgggcatccg ggtaatttca ggacctggat ttacaggtgg agatttagta
1680aggttggatc ctaacggtag cgtaagttac aattttacac ccgctaatca acaagcattg
1740caatcgaata ttagaatacg tttacgttat gcttgtcaag ggacagcttc attaagaata
1800acgtttggta acgcttctag ccaagttatt tcacttattt ctacaacttc atcaataaat
1860aatcttcaat atgaaaattt ccatgttgtt aatgttccga ataacattaa ttttcaatca
1920gtaggtactc aaataactat tcaaaatatc agtcaaaatc ctaacatatc gctagatagt
1980attgaacttt tttccaacat acctattcca caggaacccc cttttaaccc agtggttcct
2040gaaccaccta ttatttcagg aaattatcaa attgtaacac ctttagatcg tagaagtata
2100atagatttaa accctaataa taatgttaca ttatggacaa ataatagatc gagtaatcaa
2160atttggaatt tttcatatga ccaacaaaga ggtgcatatc taatacgaag tttaagaact
2220ggaagtttag ttttatctat ggattctccc cgtactagca atgtgtttgg ttatccatct
2280aattcagatg cttctcagtt ttggatttta gaacctaatc aagatggatt tatatttaga
2340agtcttagag atagaaattt agttttagat gtttcaggtg gaagagtgga ccctggaacg
2400agaataatag tttttccttt tactaattca atcaatcaaa gatttacatt gcaaagggta
246032130DNABacillus thuringiensis 3atggagtcaa tcatatgtat gtttagagtc
ctttatatca gaaattatga gcatacagga 60ggaataaaaa tgaagccgta tcaaaatgaa
aatgaatatg aaatattgga tgccttacca 120aagtattcga acatcgtcaa tgtttattca
aggtatccgt tagcaaataa tccacaagtt 180cctttacaaa atacaagtta taaagattgg
cttaatatgt gtcaaactat tactccactt 240tgtactccta tagacattga tagtaaatta
gtcgctaccg ctatagggat actaggcgct 300atattcaaag ctatgcctgg tccaggatca
gctgtaggat tatttttaaa aactttttca 360acaataatac ctattctttg gccaaatgac
aacacaccga tatggaaaga gttcacaaaa 420caaggattgc aactttttag accggaatta
ggcagagatg caatagaaat tataggcaac 480gacgtacagt ccggcttcaa tgcgttaaaa
gaccacatga acgactttga gactaagttt 540gaaatctggg acaaagatag aactcaaact
aatgcaacat atctcataac tgcatttggc 600gttgttaacg gtaaaattat cgaccttaaa
aatcaattct taataaaccc cgcaaatcaa 660cccgcatttc taaatctcta tgcacaaact
gccaatattg atttgatttt atatcaaaga 720ggggccgtat atggagataa ttgggcaaaa
gctataaatg atagttccat atctccgttt 780aatagttcgc aaatttttta tgactcttta
aaagctaaaa taaaagagta tactaattat 840tgtgcagaaa catatagaaa cagtttaact
atactcaaaa atcaacccaa tatccaatgg 900gatatatata atagatatcg tagagaggcg
actttaggtg cattagattt agttgcatta 960ttcccaaatt acgatatatg taaatatcca
atctcaacaa aaacagaact tactagaaaa 1020gtttatatgc catcattcta tttacaagca
cttcaacata gtaacataga agcattggaa 1080aaccaactta cacatccccc atcattattt
acttggttaa acgaattaaa cctttataca 1140atacgtgaaa atttcaatcc agctttacag
gtatcttcat tgtcaggtct tcaagctaaa 1200tatcgttata cccaaaattc gactatactt
cctaatccgc cggctcaagg aatcacaaat 1260ggcacaccaa taccaataat agggttaaat
aacttgttta tttataaact atcaatgtca 1320caatatcgtc atccaaatga ttgtgtacca
atagctggaa tttccgatat gaccttttat 1380aaaagtgact ataatggcaa tgcttccgca
actcaaactt atcaagcagg tagaaactcc 1440aataatgtca tagatacatt tatgaatggt
ccacaaaatg catcaagctc aaataatatt 1500tctattaacc aaacaaacca tatactatct
gatattaaaa tgaattacgc tcgatctggc 1560ggagtgtatg attttggata ttcatttgct
tggacacata ctagtgtaga tcctgataat 1620ctaattgttc cgaatagaat tacacaaatt
ccagctgtta aagctaactg tttgtcttca 1680ccagctagag taattgcagg gcctggtcat
acaggaggag atttagttgc tcttctaaac 1740ggtggtactc aagctggtag aatgcaaatc
caatgtaaaa caggtagctt tactggagct 1800tccagacgtt atggtatacg catgcgttat
gctgcaaata atgcatttac agtgagtcta 1860tcatatactt tacagggtgg taatacaata
ggtacaacat ttattaccga acgtacattt 1920tcaagaccta ataatataat accaacggat
ttaaaatacg aggagtttaa atataaagaa 1980tataatcaaa ttattacaat gacttcacct
caaaatacaa tagtaactat agctattcaa 2040caactaaatc cgatcccaaa tgatcaatta
attattgata gaatcgaatt ttatccagtg 2100gatcaaggtg tagtagcttg tccagtgaac
213041689DNABacillus thuringiensis
4gtgaattcta tgtttacaag tggggcgaaa aacaggttga aaatagaaac aacagatcat
60gaaatagatc aggctgcaat ttctataaag gctatgttag aggaagaaca ttcacaagag
120aaaatgatgt tatgggatga agtaaaacat gcaaaatacc tcagtcattc tcgtaatcta
180cttcaaaatg gtgattttga agatttattt aatggctgga ctacaagtaa taatatgtcc
240attcagaacg ataattcaac ttttaaagga caatatttaa atatgcctgg agcacgagac
300atatatggaa ccatatttcc aacatatgtt tatcaaaaaa tagaggaatc caaattaaaa
360tcatatacac gttatcgagt aagaggattt gtgggaagta gtaaagattt aaaattaatg
420gtaacacgtt acgggaaaga aattgatgct agtatggatg ttccaaatga tttggcatat
480atgcagccta acccttcatg tggggattat cgctgtgact catcatccca gtctatgatg
540aatcaagggt atcctacacc atatacagat ggatatgctt ccgatatgta tgcatgcccg
600tcaaacttag gtaaaaaaca tgtgaagtgt cacgatcgtc atccatttga ttttcatatt
660gacaccggag aagtagatat aaatacaaac ttagggattt tagtcttatt taaaatttcc
720aatcccgatg gatacgctac attaggaaat ctagaagtga ttgaagaagg gccactaaca
780ggcgaagcat tggcacatgt gaaacaaaag gaaaagaaat ggaatcaaca catggagaaa
840aaacgttggg aaacacaaca agcctatgat ccagcaaaac aagcagtaaa tgcattattc
900acaaatgcac aaggagacga attacactat catattactt tagatcatat tcagaacgcc
960gatcagttgg tacagtcgat tccttatgta caccatgctt ggttaccgga tgctccaggt
1020atgaactatg atgtatatca aggcttaaac gcacgtatca tgcaggctta caatttatat
1080gatgcacgaa atgtcataac aaatggcgac tttacacaag gattaatggg atggcacgca
1140acaggaaagg cagcggtaca acagatggat ggagcttctg tattagttct atcaaactgg
1200agtgcggggg tatctcaaaa cttgcatgcc caagatcatc atggatatgt gttacgtgtg
1260attgccaaaa aagaaggacc tggaaaaggg tatgtaacga tgatggattg taacggtaat
1320caggaaacgc tgaagttcac ttcttgtgaa gaaggataca tgacaaaaac agtagaggta
1380ttcccagaaa gtgatcgtgt acggattgaa ataggagaaa ccgaaggtac attttatata
1440gatagcatcg agttgctttg tatgcaagga tatgctaaca ataataatcc gcagacaggt
1500aatatgtatg agcaaagtaa taatcagaat acgagcgatg tgtatcatca agggtataca
1560aacaactata accaagactc tagtagtatg tataatcaaa attatactaa caatgatgac
1620ctgcattccg gttgtacatg taaccaaggg cataactttg gctgtacatg taatcaagga
1680tataaccgt
168952070DNABacillus thuringiensis 5gtgaaaagta tgaattcata tcaaaataaa
aatgaatatg aaatattgga tgcttcacaa 60aataactcta ctatgtctaa tcgttatcca
aggtatccac tagcaaatga cccacaagcc 120tctatgcaga atacgaatta taaagattgg
ttaaatatgt gcgattcaaa cacacaattt 180gttggtgata taagcacgta ttctagtcct
gaagctgctt taagtgtacg agatgctgtt 240ttaacgggta ttaacactgc tgggactata
ctttcgaatt taggggtccc ttttgctagt 300caatcatttg gaatgattgg taggataata
ggtattttat ggccgggacc tgacccattt 360gcagcactta tggttcttgt tgaagagctt
attaatcaaa ggataaatga tgagataaga 420aaccatgctc ttttagaatt agcgggttta
aagggcatta tggatctata tcgaactaga 480tggcgtgcat gggaccttaa taaagataat
cctgaaactc gagaagcagt acgagcgcag 540tatcgaaccg ctgataactt ttttatacaa
aacatgccaa aatttgggcg tgaagaccat 600ggggttttat tgttaccagt atatgcgcaa
gccgcgaata tgcatttaat tttattaaga 660gatgcttatg tatttggaac agggtgggga
ttaggtcctg gtgaagttcg tgataattat 720acaagactac aggagaagat tagagagtat
aaagatcatt gtgtaacatt ctataatcag 780ggtttaaatc gatttaatcg ctcaaatgct
caagattggg tgagctttaa taggtttcgt 840acagatatga cattaacagt attggatcta
gcaatattat ttccaaacta tgatccgcgc 900atatatccat cggcagtaaa aacagaattg
actagggaaa tttatacaga tccagtaggg 960tttactgggg tattagggag tggaggtagg
acttaccctt ggtataatcc taatgatacg 1020tcctttgcta ctatggaaaa tagcgccaga
cgacgacctt cttttaccac ttggcttaat 1080cgcattcgta tatttacagg tcatataggt
aatttttctg ctgcgggaaa tgtttgggga 1140gggcatgaat tatttgaacg tagcaataac
ggttctgaaa taattcagag atttggtaat 1200acgaatacct cttatactcc tgttagaaat
tgggatttca cgaatcagaa tcgtactgtt 1260ttcagtattg cttcaaccgc tcgcgtgtta
ttagcgggat cagagggaaa tgctcatcgt 1320ccgagtcagt atggcgtctc gagagttgat
atgcatacag caataggtgg taatacttct 1380ggtggacagt ttatatacga agtacctaat
gttcattcat cccaaaatat tttatcagaa 1440ttaccaggag agaatcagca aagaccagac
gcaagaaatc acagccacat attatcttat 1500atatcaaatt ttgatgcaaa acgaggtggt
actgtcggca acgttagact tttaacgtat 1560ggttggacgc ataccagtat ggatcgtaat
aatcgtcttg aacgagatag aattactcaa 1620atagatgctg ttaaaggttg ggggggagtt
actgggtctg tcataccagg acctactgga 1680gggagtttgg taacgatccc tagtaatcct
tggagcgttt cccttagagt tcaagcacca 1740caaatacaaa caaattatcg tattcgtttg
cgttttgctt gtgtatggcc gggggcgcat 1800catatgtggg taacctacgg cggtatttcc
caccctgttc aattatgcaa taatccatca 1860tcaggtcgcc catcaaacaa tcttctagag
agcgattttg gctatgttgt tgttccaggt 1920actttttcgc catcaataaa tcccgaaata
cgattttcag ctatcagtaa tgcccccgtg 1980ctagacaaaa ttgaatttat tccacttgac
atttataatg agcattttgt agaagaaaga 2040gcaaagacaa taaatgatct atttattaat
207061713DNABacillus thuringiensis
6gtgaaaaagg ttgtgaatcc tatgtttaca agtggcgcga aaaatacgtt aaaaatagaa
60acgacagatt atgaaataga tcaagtggca aattctatag aatgtatgtc agatgaacaa
120aatccacagg aaaaaatgat gctatgggat gaaataaaac aggcaaaaca acttagtcgg
180tctcgtaatt tactccacaa tggtgatttt gaggatttat ttagaggctg gacaacaagt
240actcatatta cgattcaggc ggataatccg attttcaaag gaaattatat taatatacca
300ggggcaggag atattgatgg gacgctattc ccgagctata tctatcaaaa aatagaggaa
360tccaaattaa aatcaaatac acgttataga gtaagagggt ttgtggggag tagtaaaaat
420ctaaaattag tggtaacacg ttacgggaaa gaaattgatg ctagtatgga tgttccaaat
480gatttagcct atatgcagcc taacccttca tgtggggatt atcgctgtga atcctcgtcc
540cagtatgtga gccaagggta tccgacagca ccatatacag atggatatgc ttctgagatg
600tatgcatgcc cgtcagaccg agttaaaaaa catgtgaagt gtcacgatcg ccatccattt
660gattttcata ttgacatcgg agaattagat ataaatacaa acgtaggtat ttgggtctta
720tttaaaattt ctaatccaga tggatatgct acattaggga atttagaagt gattgaagaa
780ggaccactaa caggtgaagc attaacgcat gcgaaacaaa aggaaaagaa atggaaacaa
840cacatggaga agaagcgatt ggaaacacaa caagcctatg atccggcaaa acaggcagta
900gatgcattat tcacaaatgc acaaggagaa gagttacact atcatattac tttagatcat
960attcagaacg ccaatcagtt ggtacagtcg attccttatg tacaccatgc ttggttaccg
1020gatgctccag gtatgaacta tgatgtatat caagggttaa acgcacgtat catgcaagct
1080tataatttat atgctgcacg aaatgtcata acaaatggtg actttacaca aggattacag
1140ggatggcacg caacaggaaa ggcaacggta caacaaatgg atggagcttc tgtattagtt
1200ctgtcaaact ggagtgctgg ggtatctcag aatctgcatg cccaagatca tcatggatat
1260gtgttacgtg tgattgccaa aaaagaagga cctggaaaag ggtatgtaac gatgatggat
1320tgtaacggca atcaggagac actgaagttc acttcttgtg aagaaggata tatgacaaaa
1380acagtagaga tattcccaga aagtgatcgt gtacggattg aaataggaga aaccgaaggt
1440acgttttatg tagatagcat cgagttgctt tgtatgcaag gatatgctat caataataac
1500ccacacacgg gtaatatgta tgagcaaagt tataatggaa tttataatca gaatacgagc
1560gatgtgtatc accaagggta tacaaacaac tataaccaag actctagtag tatgtataat
1620caaaattata ctaacaatga tgaccagcat tccgattgca catgtaatca agggcataat
1680tctggctgta catgtaatca aggatataac cgt
171371881DNABacillus thuringiensis 7gtgaaaaata tgaattcata tcaaaataaa
aatgaatatg aaatattgga tacttcacca 60aacaactcta ctatgtctac tcttcatcca
aggtacccac tagcaaagga tccatacaag 120cctatgcgaa atacaaacta taaagaatgg
ctagctatgt gtgcaaataa taatcaagta 180cctattgatc cccttgataa tacctgggca
ggtgttatgg cagctctttt cgcctctgct 240gcagctatag cgggattaat gtcagcagtt
ccagttttct ctgttgtagc cacaggaaca 300gccttagcag cagccttaac acctatttta
ttccctagta atggcccaga tgtatccacc 360cagcttatga gtaatacaga agctttacta
aaaagagagc tagatactta tgttagagca 420agggcagatt cagaatttca agccttagaa
gctcaaagag aatttttcaa atcagctttt 480gattattgga aattatatcc tacaaatagc
aacgctatag ctacggttgc tgctaggttc 540cacacagtaa atggtgcttt tgtaacagca
atgcgtttat tcagaacggc aggttatgaa 600gcattactgt taccagttta tgcacaagcg
gcgcgtcttc atttactcca tttacgagat 660ggtgtcctgt ttgcgaatga atgggggcta
gctaaagacc ctggagactt acatgaccaa 720gaatttaata aatatgctgc tgaatatgcg
gattattgtg aatcaacgta taatacagag 780ctaaaccgca ttaaaactgc tccaggtaaa
acatggcttg actataatca gtaccgacga 840attatgacaa ttgctgtttt ggatatagct
gctaaatttt caattttaaa tcctcgccta 900tatagattac ctttgcaaga agaaattctc
actcgaaaaa tatatactga tcctgttaat 960ttctcacctg gtccttcaat cgcagatgat
gaaaatagat atacagtccc actatccctt 1020gttacacaat tagtcaactc aagattattt
actaacgtgg catctgctca aaatgctgga 1080tttattggaa atcaaaatcg ttataaaaat
ataggcgttg gcgacccagt tgatggtcct 1140ataattggac aatcagtata cgaaaaagtg
gatgcaggta taccgacaaa tgaatgggtt 1200tatgaagttg gtgtaaatgg tatacagaat
gattatccac gtaatatagg tttgagaaag 1260ggttctacaa ctgcatttac agatcattta
gctggaagtc agtataattt aggtccttta 1320actagggtct ctataccaac taaagacaat
gccccaataa ataatactaa ttttactcat 1380cgattatcag atataattct tcctggaaat
aaggggtcat cttttgcatg gactcatgtt 1440gatgtcgatc ctacaggaaa ctatttatca
acaactaaga ttaatttaat acctgctaca 1500aaagcatcta aaataccact ttctttctat
ctaagaaagg gaccaggatt tataggggga 1560gatttagtca gattaggaag tggcttcgaa
tgttcttata agtttaattt caaatcccca 1620ggtagctcag ctaattttag aattcgtata
cgttatgcag gtgcgggtag tggtcagggt 1680gctgatggtc aggtatattt taaattaggg
aattatacat ctccaactac tccttggggc 1740catactggat ttgactatgg aaatgtgaag
tataatcaat ttagagtatt agagcttttt 1800ggaactgcag aaaacattac agacaacgac
ttgaagatta tagtatggac aggttcaagt 1860gctcaggatt ttttatctag a
188181698DNABacillus thuringiensis
8gtgagtccta tgtatattaa tacgatgaaa aatacattaa aactagaaac gacagattat
60gaaatagatc aagccgccat ttctatagaa tgcatgtcta atgaacaaaa tccacaggaa
120aaaatgatat tatgggatga agtaaaacag gcaaaacaac tcagtcaatc tcgtaattta
180ctctacaatg gtgattttga agatgcatca aacggctgga aaacaagtta tacgattgaa
240attggaaagt atagttccat ttttaaaggg cagtaccttc atatgtttgg ggcaagagat
300gttttaggtg aagtgtttcc aacatatgtg tatcaaaaaa ttgatgaatc taaattaaaa
360ccctatacac gttatcgagt aagaggattt gtgggaagta gtaaagatct aaaattagtg
420gtaacccgtt acgggaaaga aattgacgcc attatggatg ttccagatga tttggcctat
480atgcagccta ccccttcatg tggggattat cgttgtgaat cagcgtcaca gtatgtgagc
540caagggtatc ctacaccata tggagatgga tatgcttctg ataggtatgc atgcccgtca
600gaccgagtta aaaaacatgt gaagtgtcac aatcgccatc catttgattt tcatattgac
660accggagaat tagatataaa tacaaacgta ggtatttggg tcttatttaa aatttctaat
720ccagatggat acgctacatt agggaattta gaagtgattg aagaaggacc aataacaggt
780gaagcattaa cgcatgcgaa acaaaaggaa aagaaatgga atcaacacat ggagaaagcg
840caaatcgaaa cacagcaagc ctatgatccg gcaaaacagg cagtagatgc attattcaca
900aatgcacaag gagaagagtt acactatcat attactttag atcatattca gaacgccaat
960cagttggtac agtcgattcc ttatgtacac catgcttggt taccggatgc tccaggtatg
1020aactatgatg tatatcaaga gttaaacgca cgtatcatgc aagcacgcta tttacatgat
1080gcacgaaatg tcataacaaa tggtgacttt acacaaggat tacagggatg gcacgcaaca
1140ggaaaggcaa cggtacaaca aatggatgga gcttctgtat tagttctgtc aaactggagt
1200gctggggtat ctcagaatct gcatgcccaa gatcatcatg gatatgtgtt acgtgtgatt
1260gccaaaaaag aaggacctgg aaaagggtat gtaacgatga tggattgtaa cggcaatcag
1320gagacactga agttcacttc ttgtgaagaa ggatatatga caaaaacagt agagatattc
1380ccagaaagtg atcgtgtacg gattgaaata ggagaaaccg aaggtacgtt ttatgtagat
1440agcatcgagt tgctttgtat gcaaggatat gctatcaata ataacccaca cacgggtaat
1500atgtatgagc aaagttataa tggaatttat aatcagaata cgagcgatgt gtatcaccaa
1560gggtatacaa acaactataa ccaagactct agtagtatgt ataatcaaaa ttatactaac
1620aatgatgacc agcattccga ttgcacatgt aatcaagggc ataattctgg ctgtacatgt
1680aatcaaggat ataaccgt
16989888DNABacillus thuringiensis 9atgaaaaagt taatgttttc tttagtagca
acaactatga gtatgggatt aattcttgga 60tctgcacctg taaaagcaga cgtaagcaac
aagaatagtg catatcagga tattgatgag 120agagttaaga aaatggcgca gagtgctgct
tgggggggac aagagtatag aaatcataat 180ataaaagata ttgaattaaa gggtaatctt
atagatggtt ctatgattga aaattcagaa 240gtattaactg tttcatcaga tattttagaa
aataaattag gacatacagt aaatatgcct 300agtactggtt atgaacatga atttgaagaa
acgactaata caactaatac aagtggatgg 360acatttggct ataattataa cgcaagtttt
tcagtattaa tggtttcagc ttcacaaagt 420tttagtgttg aatataatat gtctacttca
aacactcatg aaaaaaagga gaaaagaaaa 480tttactgtcc cttcaataga agttccagtt
cctgctggga aaaaatacaa agttgaatat 540gtgtttgaaa aagttaaagt ttcaggaaaa
aataaaattg atgcaaatct ctatggtgat 600gttacttatt attataataa tcagccgatg
tcaccacagc ttttatattc agtacaagga 660cttgcagctg ataagcaagg gtttgagcaa
gtcataagag attcagctgt aggaaacgat 720agatttggaa ttaagactac aggtattggt
cagtttagca ctgagtttgg aacacgtcta 780actagaactc ttacagacat tactgatact
agaaatccag taaaagtaga gacgaaaaat 840gtcccagttg agtttaaaac actttcaatt
gatactagag taattaaa 88810924DNABacillus thuringiensis
10atgaaaaaaa atagaatgtt gcttaaatgg atgtgtggtt taacaattgg aatcgggagt
60ttaacaggag gtagtttaaa tgcttttgcg gatgaagtct ctgattcttt agcagacgtg
120ggttttcttt atggggatta tctttataaa actaaacaac atccacaagg aacattacct
180atcacctatc caatgagaga aatcaataat tatcaaatta ttgataaatc tgtttcgcaa
240gttgggagta cggaatatga agaaggtcaa actctgtatg tcgatgatga tgtttttgac
300aataagacag gtactgatca aacttttaaa acaattcagt ttgaaaaaga attttcagaa
360acagccacat cgtcaacaac acattctgtg ggtacaagcc tagaagaaag tgtgaaattt
420gatttctttg tgggtgaagg ttcagccaaa ttcacagtaa actataattt tagtaaaaca
480ggttctctct caacaagcaa taaaataaaa tacacgttac cttctcaatc aattaatgta
540cctgctaaca agaaatatga agttatatgt gttcttgaaa ctaaaaaagc aaaggctaat
600gttcaattta acgtcgatgt tcttggtaat gcgaaatatg tatatagcaa taattcaccg
660tatacaccta aatacgaaag tggtgctact atgttgaaaa ctttaaatga aaaaaatcct
720actcctagcg tctcatggtt aggtaaagaa tgggaaaaat gggaatatca cgatggaaaa
780gcgagataca aaaatggaag tgggacagtt tcagctgaat atggtacaag aatggtactt
840gtaattaacg atataacaaa taataaaaca agaggtagta aagagattgc tagaatccct
900gttacaccaa tccaaaaaca aatg
924113732DNABacillus thuringiensis 11atgaatggag gagagaatat gaatcaaaat
aatcaaaatg aaatgcaaat aatagattct 60tcatccaatg attttagtca atcaaacagg
tatccaagat atccattagc taaagagtca 120aattataaag attggttagc tagttgtgat
gaatccaatg tagatacatt atcagctaca 180agtaatgtaa gggcttcagt atcgagagct
ctgggtattg tgaatcagat tttgggtttt 240cttggtcttg gatttattgg aacagggctt
ggtgtattaa gtgatttatt taattcattt 300tggccatcaa atgataactc gatttgggaa
tcttttctac gtagtgttga agaactcatt 360gatcgacgag tacgtgaagt cgagagattt
cgtatcgaat cccaatttac tggtttaaga 420aacgttatgt cgaattataa tggtgctctt
cgtgattgga atggaaatcg taataatcta 480gcacttcaaa gtgaagtaag aagccgcttt
gataattcag atgatgcttt tgcatctcgt 540atgcctgaat ttagaataga aggttttgaa
atacaatcac tagctgtata tgcacaggct 600gcaactctcc atttattatt attaagggat
ggcgttgtta atgggctgca atggggattc 660gatatcgtta cggttaaccg tctttatgaa
aaactagtat gtctgagtgg tgcatatgca 720gatcattgta cattatttta tagacaaggt
ttggaggagt taaggaacag agggaattgg 780aatgcattca ataattatcg tagagacatg
actcttcaag tattggatgt catttcttta 840tttccaaatt atgaccctcg cctatatgac
attaatacaa acacacaact tacaagagaa 900atatacactg aaccacttgc tattcctgga
tggcttaatt ctcattccaa tccaactcag 960ttccaacaaa tagaaaatga tcttatccgc
tcaccttcag tgttttctaa tctcgagact 1020ctttttatgg aagctggttt tgcattcttc
caagccggca tagctagaca agcagtatta 1080agaacacgta cgtctagttt aaatatgaat
cgcactgctg tcatcgtaac tccttggcaa 1140ggggcacctc atcctaatgt ttcacatgaa
cttcaagtga cccttcaaga tcggaatgtt 1200tttaatatca attcagtagt aggtagggaa
atttctagtc aaaccggctt attatttggg 1260gtccaacaag ccacttttca ctttgtatgg
gcaggcggaa acgcagctac gacaacacaa 1320ttcaatctac caccgatttc tggacattca
acttctataa catctaatat acccggaaca 1380aactcaacaa ctccaactgg ttcagattat
acccatagac tatcttcgat aacttcaact 1440tcagtaggaa cgtggcagag agatagaacg
aatattatgg catatggatg gactcatgtt 1500agtgcagagc gtactaatag gattataccg
aatagaatta cacaaatccc agctgtaaaa 1560ggatcactgt ttagtgataa tccaccaaac
acatcacgaa cacgtgtaga aaatggccct 1620ggtcatactg ggggtggact cgtagttatg
gacggaggaa ctagtgtatt acagatgaga 1680gtcacttctt cagcaaggca aaggtatgat
atgcgtttac gttatgtagc tcttgctcca 1740gctactgttg aagtaagaat tccggaatta
ggggggcatg ttaggtttca gatgccaatg 1800actgcaacgg ggttacctgc gcctctacca
tacagccatt tgcgatatgt ggatatcccg 1860ctgaggtttg agacacccca tggtgaaaat
acttggacgt ttgaactaca gactacgttt 1920gcagcagttg caattgacag agtcgaattc
ataccagtta atgctacagc tttagaatat 1980gaaggaaaac gacatctaga aaaggcaaag
aaagccgtgg gtgatctgtt tatcaataat 2040ggaaaagagg ctttaaaagt agatacgacg
gattatgatg tggatcaagc tgccaatcta 2100gtagaatgta tgccagagga actgtacaca
aaagaaaaaa tgatcctact tgatgaagtg 2160aaacatgcga agagattcag tcaatcccgt
aatctcattc aaaatggaga ctttgaattt 2220gctacagatg gatggatgac aagtagtaat
gtcatcgttc aggcggataa tacagtattc 2280aaagggaaat atctcaatat gccaggggca
atagaaacag atacaagtac gttcccgact 2340tatatatacc aaaaaataga tgaatccaga
ttaaagccat atacacgcta taaagtcagg 2400ggctttgtcg gaagtagtca tgatgtgagg
ctaattgtgg aacgcaatgg taaagaagtg 2460gatgcactcc taaatgtaag aaatgatttg
tcccttgata ccgtagctcc ttcctgtatt 2520gaagctaatc aaccttatcc tatcatccat
gatggatgtc tcacgaatgt aatagataca 2580aattcttatg aagaggctca gtccggtcat
gctaactgca aaaaagaaca tggaatgtgc 2640catcagtctc atcaatttga ttttcacatt
gatacagggg aaatacacac aaacaagaat 2700ccaggtattt gggttctgtt taaaatttct
tcgccagaag gacacgcaac cttagataac 2760attgagttaa ttgaagatgg tccgttagta
ggagaatcgc tagccttcgt gaaaaaacaa 2820gaaaagaaat gggaaaatga gatggaaaca
agatggctcc aaacaaaaga agtatatgaa 2880aaggcaaaag gggaaataga ttccttattt
acagatgcac aagatcaagc tttaaaattc 2940gacacaaaca tttctcatat tatttcagca
gagcatcttg tacaatccat gccttatgta 3000tataacaatt ggttatcaga tgtgccaggt
atgaattatg acatctatac agaattagaa 3060cgtcgtatta cgcaggcata ctctttatat
gaacatagaa atatcattaa aaatggagac 3120tttgattatg gtttaaatca ttggcacgcg
acgcctcatg cgaaagtgca acaaatagat 3180ggtacagctg tattagtact tccaaactgg
agttccaatg tgtctcaaaa tctatgtgta 3240gagcacaacc gcggttatat attacgtgta
acagcaaaaa aagaagacat gggcaaagga 3300tatgtgacta ttagtgactg caatggaaat
caggaaacac ttacgttcac ttcttgtgct 3360aattatgtag caaacgaaat cacaaatgac
caatcggagt atcacttcag tcaagagatg 3420aatgaacaac gtggttataa tccaaatgaa
accataaaca agcaattaga ttatagtcta 3480gatcaagtaa gaaatgaaca acgttgttat
aatccaaatg aaatcacaaa tgaccagtcg 3540gaatatcatt acagtcaaga gatgaatgaa
caacgttgtt ataatccaaa tgaaatcata 3600aacgagcaca ggaattatgt aacaagaacc
attgatttct tcccagatac aaatcaagtg 3660cgcattgata ttggagaaac tgaaggtact
ttcaaagtag aaagtataga attgatttgt 3720atgaagagcc aa
3732121563DNABacillus thuringiensis
12gtgaccaaat cagaatttat ccaaaattca tgcagaaaaa tgcaatcaaa agtgagggtt
60attattctaa gtactaatga tcctgtagtc aataataata ctttagatat aacagaaata
120aaagacctag ctcatttatc tcaagctatc atgttagcta ataattttca agctgctctt
180gtacccactt catcagaatt tggacaggat gtattaagat ttgatgtaaa tcaaggaatt
240agcatagcta ataacattta tcctaaagct gtagatataa attatatatc acgtacgctt
300tctcaaagta acaaccaagt aaattctatg ataaatatgg tggtaaatga attgaaatta
360ttattaggga taaatcttgc tgattcagtg ttacaacaat taacatcttt agttgcgtat
420acatttacaa atttatatac acagcaaaat tctgcttggg ttttttgggg gaaacaagct
480tctaatcaaa caaattatac ttataatatt gtgtttgcaa ttcaaaatgc tcaaacaggt
540aactttatga aagctattcc tatgggattt gaaatttcag cttatgctgt caaagaacag
600gtattattct ttaccattca agattacgca agttatagcg ttaaaataca ggcaattaat
660gtcacgcagc ctttaattaa tagtagctat ggaagtttaa gtggcgtgta taatataata
720accgctctaa ataatataag tgttataact atgtcaaatt cggatgaaaa tgttaattta
780tggtatgata atgatgattt aaaccaaaaa tggattcttg aatttaatca taatcactat
840gcttatataa tccgaaacct tagcaatcgc tctttagtat taacatggga tagtacttca
900ggttctaata atgtttttgc tacaaactat caaggaaacg atgaacaatt ttggattatt
960caagatacgg ataatgatta tttttattta tcaaatatga gagatactca atatgtatta
1020gagatagctg gctctgtgtt ttataacgga acaaatgtta tagttaataa aaaaacaagc
1080agtttaaatc aaaaattttc aattaatcgt ataaatcgtc aaattcagaa tggtatatat
1140aatattacaa cctacctaaa tgctagcagt gttataacta tgtcaacaga ttataatatt
1200aatgtacacg attatcctgt taatttatgg tttaaaaatg atagtattaa tcaaaaatgg
1260atttttgaat ttgatagtga taaatccgct tatcgagtta gaagtgtcag taatccatct
1320ttatttctat catggccggt agcttctttt acaaatcgcg ctgctgttac acctaatcca
1380agagacaatg aatatttttg gtttcttcaa agcgctggat tgggtacttt ttatttagta
1440agtatgagag acactcgata tgtattagaa gtggaaaatt ccaatattga taacggaaca
1500aatattatag ttaatcaaag aacaggtaat tttaatcaga gattttatat agaaaatatt
1560aat
1563131249PRTBacillus thuringiensis 13Met Asn Gly Gly Glu Asn Met Asn Gln
Asn Asn Gln Asn Glu Met His1 5 10
15 Ile Ile Asp Ser Ser Ser Asn Asp Phe Ser Gln Ser Asn Arg
Tyr Pro 20 25 30
Arg Tyr Pro Leu Ala Lys Glu Ser Asn Tyr Lys Asp Trp Leu Ala Ser 35
40 45 Cys Asp Glu Ser Asn
Leu Asp Arg Leu Ser Thr Pro Ser Ser Val Gln 50 55
60 Asp Ala Val Val Thr Ser Leu Asn Ile Phe
Ser Tyr Ile Phe Gly Phe65 70 75
80 Leu Asp Ala Gly Ala Thr Ser Ala Gly Leu Gly Ile Leu Gly Val
Leu 85 90 95 Phe
Gly Gln Phe Trp Pro Ser Asn Asn Asn Ala Val Trp Glu Thr Phe
100 105 110 Leu Arg Ser Val Glu
Glu Leu Ile Ala Arg Glu Ile Asp Ile Val Glu 115
120 125 Arg Asn Arg Ile Met Ala Gln Phe Asp
Gly Leu Arg Asn Val Met Ser 130 135
140 Asn Tyr Asn Gly Ala Leu Ile Asp Trp Asp Gly Asn Arg
Asp Asn Thr145 150 155
160 Ala Leu Gln Ser Glu Val Arg Ser Arg Phe Asp Asn Ala Asp Asp Ala
165 170 175 Phe Ala Leu Arg
Ile Pro Glu Phe Arg Ile Lys Asp Phe Glu Ile Gln 180
185 190 Ser Leu Ala Val Tyr Ala Gln Ala Ala
Thr Leu His Leu Leu Leu Leu 195 200
205 Arg Asp Ala Val Val Asn Gly Gln Leu Trp Gly Val Asp Pro
Val Thr 210 215 220
Thr Gln Arg Arg Tyr Glu Lys Leu Val Cys Leu Ser Gly Ala Tyr Ala225
230 235 240 Asp His Cys Thr Phe
Phe Tyr Arg Gln Gly Leu Glu Glu Leu Arg Gly 245
250 255 Lys Gly Asn Trp Thr Ala Phe Asn Asn Tyr
Arg Arg Asn Met Asn Ile 260 265
270 Gln Val Leu Asp Val Ile Ser Leu Phe Ser Asn Tyr Asp Pro Arg
Leu 275 280 285 Tyr
Gly Asn Asn Thr Asn Thr Gln Leu Thr Arg Glu Ile Phe Thr Glu 290
295 300 Pro Leu Ala Thr Pro Gly
Trp Leu Asp Arg Tyr Ser Asn Pro Asp Gln305 310
315 320 Phe Gln Gln Ile Glu Tyr Asn Leu Asn Pro Ser
Pro Ser Leu Ser Ser 325 330
335 Thr Leu Leu Asn Leu Ala Ala Asp Thr Gly Leu Gly Tyr Tyr Gly Ala
340 345 350 Gly Ala Val
Thr Pro Arg Pro Ile Ile Gln Arg Thr Ser Met Arg Arg 355
360 365 Leu Asn Thr Gly Ala Thr Val Pro
Phe Thr Thr Ala Trp Gln Gly Ala 370 375
380 Pro Asn Pro Leu Ile Ser Gln Gln Lys Gln Leu Ser Phe
Glu Gly Phe385 390 395
400 Asp Val Phe Asn Ile Asn Ser Val Val Ser Arg Glu Val Ser Ser Gln
405 410 415 Thr Ser Ser Leu
Phe Gly Val Gln Gln Ala Val Phe His Thr Val Ile 420
425 430 Ala Gly Gly Asn Ile Pro Pro Thr Met
Lys Ile Ile Asp Leu Gln Pro 435 440
445 Arg Gly Asn Tyr Ser Thr Ser Val Thr Ser Asn Ile Pro Gly
Lys Arg 450 455 460
Ser Ala Thr Ala Thr Ala Ser Asp Tyr Thr His Arg Leu Ser Ser Ile465
470 475 480 Thr Ser Thr Ser Val
Gly Thr Ala Tyr Arg Asp Arg Thr Asn Ile Met 485
490 495 Ala Tyr Gly Trp Thr His Val Ser Ser Glu
Lys Thr Asn Arg Ile Leu 500 505
510 Pro Asn Arg Ile Thr Gln Ile Pro Phe Val Lys Gly Ile Ile Thr
Ser 515 520 525 Ser
Gly Thr His Val Arg Ser Gly Pro Asp His Thr Gly Gly Ser Leu 530
535 540 Val Ser Met Gly Gly Asp
Ala Gln Phe Gly Met Val Val Thr Ser Ser545 550
555 560 Ala Arg Gln Arg Tyr Arg Val Arg Leu Arg Tyr
Ala Ala Ser Asn Ser 565 570
575 Val Asp Phe Arg Leu Arg Ile Ser Pro Leu Gly Val Asp Tyr Asn Phe
580 585 590 Thr Leu Pro
Gly Gly Gly Thr Ser Phe Asn Pro Asp Leu Arg Tyr Ser 595
600 605 Ser Phe Arg Tyr Ile Thr Leu Pro
Ile Glu Phe Glu Thr Pro Asn Ser 610 615
620 Leu Leu Asn Phe Ser Phe Asp Leu Asp Thr Leu Thr Leu
Met Asn Gly625 630 635
640 Thr Cys Phe Phe Asp Arg Val Glu Phe Leu Pro Val Asn Ser Ile Ala
645 650 655 Leu Glu Tyr Glu
Gly Lys Gln Lys Leu Glu Lys Ala Lys Gln Ala Val 660
665 670 Asp Asn Leu Phe Thr Asn Ile Gly Lys
Asn Ala Leu Lys Val Asp Thr 675 680
685 Thr Asp Tyr Asp Val Asp Gln Ala Ala Asn Leu Val Glu Cys
Val Pro 690 695 700
Gly Glu Leu Tyr Thr Lys Glu Lys Met Ile Leu Leu Asp Glu Val Lys705
710 715 720 His Ala Lys Gln Leu
Ser Ala Ser Arg Asn Leu Ile Gln Asn Gly Asn 725
730 735 Phe Ala Phe Tyr Thr Asp Glu Trp Thr Thr
Ser Asn Asn Val Ser Ile 740 745
750 Gln Thr Asp Asn Gln Ile Phe Lys Gly Asn Tyr Leu Lys Met Pro
Gly 755 760 765 Ala
Arg Glu Thr Glu Gly Gly Thr Thr Arg Phe Pro Thr Tyr Val Leu 770
775 780 Gln Lys Ile Asp Glu Ser
Lys Leu Lys Pro Tyr Thr Arg Tyr Lys Val785 790
795 800 Arg Gly Phe Val Gly Ser Ser His Asp Val Lys
Leu Ile Val Glu Arg 805 810
815 Tyr Gly Lys Glu Val Asp Ala Ile Leu Asn Val Arg Asn Asp Leu Ala
820 825 830 Leu Asp Thr
Val Ser Ser Ser Cys Val Glu Val Asn Gln Cys Gln Ser 835
840 845 Gln Met Tyr Pro Ile Met His Asp
Gly Tyr Leu Ala Asn Val Ile Asp 850 855
860 Thr Asn Ser Tyr Glu Glu Ala Gln Ser Asp His Ala Asn
Phe Lys Lys865 870 875
880 Glu Gln Gly Met Cys His Gln Ser His Gln Phe Asp Phe His Ile Asp
885 890 895 Thr Gly Glu Val
His Leu Asn Lys Asn Pro Gly Ile Trp Val Leu Phe 900
905 910 Lys Ile Ser Ser Pro Glu Gly His Ala
Thr Leu Asp Asn Ile Glu Leu 915 920
925 Ile Glu Asp Gly Pro Leu Val Gly Glu Ser Leu Ala Leu Val
Lys Lys 930 935 940
Arg Glu Lys Lys Trp Lys His Glu Met Lys Thr Arg Trp Leu Gln Thr945
950 955 960 Lys Glu Val Tyr Glu
Lys Ala Lys Gly Ala Ile Asp Ala Leu Phe Thr 965
970 975 Asp Ala Gln Asp His Ala Ile Lys Phe Asp
Thr Asn Ile Ser His Ile 980 985
990 Ile Ser Ala Glu His Leu Val Gln Ser Met Pro Tyr Val Tyr Asn
Lys 995 1000 1005 Trp
Leu Ser Asp Val Pro Gly Met Asn Tyr Asp Ile Tyr Thr Glu Leu 1010
1015 1020 Glu Arg Arg Ile Thr Gln
Ala Tyr Ser Leu Tyr Glu Arg Arg Asn Ile1025 1030
1035 1040 Ile Arg Asn Gly Asp Phe Asn Tyr Asp Leu Asn
His Trp Tyr Ala Thr 1045 1050
1055 Pro His Ala Lys Val Gln Gln Ile Asp Ser Thr Ala Val Leu Val Leu
1060 1065 1070 Pro Asn Trp
Ser Ser Asn Val Ser Gln Asn Leu Cys Val Glu His Asn 1075
1080 1085 Arg Gly Tyr Ile Leu Arg Val Thr
Ala Lys Lys Glu Asp Met Gly Lys 1090 1095
1100 Gly Tyr Val Thr Ile Ser Asp Cys Asn Gly Asn Gln Glu
Thr Leu Thr1105 1110 1115
1120 Phe Thr Ser Cys Asp Asn Tyr Val Ser Asn Glu Ile Thr Asn Asp Gln
1125 1130 1135 Ser Glu Tyr His
Phe Ser Gln Glu Met Asn Glu Gln Arg Ser Tyr Asn 1140
1145 1150 Pro Asn Glu Ala Ile Asn Glu Gln Leu
Asp Tyr Ser Leu Gly Gln Val 1155 1160
1165 Arg Asn Glu Gln Arg Cys Tyr Thr Arg Asn Ala Ile Thr Asn
Asp Gln 1170 1175 1180
Ser Glu Tyr His Phe Ser Gln Glu Met Asn Glu Gln Arg Ser Tyr Asn1185
1190 1195 1200 Pro Asn Glu Thr Ile
Asn Glu Gln Arg Asn Tyr Val Thr Arg Thr Ile 1205
1210 1215 Asp Phe Phe Pro Asp Thr Asp Gln Val Arg
Ile Asp Ile Gly Glu Thr 1220 1225
1230 Glu Gly Thr Phe Lys Val Glu Ser Ile Glu Leu Ile Cys Met Lys
Ser 1235 1240 1245 Gln
14820PRTBacillus thuringiensis 14Met Val Cys Leu Ile Thr Ile Gly Gly
Ile Val Met Asn Ser Tyr Gln1 5 10
15 Asn Arg Asn Glu Tyr Glu Ile Leu Asp Ala Ser Pro Asn His
Gly Asn 20 25 30
Ile Ser Asn Arg Tyr Pro Phe Ala Lys Asn Pro Asn Ala Met Glu Asn 35
40 45 Val Asn Tyr Lys Asn
Trp Leu Asn Val Arg Glu Asp Val Ala Pro Ser 50 55
60 Phe Phe Gly Gly Ala Leu Gly Ile Ile Val
Asn Leu Phe Lys Gln Tyr65 70 75
80 Val Ser Phe Ile Lys Ala Pro Ser Val Ser Gly Gly Val Gly Phe
Leu 85 90 95 Arg
Thr Ile Ile Gly Leu Met Thr Asn Arg Asn Val Ile Asn Leu Thr
100 105 110 Ile Asp Asp Val Gln
Arg Leu Ile Asn Gln Ser Leu Asp Asn Ile Thr 115
120 125 Arg Asp Ala Ala Asn Thr Lys Phe Thr
Ser Ile Gln Asn Asn Tyr Asn 130 135
140 Gln Tyr Leu Leu Asn Arg Gln Asn Tyr Arg Asn Arg Ser
Leu Pro Arg145 150 155
160 Asn Ile Phe Val Gln Ser Leu Gln Asn Ile Glu Arg Glu Leu Arg Ser
165 170 175 Ala Leu Asp Ser
Thr Phe Ser Leu Gln Asn Arg Glu Leu Leu Leu Leu 180
185 190 Pro Asn Phe Thr Gln Ile Ala Met Leu
His Leu Thr Val Leu Arg Asp 195 200
205 Ala Val Ile Phe Gln Gly Asn Asp Leu Ile Val Pro Thr Ile
Ser Glu 210 215 220
Gly Pro Ile Asn Pro Leu Leu Thr Arg Pro Pro Ser Asn Thr Phe Glu225
230 235 240 Glu Ala Leu Leu Thr
Ser Ile Arg Ile Tyr Ser Asn Tyr Cys Val Arg 245
250 255 Gln Tyr Glu Val Gly Leu Asn Leu Leu Arg
Asn Arg Gly Asn Thr Ser 260 265
270 Arg Asn Trp Leu Asp Phe Asn Ala Tyr Arg Leu Glu Met Thr Phe
Lys 275 280 285 Val
Leu Asp Phe Val Thr Leu Phe Ser Leu Phe Asp Thr Val Lys Tyr 290
295 300 Pro Val Ser Ile Val Ser
Glu Thr Asp Ser Asp Ser Thr Ser Pro Val305 310
315 320 Val Tyr Gln Leu Ser Arg Val Ile Tyr Thr Asp
Pro Val Gly Ala Ile 325 330
335 Arg Ser Asp Gly Arg Gly Trp Phe Asp Pro Pro Val Gly Thr Asp Arg
340 345 350 Val Thr Phe
Thr Ser Ile Glu Asn Glu Ile Pro Ala Pro Thr Thr Ser 355
360 365 Arg His Leu Ser Glu Leu Thr Ile
Ser Ser Gly Pro Leu Gly Phe Gly 370 375
380 Val Asn Pro Ala Arg Thr His Ser Trp Gln Gly Asn Arg
Asn Val Asn385 390 395
400 Ile Ser Ala Pro Thr Asp Val Ser Gly Ala Ile Ser Asn Arg Thr Arg
405 410 415 Thr Ile Pro Ala
Arg Asn Ile Phe Arg Val Asn Ser Arg Val Tyr Thr 420
425 430 Leu Asp Trp Arg Leu Tyr Gly Val Tyr
Arg Ala Glu Phe Phe Gln Gly 435 440
445 Ala His Ser Gln Val Phe Ser Glu Asn Pro Pro Thr Gly Ile
Gly Ala 450 455 460
Gln Ser Ala Asn Asn Phe Arg Phe Leu Pro Gly Glu Asn Ser Glu Thr465
470 475 480 Pro Thr Pro Gln Asp
Tyr Thr His Val Leu Ser Arg Val Val Asn Ala 485
490 495 Thr Val Gly Leu Thr Pro Ala Thr Gly Asn
Gln Arg Asn Ser Val Leu 500 505
510 Ile Phe Gly Trp Thr His Lys Ser Leu Thr Ser Glu Asn Ile Tyr
Arg 515 520 525 Ile
Asn Glu Ile Thr Lys Val Ala Ala Val Asn Thr Arg Gly Asn Ser 530
535 540 Gly Ile Arg Val Ile Ser
Gly Pro Gly Phe Thr Gly Gly Asp Leu Val545 550
555 560 Arg Leu Asp Pro Asn Gly Ser Val Ser Tyr Asn
Phe Thr Pro Ala Asn 565 570
575 Gln Gln Ala Leu Gln Ser Asn Ile Arg Ile Arg Leu Arg Tyr Ala Cys
580 585 590 Gln Gly Thr
Ala Ser Leu Arg Ile Thr Phe Gly Asn Ala Ser Ser Gln 595
600 605 Val Ile Ser Leu Ile Ser Thr Thr
Ser Ser Ile Asn Asn Leu Gln Tyr 610 615
620 Glu Asn Phe His Val Val Asn Val Pro Asn Asn Ile Asn
Phe Gln Ser625 630 635
640 Val Gly Thr Gln Ile Thr Ile Gln Asn Ile Ser Gln Asn Pro Asn Ile
645 650 655 Ser Leu Asp Ser
Ile Glu Leu Phe Ser Asn Ile Pro Ile Pro Gln Glu 660
665 670 Pro Pro Phe Asn Pro Val Val Pro Glu
Pro Pro Ile Ile Ser Gly Asn 675 680
685 Tyr Gln Ile Val Thr Pro Leu Asp Arg Arg Ser Ile Ile Asp
Leu Asn 690 695 700
Pro Asn Asn Asn Val Thr Leu Trp Thr Asn Asn Arg Ser Ser Asn Gln705
710 715 720 Ile Trp Asn Phe Ser
Tyr Asp Gln Gln Arg Gly Ala Tyr Leu Ile Arg 725
730 735 Ser Leu Arg Thr Gly Ser Leu Val Leu Ser
Met Asp Ser Pro Arg Thr 740 745
750 Ser Asn Val Phe Gly Tyr Pro Ser Asn Ser Asp Ala Ser Gln Phe
Trp 755 760 765 Ile
Leu Glu Pro Asn Gln Asp Gly Phe Ile Phe Arg Ser Leu Arg Asp 770
775 780 Arg Asn Leu Val Leu Asp
Val Ser Gly Gly Arg Val Asp Pro Gly Thr785 790
795 800 Arg Ile Ile Val Phe Pro Phe Thr Asn Ser Ile
Asn Gln Arg Phe Thr 805 810
815 Leu Gln Arg Val 820 15710PRTBacillus thuringiensis
15Met Glu Ser Ile Ile Cys Met Phe Arg Val Leu Tyr Ile Arg Asn Tyr1
5 10 15 Glu His Thr Gly
Gly Ile Lys Met Lys Pro Tyr Gln Asn Glu Asn Glu 20
25 30 Tyr Glu Ile Leu Asp Ala Leu Pro Lys
Tyr Ser Asn Ile Val Asn Val 35 40
45 Tyr Ser Arg Tyr Pro Leu Ala Asn Asn Pro Gln Val Pro Leu
Gln Asn 50 55 60
Thr Ser Tyr Lys Asp Trp Leu Asn Met Cys Gln Thr Ile Thr Pro Leu65
70 75 80 Cys Thr Pro Ile Asp
Ile Asp Ser Lys Leu Val Ala Thr Ala Ile Gly 85
90 95 Ile Leu Gly Ala Ile Phe Lys Ala Met Pro
Gly Pro Gly Ser Ala Val 100 105
110 Gly Leu Phe Leu Lys Thr Phe Ser Thr Ile Ile Pro Ile Leu Trp
Pro 115 120 125 Asn
Asp Asn Thr Pro Ile Trp Lys Glu Phe Thr Lys Gln Gly Leu Gln 130
135 140 Leu Phe Arg Pro Glu Leu
Gly Arg Asp Ala Ile Glu Ile Ile Gly Asn145 150
155 160 Asp Val Gln Ser Gly Phe Asn Ala Leu Lys Asp
His Met Asn Asp Phe 165 170
175 Glu Thr Lys Phe Glu Ile Trp Asp Lys Asp Arg Thr Gln Thr Asn Ala
180 185 190 Thr Tyr Leu
Ile Thr Ala Phe Gly Val Val Asn Gly Lys Ile Ile Asp 195
200 205 Leu Lys Asn Gln Phe Leu Ile Asn
Pro Ala Asn Gln Pro Ala Phe Leu 210 215
220 Asn Leu Tyr Ala Gln Thr Ala Asn Ile Asp Leu Ile Leu
Tyr Gln Arg225 230 235
240 Gly Ala Val Tyr Gly Asp Asn Trp Ala Lys Ala Ile Asn Asp Ser Ser
245 250 255 Ile Ser Pro Phe
Asn Ser Ser Gln Ile Phe Tyr Asp Ser Leu Lys Ala 260
265 270 Lys Ile Lys Glu Tyr Thr Asn Tyr Cys
Ala Glu Thr Tyr Arg Asn Ser 275 280
285 Leu Thr Ile Leu Lys Asn Gln Pro Asn Ile Gln Trp Asp Ile
Tyr Asn 290 295 300
Arg Tyr Arg Arg Glu Ala Thr Leu Gly Ala Leu Asp Leu Val Ala Leu305
310 315 320 Phe Pro Asn Tyr Asp
Ile Cys Lys Tyr Pro Ile Ser Thr Lys Thr Glu 325
330 335 Leu Thr Arg Lys Val Tyr Met Pro Ser Phe
Tyr Leu Gln Ala Leu Gln 340 345
350 His Ser Asn Ile Glu Ala Leu Glu Asn Gln Leu Thr His Pro Pro
Ser 355 360 365 Leu
Phe Thr Trp Leu Asn Glu Leu Asn Leu Tyr Thr Ile Arg Glu Asn 370
375 380 Phe Asn Pro Ala Leu Gln
Val Ser Ser Leu Ser Gly Leu Gln Ala Lys385 390
395 400 Tyr Arg Tyr Thr Gln Asn Ser Thr Ile Leu Pro
Asn Pro Pro Ala Gln 405 410
415 Gly Ile Thr Asn Gly Thr Pro Ile Pro Ile Ile Gly Leu Asn Asn Leu
420 425 430 Phe Ile Tyr
Lys Leu Ser Met Ser Gln Tyr Arg His Pro Asn Asp Cys 435
440 445 Val Pro Ile Ala Gly Ile Ser Asp
Met Thr Phe Tyr Lys Ser Asp Tyr 450 455
460 Asn Gly Asn Ala Ser Ala Thr Gln Thr Tyr Gln Ala Gly
Arg Asn Ser465 470 475
480 Asn Asn Val Ile Asp Thr Phe Met Asn Gly Pro Gln Asn Ala Ser Ser
485 490 495 Ser Asn Asn Ile
Ser Ile Asn Gln Thr Asn His Ile Leu Ser Asp Ile 500
505 510 Lys Met Asn Tyr Ala Arg Ser Gly Gly
Val Tyr Asp Phe Gly Tyr Ser 515 520
525 Phe Ala Trp Thr His Thr Ser Val Asp Pro Asp Asn Leu Ile
Val Pro 530 535 540
Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Ala Asn Cys Leu Ser Ser545
550 555 560 Pro Ala Arg Val Ile
Ala Gly Pro Gly His Thr Gly Gly Asp Leu Val 565
570 575 Ala Leu Leu Asn Gly Gly Thr Gln Ala Gly
Arg Met Gln Ile Gln Cys 580 585
590 Lys Thr Gly Ser Phe Thr Gly Ala Ser Arg Arg Tyr Gly Ile Arg
Met 595 600 605 Arg
Tyr Ala Ala Asn Asn Ala Phe Thr Val Ser Leu Ser Tyr Thr Leu 610
615 620 Gln Gly Gly Asn Thr Ile
Gly Thr Thr Phe Ile Thr Glu Arg Thr Phe625 630
635 640 Ser Arg Pro Asn Asn Ile Ile Pro Thr Asp Leu
Lys Tyr Glu Glu Phe 645 650
655 Lys Tyr Lys Glu Tyr Asn Gln Ile Ile Thr Met Thr Ser Pro Gln Asn
660 665 670 Thr Ile Val
Thr Ile Ala Ile Gln Gln Leu Asn Pro Ile Pro Asn Asp 675
680 685 Gln Leu Ile Ile Asp Arg Ile Glu
Phe Tyr Pro Val Asp Gln Gly Val 690 695
700 Val Ala Cys Pro Val Asn705 710
16563PRTBacillus thuringiensis 16Met Asn Ser Met Phe Thr Ser Gly Ala Lys
Asn Arg Leu Lys Ile Glu1 5 10
15 Thr Thr Asp His Glu Ile Asp Gln Ala Ala Ile Ser Ile Lys Ala
Met 20 25 30 Leu
Glu Glu Glu His Ser Gln Glu Lys Met Met Leu Trp Asp Glu Val 35
40 45 Lys His Ala Lys Tyr Leu
Ser His Ser Arg Asn Leu Leu Gln Asn Gly 50 55
60 Asp Phe Glu Asp Leu Phe Asn Gly Trp Thr Thr
Ser Asn Asn Met Ser65 70 75
80 Ile Gln Asn Asp Asn Ser Thr Phe Lys Gly Gln Tyr Leu Asn Met Pro
85 90 95 Gly Ala Arg
Asp Ile Tyr Gly Thr Ile Phe Pro Thr Tyr Val Tyr Gln 100
105 110 Lys Ile Glu Glu Ser Lys Leu Lys
Ser Tyr Thr Arg Tyr Arg Val Arg 115 120
125 Gly Phe Val Gly Ser Ser Lys Asp Leu Lys Leu Met Val
Thr Arg Tyr 130 135 140
Gly Lys Glu Ile Asp Ala Ser Met Asp Val Pro Asn Asp Leu Ala Tyr145
150 155 160 Met Gln Pro Asn Pro
Ser Cys Gly Asp Tyr Arg Cys Asp Ser Ser Ser 165
170 175 Gln Ser Met Met Asn Gln Gly Tyr Pro Thr
Pro Tyr Thr Asp Gly Tyr 180 185
190 Ala Ser Asp Met Tyr Ala Cys Pro Ser Asn Leu Gly Lys Lys His
Val 195 200 205 Lys
Cys His Asp Arg His Pro Phe Asp Phe His Ile Asp Thr Gly Glu 210
215 220 Val Asp Ile Asn Thr Asn
Leu Gly Ile Leu Val Leu Phe Lys Ile Ser225 230
235 240 Asn Pro Asp Gly Tyr Ala Thr Leu Gly Asn Leu
Glu Val Ile Glu Glu 245 250
255 Gly Pro Leu Thr Gly Glu Ala Leu Ala His Val Lys Gln Lys Glu Lys
260 265 270 Lys Trp Asn
Gln His Met Glu Lys Lys Arg Trp Glu Thr Gln Gln Ala 275
280 285 Tyr Asp Pro Ala Lys Gln Ala Val
Asn Ala Leu Phe Thr Asn Ala Gln 290 295
300 Gly Asp Glu Leu His Tyr His Ile Thr Leu Asp His Ile
Gln Asn Ala305 310 315
320 Asp Gln Leu Val Gln Ser Ile Pro Tyr Val His His Ala Trp Leu Pro
325 330 335 Asp Ala Pro Gly
Met Asn Tyr Asp Val Tyr Gln Gly Leu Asn Ala Arg 340
345 350 Ile Met Gln Ala Tyr Asn Leu Tyr Asp
Ala Arg Asn Val Ile Thr Asn 355 360
365 Gly Asp Phe Thr Gln Gly Leu Met Gly Trp His Ala Thr Gly
Lys Ala 370 375 380
Ala Val Gln Gln Met Asp Gly Ala Ser Val Leu Val Leu Ser Asn Trp385
390 395 400 Ser Ala Gly Val Ser
Gln Asn Leu His Ala Gln Asp His His Gly Tyr 405
410 415 Val Leu Arg Val Ile Ala Lys Lys Glu Gly
Pro Gly Lys Gly Tyr Val 420 425
430 Thr Met Met Asp Cys Asn Gly Asn Gln Glu Thr Leu Lys Phe Thr
Ser 435 440 445 Cys
Glu Glu Gly Tyr Met Thr Lys Thr Val Glu Val Phe Pro Glu Ser 450
455 460 Asp Arg Val Arg Ile Glu
Ile Gly Glu Thr Glu Gly Thr Phe Tyr Ile465 470
475 480 Asp Ser Ile Glu Leu Leu Cys Met Gln Gly Tyr
Ala Asn Asn Asn Asn 485 490
495 Pro Gln Thr Gly Asn Met Tyr Glu Gln Ser Asn Asn Gln Asn Thr Ser
500 505 510 Asp Val Tyr
His Gln Gly Tyr Thr Asn Asn Tyr Asn Gln Asp Ser Ser 515
520 525 Ser Met Tyr Asn Gln Asn Tyr Thr
Asn Asn Asp Asp Leu His Ser Gly 530 535
540 Cys Thr Cys Asn Gln Gly His Asn Phe Gly Cys Thr Cys
Asn Gln Gly545 550 555
560 Tyr Asn Arg17690PRTBacillus thuringiensis 17Met Lys Ser Met Asn Ser
Tyr Gln Asn Lys Asn Glu Tyr Glu Ile Leu1 5
10 15 Asp Ala Ser Gln Asn Asn Ser Thr Met Ser Asn
Arg Tyr Pro Arg Tyr 20 25 30
Pro Leu Ala Asn Asp Pro Gln Ala Ser Met Gln Asn Thr Asn Tyr Lys
35 40 45 Asp Trp Leu
Asn Met Cys Asp Ser Asn Thr Gln Phe Val Gly Asp Ile 50
55 60 Ser Thr Tyr Ser Ser Pro Glu Ala
Ala Leu Ser Val Arg Asp Ala Val65 70 75
80 Leu Thr Gly Ile Asn Thr Ala Gly Thr Ile Leu Ser Asn
Leu Gly Val 85 90 95
Pro Phe Ala Ser Gln Ser Phe Gly Met Ile Gly Arg Ile Ile Gly Ile
100 105 110 Leu Trp Pro Gly Pro
Asp Pro Phe Ala Ala Leu Met Val Leu Val Glu 115
120 125 Glu Leu Ile Asn Gln Arg Ile Asn Asp
Glu Ile Arg Asn His Ala Leu 130 135
140 Leu Glu Leu Ala Gly Leu Lys Gly Ile Met Asp Leu Tyr
Arg Thr Arg145 150 155
160 Trp Arg Ala Trp Asp Leu Asn Lys Asp Asn Pro Glu Thr Arg Glu Ala
165 170 175 Val Arg Ala Gln
Tyr Arg Thr Ala Asp Asn Phe Phe Ile Gln Asn Met 180
185 190 Pro Lys Phe Gly Arg Glu Asp His Gly
Val Leu Leu Leu Pro Val Tyr 195 200
205 Ala Gln Ala Ala Asn Met His Leu Ile Leu Leu Arg Asp Ala
Tyr Val 210 215 220
Phe Gly Thr Gly Trp Gly Leu Gly Pro Gly Glu Val Arg Asp Asn Tyr225
230 235 240 Thr Arg Leu Gln Glu
Lys Ile Arg Glu Tyr Lys Asp His Cys Val Thr 245
250 255 Phe Tyr Asn Gln Gly Leu Asn Arg Phe Asn
Arg Ser Asn Ala Gln Asp 260 265
270 Trp Val Ser Phe Asn Arg Phe Arg Thr Asp Met Thr Leu Thr Val
Leu 275 280 285 Asp
Leu Ala Ile Leu Phe Pro Asn Tyr Asp Pro Arg Ile Tyr Pro Ser 290
295 300 Ala Val Lys Thr Glu Leu
Thr Arg Glu Ile Tyr Thr Asp Pro Val Gly305 310
315 320 Phe Thr Gly Val Leu Gly Ser Gly Gly Arg Thr
Tyr Pro Trp Tyr Asn 325 330
335 Pro Asn Asp Thr Ser Phe Ala Thr Met Glu Asn Ser Ala Arg Arg Arg
340 345 350 Pro Ser Phe
Thr Thr Trp Leu Asn Arg Ile Arg Ile Phe Thr Gly His 355
360 365 Ile Gly Asn Phe Ser Ala Ala Gly
Asn Val Trp Gly Gly His Glu Leu 370 375
380 Phe Glu Arg Ser Asn Asn Gly Ser Glu Ile Ile Gln Arg
Phe Gly Asn385 390 395
400 Thr Asn Thr Ser Tyr Thr Pro Val Arg Asn Trp Asp Phe Thr Asn Gln
405 410 415 Asn Arg Thr Val
Phe Ser Ile Ala Ser Thr Ala Arg Val Leu Leu Ala 420
425 430 Gly Ser Glu Gly Asn Ala His Arg Pro
Ser Gln Tyr Gly Val Ser Arg 435 440
445 Val Asp Met His Thr Ala Ile Gly Gly Asn Thr Ser Gly Gly
Gln Phe 450 455 460
Ile Tyr Glu Val Pro Asn Val His Ser Ser Gln Asn Ile Leu Ser Glu465
470 475 480 Leu Pro Gly Glu Asn
Gln Gln Arg Pro Asp Ala Arg Asn His Ser His 485
490 495 Ile Leu Ser Tyr Ile Ser Asn Phe Asp Ala
Lys Arg Gly Gly Thr Val 500 505
510 Gly Asn Val Arg Leu Leu Thr Tyr Gly Trp Thr His Thr Ser Met
Asp 515 520 525 Arg
Asn Asn Arg Leu Glu Arg Asp Arg Ile Thr Gln Ile Asp Ala Val 530
535 540 Lys Gly Trp Gly Gly Val
Thr Gly Ser Val Ile Pro Gly Pro Thr Gly545 550
555 560 Gly Ser Leu Val Thr Ile Pro Ser Asn Pro Trp
Ser Val Ser Leu Arg 565 570
575 Val Gln Ala Pro Gln Ile Gln Thr Asn Tyr Arg Ile Arg Leu Arg Phe
580 585 590 Ala Cys Val
Trp Pro Gly Ala His His Met Trp Val Thr Tyr Gly Gly 595
600 605 Ile Ser His Pro Val Gln Leu Cys
Asn Asn Pro Ser Ser Gly Arg Pro 610 615
620 Ser Asn Asn Leu Leu Glu Ser Asp Phe Gly Tyr Val Val
Val Pro Gly625 630 635
640 Thr Phe Ser Pro Ser Ile Asn Pro Glu Ile Arg Phe Ser Ala Ile Ser
645 650 655 Asn Ala Pro Val
Leu Asp Lys Ile Glu Phe Ile Pro Leu Asp Ile Tyr 660
665 670 Asn Glu His Phe Val Glu Glu Arg Ala
Lys Thr Ile Asn Asp Leu Phe 675 680
685 Ile Asn 690 18571PRTBacillus thuringiensis 18Met Lys
Lys Val Val Asn Pro Met Phe Thr Ser Gly Ala Lys Asn Thr1 5
10 15 Leu Lys Ile Glu Thr Thr Asp
Tyr Glu Ile Asp Gln Val Ala Asn Ser 20 25
30 Ile Glu Cys Met Ser Asp Glu Gln Asn Pro Gln Glu
Lys Met Met Leu 35 40 45
Trp Asp Glu Ile Lys Gln Ala Lys Gln Leu Ser Arg Ser Arg Asn Leu
50 55 60 Leu His Asn
Gly Asp Phe Glu Asp Leu Phe Arg Gly Trp Thr Thr Ser65 70
75 80 Thr His Ile Thr Ile Gln Ala Asp
Asn Pro Ile Phe Lys Gly Asn Tyr 85 90
95 Ile Asn Ile Pro Gly Ala Gly Asp Ile Asp Gly Thr Leu
Phe Pro Ser 100 105 110
Tyr Ile Tyr Gln Lys Ile Glu Glu Ser Lys Leu Lys Ser Asn Thr Arg
115 120 125 Tyr Arg Val Arg
Gly Phe Val Gly Ser Ser Lys Asn Leu Lys Leu Val 130
135 140 Val Thr Arg Tyr Gly Lys Glu Ile
Asp Ala Ser Met Asp Val Pro Asn145 150
155 160 Asp Leu Ala Tyr Met Gln Pro Asn Pro Ser Cys Gly
Asp Tyr Arg Cys 165 170
175 Glu Ser Ser Ser Gln Tyr Val Ser Gln Gly Tyr Pro Thr Ala Pro Tyr
180 185 190 Thr Asp Gly
Tyr Ala Ser Glu Met Tyr Ala Cys Pro Ser Asp Arg Val 195
200 205 Lys Lys His Val Lys Cys His Asp
Arg His Pro Phe Asp Phe His Ile 210 215
220 Asp Ile Gly Glu Leu Asp Ile Asn Thr Asn Val Gly Ile
Trp Val Leu225 230 235
240 Phe Lys Ile Ser Asn Pro Asp Gly Tyr Ala Thr Leu Gly Asn Leu Glu
245 250 255 Val Ile Glu Glu
Gly Pro Leu Thr Gly Glu Ala Leu Thr His Ala Lys 260
265 270 Gln Lys Glu Lys Lys Trp Lys Gln His
Met Glu Lys Lys Arg Leu Glu 275 280
285 Thr Gln Gln Ala Tyr Asp Pro Ala Lys Gln Ala Val Asp Ala
Leu Phe 290 295 300
Thr Asn Ala Gln Gly Glu Glu Leu His Tyr His Ile Thr Leu Asp His305
310 315 320 Ile Gln Asn Ala Asn
Gln Leu Val Gln Ser Ile Pro Tyr Val His His 325
330 335 Ala Trp Leu Pro Asp Ala Pro Gly Met Asn
Tyr Asp Val Tyr Gln Gly 340 345
350 Leu Asn Ala Arg Ile Met Gln Ala Tyr Asn Leu Tyr Ala Ala Arg
Asn 355 360 365 Val
Ile Thr Asn Gly Asp Phe Thr Gln Gly Leu Gln Gly Trp His Ala 370
375 380 Thr Gly Lys Ala Thr Val
Gln Gln Met Asp Gly Ala Ser Val Leu Val385 390
395 400 Leu Ser Asn Trp Ser Ala Gly Val Ser Gln Asn
Leu His Ala Gln Asp 405 410
415 His His Gly Tyr Val Leu Arg Val Ile Ala Lys Lys Glu Gly Pro Gly
420 425 430 Lys Gly Tyr
Val Thr Met Met Asp Cys Asn Gly Asn Gln Glu Thr Leu 435
440 445 Lys Phe Thr Ser Cys Glu Glu Gly
Tyr Met Thr Lys Thr Val Glu Ile 450 455
460 Phe Pro Glu Ser Asp Arg Val Arg Ile Glu Ile Gly Glu
Thr Glu Gly465 470 475
480 Thr Phe Tyr Val Asp Ser Ile Glu Leu Leu Cys Met Gln Gly Tyr Ala
485 490 495 Ile Asn Asn Asn
Pro His Thr Gly Asn Met Tyr Glu Gln Ser Tyr Asn 500
505 510 Gly Ile Tyr Asn Gln Asn Thr Ser Asp
Val Tyr His Gln Gly Tyr Thr 515 520
525 Asn Asn Tyr Asn Gln Asp Ser Ser Ser Met Tyr Asn Gln Asn
Tyr Thr 530 535 540
Asn Asn Asp Asp Gln His Ser Asp Cys Thr Cys Asn Gln Gly His Asn545
550 555 560 Ser Gly Cys Thr Cys
Asn Gln Gly Tyr Asn Arg 565 570
19627PRTBacillus thuringiensis 19Val Lys Asn Met Asn Ser Tyr Gln Asn Lys
Asn Glu Tyr Glu Ile Leu1 5 10
15 Asp Thr Ser Pro Asn Asn Ser Thr Met Ser Thr Leu His Pro Arg
Tyr 20 25 30 Pro
Leu Ala Lys Asp Pro Tyr Lys Pro Met Arg Asn Thr Asn Tyr Lys 35
40 45 Glu Trp Leu Ala Met Cys
Ala Asn Asn Asn Gln Val Pro Ile Asp Pro 50 55
60 Leu Asp Asn Thr Trp Ala Gly Val Met Ala Ala
Leu Phe Ala Ser Ala65 70 75
80 Ala Ala Ile Ala Gly Leu Met Ser Ala Val Pro Val Phe Ser Val Val
85 90 95 Ala Thr Gly
Thr Ala Leu Ala Ala Ala Leu Thr Pro Ile Leu Phe Pro 100
105 110 Ser Asn Gly Pro Asp Val Ser Thr
Gln Leu Met Ser Asn Thr Glu Ala 115 120
125 Leu Leu Lys Arg Glu Leu Asp Thr Tyr Val Arg Ala Arg
Ala Asp Ser 130 135 140
Glu Phe Gln Ala Leu Glu Ala Gln Arg Glu Phe Phe Lys Ser Ala Phe145
150 155 160 Asp Tyr Trp Lys Leu
Tyr Pro Thr Asn Ser Asn Ala Ile Ala Thr Val 165
170 175 Ala Ala Arg Phe His Thr Val Asn Gly Ala
Phe Val Thr Ala Met Arg 180 185
190 Leu Phe Arg Thr Ala Gly Tyr Glu Ala Leu Leu Leu Pro Val Tyr
Ala 195 200 205 Gln
Ala Ala Arg Leu His Leu Leu His Leu Arg Asp Gly Val Leu Phe 210
215 220 Ala Asn Glu Trp Gly Leu
Ala Lys Asp Pro Gly Asp Leu His Asp Gln225 230
235 240 Glu Phe Asn Lys Tyr Ala Ala Glu Tyr Ala Asp
Tyr Cys Glu Ser Thr 245 250
255 Tyr Asn Thr Glu Leu Asn Arg Ile Lys Thr Ala Pro Gly Lys Thr Trp
260 265 270 Leu Asp Tyr
Asn Gln Tyr Arg Arg Ile Met Thr Ile Ala Val Leu Asp 275
280 285 Ile Ala Ala Lys Phe Ser Ile Leu
Asn Pro Arg Leu Tyr Arg Leu Pro 290 295
300 Leu Gln Glu Glu Ile Leu Thr Arg Lys Ile Tyr Thr Asp
Pro Val Asn305 310 315
320 Phe Ser Pro Gly Pro Ser Ile Ala Asp Asp Glu Asn Arg Tyr Thr Val
325 330 335 Pro Leu Ser Leu
Val Thr Gln Leu Val Asn Ser Arg Leu Phe Thr Asn 340
345 350 Val Ala Ser Ala Gln Asn Ala Gly Phe
Ile Gly Asn Gln Asn Arg Tyr 355 360
365 Lys Asn Ile Gly Val Gly Asp Pro Val Asp Gly Pro Ile Ile
Gly Gln 370 375 380
Ser Val Tyr Glu Lys Val Asp Ala Gly Ile Pro Thr Asn Glu Trp Val385
390 395 400 Tyr Glu Val Gly Val
Asn Gly Ile Gln Asn Asp Tyr Pro Arg Asn Ile 405
410 415 Gly Leu Arg Lys Gly Ser Thr Thr Ala Phe
Thr Asp His Leu Ala Gly 420 425
430 Ser Gln Tyr Asn Leu Gly Pro Leu Thr Arg Val Ser Ile Pro Thr
Lys 435 440 445 Asp
Asn Ala Pro Ile Asn Asn Thr Asn Phe Thr His Arg Leu Ser Asp 450
455 460 Ile Ile Leu Pro Gly Asn
Lys Gly Ser Ser Phe Ala Trp Thr His Val465 470
475 480 Asp Val Asp Pro Thr Gly Asn Tyr Leu Ser Thr
Thr Lys Ile Asn Leu 485 490
495 Ile Pro Ala Thr Lys Ala Ser Lys Ile Pro Leu Ser Phe Tyr Leu Arg
500 505 510 Lys Gly Pro
Gly Phe Ile Gly Gly Asp Leu Val Arg Leu Gly Ser Gly 515
520 525 Phe Glu Cys Ser Tyr Lys Phe Asn
Phe Lys Ser Pro Gly Ser Ser Ala 530 535
540 Asn Phe Arg Ile Arg Ile Arg Tyr Ala Gly Ala Gly Ser
Gly Gln Gly545 550 555
560 Ala Asp Gly Gln Val Tyr Phe Lys Leu Gly Asn Tyr Thr Ser Pro Thr
565 570 575 Thr Pro Trp Gly
His Thr Gly Phe Asp Tyr Gly Asn Val Lys Tyr Asn 580
585 590 Gln Phe Arg Val Leu Glu Leu Phe Gly
Thr Ala Glu Asn Ile Thr Asp 595 600
605 Asn Asp Leu Lys Ile Ile Val Trp Thr Gly Ser Ser Ala Gln
Asp Phe 610 615 620
Leu Ser Arg625 20566PRTBacillus thuringiensis 20Val Ser Pro Met
Tyr Ile Asn Thr Met Lys Asn Thr Leu Lys Leu Glu1 5
10 15 Thr Thr Asp Tyr Glu Ile Asp Gln Ala
Ala Ile Ser Ile Glu Cys Met 20 25
30 Ser Asn Glu Gln Asn Pro Gln Glu Lys Met Ile Leu Trp Asp
Glu Val 35 40 45
Lys Gln Ala Lys Gln Leu Ser Gln Ser Arg Asn Leu Leu Tyr Asn Gly 50
55 60 Asp Phe Glu Asp Ala
Ser Asn Gly Trp Lys Thr Ser Tyr Thr Ile Glu65 70
75 80 Ile Gly Lys Tyr Ser Ser Ile Phe Lys Gly
Gln Tyr Leu His Met Phe 85 90
95 Gly Ala Arg Asp Val Leu Gly Glu Val Phe Pro Thr Tyr Val Tyr
Gln 100 105 110 Lys
Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg Tyr Arg Val Arg 115
120 125 Gly Phe Val Gly Ser Ser
Lys Asp Leu Lys Leu Val Val Thr Arg Tyr 130 135
140 Gly Lys Glu Ile Asp Ala Ile Met Asp Val Pro
Asp Asp Leu Ala Tyr145 150 155
160 Met Gln Pro Thr Pro Ser Cys Gly Asp Tyr Arg Cys Glu Ser Ala Ser
165 170 175 Gln Tyr Val
Ser Gln Gly Tyr Pro Thr Pro Tyr Gly Asp Gly Tyr Ala 180
185 190 Ser Asp Arg Tyr Ala Cys Pro Ser
Asp Arg Val Lys Lys His Val Lys 195 200
205 Cys His Asn Arg His Pro Phe Asp Phe His Ile Asp Thr
Gly Glu Leu 210 215 220
Asp Ile Asn Thr Asn Val Gly Ile Trp Val Leu Phe Lys Ile Ser Asn225
230 235 240 Pro Asp Gly Tyr Ala
Thr Leu Gly Asn Leu Glu Val Ile Glu Glu Gly 245
250 255 Pro Ile Thr Gly Glu Ala Leu Thr His Ala
Lys Gln Lys Glu Lys Lys 260 265
270 Trp Asn Gln His Met Glu Lys Ala Gln Ile Glu Thr Gln Gln Ala
Tyr 275 280 285 Asp
Pro Ala Lys Gln Ala Val Asp Ala Leu Phe Thr Asn Ala Gln Gly 290
295 300 Glu Glu Leu His Tyr His
Ile Thr Leu Asp His Ile Gln Asn Ala Asn305 310
315 320 Gln Leu Val Gln Ser Ile Pro Tyr Val His His
Ala Trp Leu Pro Asp 325 330
335 Ala Pro Gly Met Asn Tyr Asp Val Tyr Gln Glu Leu Asn Ala Arg Ile
340 345 350 Met Gln Ala
Arg Tyr Leu His Asp Ala Arg Asn Val Ile Thr Asn Gly 355
360 365 Asp Phe Thr Gln Gly Leu Gln Gly
Trp His Ala Thr Gly Lys Ala Thr 370 375
380 Val Gln Gln Met Asp Gly Ala Ser Val Leu Val Leu Ser
Asn Trp Ser385 390 395
400 Ala Gly Val Ser Gln Asn Leu His Ala Gln Asp His His Gly Tyr Val
405 410 415 Leu Arg Val Ile
Ala Lys Lys Glu Gly Pro Gly Lys Gly Tyr Val Thr 420
425 430 Met Met Asp Cys Asn Gly Asn Gln Glu
Thr Leu Lys Phe Thr Ser Cys 435 440
445 Glu Glu Gly Tyr Met Thr Lys Thr Val Glu Ile Phe Pro Glu
Ser Asp 450 455 460
Arg Val Arg Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Tyr Val Asp465
470 475 480 Ser Ile Glu Leu Leu
Cys Met Gln Gly Tyr Ala Ile Asn Asn Asn Pro 485
490 495 His Thr Gly Asn Met Tyr Glu Gln Ser Tyr
Asn Gly Ile Tyr Asn Gln 500 505
510 Asn Thr Ser Asp Val Tyr His Gln Gly Tyr Thr Asn Asn Tyr Asn
Gln 515 520 525 Asp
Ser Ser Ser Met Tyr Asn Gln Asn Tyr Thr Asn Asn Asp Asp Gln 530
535 540 His Ser Asp Cys Thr Cys
Asn Gln Gly His Asn Ser Gly Cys Thr Cys545 550
555 560 Asn Gln Gly Tyr Asn Arg 565
21296PRTBacillus thuringiensis 21Met Lys Lys Leu Met Phe Ser Leu Val
Ala Thr Thr Met Ser Met Gly1 5 10
15 Leu Ile Leu Gly Ser Ala Pro Val Lys Ala Asp Val Ser Asn
Lys Asn 20 25 30
Ser Ala Tyr Gln Asp Ile Asp Glu Arg Val Lys Lys Met Ala Gln Ser 35
40 45 Ala Ala Trp Gly Gly
Gln Glu Tyr Arg Asn His Asn Ile Lys Asp Ile 50 55
60 Glu Leu Lys Gly Asn Leu Ile Asp Gly Ser
Met Ile Glu Asn Ser Glu65 70 75
80 Val Leu Thr Val Ser Ser Asp Ile Leu Glu Asn Lys Leu Gly His
Thr 85 90 95 Val
Asn Met Pro Ser Thr Gly Tyr Glu His Glu Phe Glu Glu Thr Thr
100 105 110 Asn Thr Thr Asn Thr
Ser Gly Trp Thr Phe Gly Tyr Asn Tyr Asn Ala 115
120 125 Ser Phe Ser Val Leu Met Val Ser Ala
Ser Gln Ser Phe Ser Val Glu 130 135
140 Tyr Asn Met Ser Thr Ser Asn Thr His Glu Lys Lys Glu
Lys Arg Lys145 150 155
160 Phe Thr Val Pro Ser Ile Glu Val Pro Val Pro Ala Gly Lys Lys Tyr
165 170 175 Lys Val Glu Tyr
Val Phe Glu Lys Val Lys Val Ser Gly Lys Asn Lys 180
185 190 Ile Asp Ala Asn Leu Tyr Gly Asp Val
Thr Tyr Tyr Tyr Asn Asn Gln 195 200
205 Pro Met Ser Pro Gln Leu Leu Tyr Ser Val Gln Gly Leu Ala
Ala Asp 210 215 220
Lys Gln Gly Phe Glu Gln Val Ile Arg Asp Ser Ala Val Gly Asn Asp225
230 235 240 Arg Phe Gly Ile Lys
Thr Thr Gly Ile Gly Gln Phe Ser Thr Glu Phe 245
250 255 Gly Thr Arg Leu Thr Arg Thr Leu Thr Asp
Ile Thr Asp Thr Arg Asn 260 265
270 Pro Val Lys Val Glu Thr Lys Asn Val Pro Val Glu Phe Lys Thr
Leu 275 280 285 Ser
Ile Asp Thr Arg Val Ile Lys 290 295
22308PRTBacillus thuringiensis 22Met Lys Lys Asn Arg Met Leu Leu Lys Trp
Met Cys Gly Leu Thr Ile1 5 10
15 Gly Ile Gly Ser Leu Thr Gly Gly Ser Leu Asn Ala Phe Ala Asp
Glu 20 25 30 Val
Ser Asp Ser Leu Ala Asp Val Gly Phe Leu Tyr Gly Asp Tyr Leu 35
40 45 Tyr Lys Thr Lys Gln His
Pro Gln Gly Thr Leu Pro Ile Thr Tyr Pro 50 55
60 Met Arg Glu Ile Asn Asn Tyr Gln Ile Ile Asp
Lys Ser Val Ser Gln65 70 75
80 Val Gly Ser Thr Glu Tyr Glu Glu Gly Gln Thr Leu Tyr Val Asp Asp
85 90 95 Asp Val Phe
Asp Asn Lys Thr Gly Thr Asp Gln Thr Phe Lys Thr Ile 100
105 110 Gln Phe Glu Lys Glu Phe Ser Glu
Thr Ala Thr Ser Ser Thr Thr His 115 120
125 Ser Val Gly Thr Ser Leu Glu Glu Ser Val Lys Phe Asp
Phe Phe Val 130 135 140
Gly Glu Gly Ser Ala Lys Phe Thr Val Asn Tyr Asn Phe Ser Lys Thr145
150 155 160 Gly Ser Leu Ser Thr
Ser Asn Lys Ile Lys Tyr Thr Leu Pro Ser Gln 165
170 175 Ser Ile Asn Val Pro Ala Asn Lys Lys Tyr
Glu Val Ile Cys Val Leu 180 185
190 Glu Thr Lys Lys Ala Lys Ala Asn Val Gln Phe Asn Val Asp Val
Leu 195 200 205 Gly
Asn Ala Lys Tyr Val Tyr Ser Asn Asn Ser Pro Tyr Thr Pro Lys 210
215 220 Tyr Glu Ser Gly Ala Thr
Met Leu Lys Thr Leu Asn Glu Lys Asn Pro225 230
235 240 Thr Pro Ser Val Ser Trp Leu Gly Lys Glu Trp
Glu Lys Trp Glu Tyr 245 250
255 His Asp Gly Lys Ala Arg Tyr Lys Asn Gly Ser Gly Thr Val Ser Ala
260 265 270 Glu Tyr Gly
Thr Arg Met Val Leu Val Ile Asn Asp Ile Thr Asn Asn 275
280 285 Lys Thr Arg Gly Ser Lys Glu Ile
Ala Arg Ile Pro Val Thr Pro Ile 290 295
300 Gln Lys Gln Met305 231244PRTBacillus
thuringiensis 23Met Asn Gly Gly Glu Asn Met Asn Gln Asn Asn Gln Asn Glu
Met Gln1 5 10 15
Ile Ile Asp Ser Ser Ser Asn Asp Phe Ser Gln Ser Asn Arg Tyr Pro
20 25 30 Arg Tyr Pro Leu Ala
Lys Glu Ser Asn Tyr Lys Asp Trp Leu Ala Ser 35 40
45 Cys Asp Glu Ser Asn Val Asp Thr Leu Ser
Ala Thr Ser Asn Val Arg 50 55 60
Ala Ser Val Ser Arg Ala Leu Gly Ile Val Asn Gln Ile Leu Gly
Phe65 70 75 80 Leu
Gly Leu Gly Phe Ile Gly Thr Gly Leu Gly Val Leu Ser Asp Leu
85 90 95 Phe Asn Ser Phe Trp Pro
Ser Asn Asp Asn Ser Ile Trp Glu Ser Phe 100
105 110 Leu Arg Ser Val Glu Glu Leu Ile Asp Arg
Arg Val Arg Glu Val Glu 115 120
125 Arg Phe Arg Ile Glu Ser Gln Phe Thr Gly Leu Arg Asn Val
Met Ser 130 135 140
Asn Tyr Asn Gly Ala Leu Arg Asp Trp Asn Gly Asn Arg Asn Asn Leu145
150 155 160 Ala Leu Gln Ser Glu
Val Arg Ser Arg Phe Asp Asn Ser Asp Asp Ala 165
170 175 Phe Ala Ser Arg Met Pro Glu Phe Arg Ile
Glu Gly Phe Glu Ile Gln 180 185
190 Ser Leu Ala Val Tyr Ala Gln Ala Ala Thr Leu His Leu Leu Leu
Leu 195 200 205 Arg
Asp Gly Val Val Asn Gly Leu Gln Trp Gly Phe Asp Ile Val Thr 210
215 220 Val Asn Arg Leu Tyr Glu
Lys Leu Val Cys Leu Ser Gly Ala Tyr Ala225 230
235 240 Asp His Cys Thr Leu Phe Tyr Arg Gln Gly Leu
Glu Glu Leu Arg Asn 245 250
255 Arg Gly Asn Trp Asn Ala Phe Asn Asn Tyr Arg Arg Asp Met Thr Leu
260 265 270 Gln Val Leu
Asp Val Ile Ser Leu Phe Pro Asn Tyr Asp Pro Arg Leu 275
280 285 Tyr Asp Ile Asn Thr Asn Thr Gln
Leu Thr Arg Glu Ile Tyr Thr Glu 290 295
300 Pro Leu Ala Ile Pro Gly Trp Leu Asn Ser His Ser Asn
Pro Thr Gln305 310 315
320 Phe Gln Gln Ile Glu Asn Asp Leu Ile Arg Ser Pro Ser Val Phe Ser
325 330 335 Asn Leu Glu Thr
Leu Phe Met Glu Ala Gly Phe Ala Phe Phe Gln Ala 340
345 350 Gly Ile Ala Arg Gln Ala Val Leu Arg
Thr Arg Thr Ser Ser Leu Asn 355 360
365 Met Asn Arg Thr Ala Val Ile Val Thr Pro Trp Gln Gly Ala
Pro His 370 375 380
Pro Asn Val Ser His Glu Leu Gln Val Thr Leu Gln Asp Arg Asn Val385
390 395 400 Phe Asn Ile Asn Ser
Val Val Gly Arg Glu Ile Ser Ser Gln Thr Gly 405
410 415 Leu Leu Phe Gly Val Gln Gln Ala Thr Phe
His Phe Val Trp Ala Gly 420 425
430 Gly Asn Ala Ala Thr Thr Thr Gln Phe Asn Leu Pro Pro Ile Ser
Gly 435 440 445 His
Ser Thr Ser Ile Thr Ser Asn Ile Pro Gly Thr Asn Ser Thr Thr 450
455 460 Pro Thr Gly Ser Asp Tyr
Thr His Arg Leu Ser Ser Ile Thr Ser Thr465 470
475 480 Ser Val Gly Thr Trp Gln Arg Asp Arg Thr Asn
Ile Met Ala Tyr Gly 485 490
495 Trp Thr His Val Ser Ala Glu Arg Thr Asn Arg Ile Ile Pro Asn Arg
500 505 510 Ile Thr Gln
Ile Pro Ala Val Lys Gly Ser Leu Phe Ser Asp Asn Pro 515
520 525 Pro Asn Thr Ser Arg Thr Arg Val
Glu Asn Gly Pro Gly His Thr Gly 530 535
540 Gly Gly Leu Val Val Met Asp Gly Gly Thr Ser Val Leu
Gln Met Arg545 550 555
560 Val Thr Ser Ser Ala Arg Gln Arg Tyr Asp Met Arg Leu Arg Tyr Val
565 570 575 Ala Leu Ala Pro
Ala Thr Val Glu Val Arg Ile Pro Glu Leu Gly Gly 580
585 590 His Val Arg Phe Gln Met Pro Met Thr
Ala Thr Gly Leu Pro Ala Pro 595 600
605 Leu Pro Tyr Ser His Leu Arg Tyr Val Asp Ile Pro Leu Arg
Phe Glu 610 615 620
Thr Pro His Gly Glu Asn Thr Trp Thr Phe Glu Leu Gln Thr Thr Phe625
630 635 640 Ala Ala Val Ala Ile
Asp Arg Val Glu Phe Ile Pro Val Asn Ala Thr 645
650 655 Ala Leu Glu Tyr Glu Gly Lys Arg His Leu
Glu Lys Ala Lys Lys Ala 660 665
670 Val Gly Asp Leu Phe Ile Asn Asn Gly Lys Glu Ala Leu Lys Val
Asp 675 680 685 Thr
Thr Asp Tyr Asp Val Asp Gln Ala Ala Asn Leu Val Glu Cys Met 690
695 700 Pro Glu Glu Leu Tyr Thr
Lys Glu Lys Met Ile Leu Leu Asp Glu Val705 710
715 720 Lys His Ala Lys Arg Phe Ser Gln Ser Arg Asn
Leu Ile Gln Asn Gly 725 730
735 Asp Phe Glu Phe Ala Thr Asp Gly Trp Met Thr Ser Ser Asn Val Ile
740 745 750 Val Gln Ala
Asp Asn Thr Val Phe Lys Gly Lys Tyr Leu Asn Met Pro 755
760 765 Gly Ala Ile Glu Thr Asp Thr Ser
Thr Phe Pro Thr Tyr Ile Tyr Gln 770 775
780 Lys Ile Asp Glu Ser Arg Leu Lys Pro Tyr Thr Arg Tyr
Lys Val Arg785 790 795
800 Gly Phe Val Gly Ser Ser His Asp Val Arg Leu Ile Val Glu Arg Asn
805 810 815 Gly Lys Glu Val
Asp Ala Leu Leu Asn Val Arg Asn Asp Leu Ser Leu 820
825 830 Asp Thr Val Ala Pro Ser Cys Ile Glu
Ala Asn Gln Pro Tyr Pro Ile 835 840
845 Ile His Asp Gly Cys Leu Thr Asn Val Ile Asp Thr Asn Ser
Tyr Glu 850 855 860
Glu Ala Gln Ser Gly His Ala Asn Cys Lys Lys Glu His Gly Met Cys865
870 875 880 His Gln Ser His Gln
Phe Asp Phe His Ile Asp Thr Gly Glu Ile His 885
890 895 Thr Asn Lys Asn Pro Gly Ile Trp Val Leu
Phe Lys Ile Ser Ser Pro 900 905
910 Glu Gly His Ala Thr Leu Asp Asn Ile Glu Leu Ile Glu Asp Gly
Pro 915 920 925 Leu
Val Gly Glu Ser Leu Ala Phe Val Lys Lys Gln Glu Lys Lys Trp 930
935 940 Glu Asn Glu Met Glu Thr
Arg Trp Leu Gln Thr Lys Glu Val Tyr Glu945 950
955 960 Lys Ala Lys Gly Glu Ile Asp Ser Leu Phe Thr
Asp Ala Gln Asp Gln 965 970
975 Ala Leu Lys Phe Asp Thr Asn Ile Ser His Ile Ile Ser Ala Glu His
980 985 990 Leu Val Gln
Ser Met Pro Tyr Val Tyr Asn Asn Trp Leu Ser Asp Val 995
1000 1005 Pro Gly Met Asn Tyr Asp Ile Tyr
Thr Glu Leu Glu Arg Arg Ile Thr 1010 1015
1020 Gln Ala Tyr Ser Leu Tyr Glu His Arg Asn Ile Ile Lys
Asn Gly Asp1025 1030 1035
1040 Phe Asp Tyr Gly Leu Asn His Trp His Ala Thr Pro His Ala Lys Val
1045 1050 1055 Gln Gln Ile Asp
Gly Thr Ala Val Leu Val Leu Pro Asn Trp Ser Ser 1060
1065 1070 Asn Val Ser Gln Asn Leu Cys Val Glu
His Asn Arg Gly Tyr Ile Leu 1075 1080
1085 Arg Val Thr Ala Lys Lys Glu Asp Met Gly Lys Gly Tyr Val
Thr Ile 1090 1095 1100
Ser Asp Cys Asn Gly Asn Gln Glu Thr Leu Thr Phe Thr Ser Cys Ala1105
1110 1115 1120 Asn Tyr Val Ala Asn
Glu Ile Thr Asn Asp Gln Ser Glu Tyr His Phe 1125
1130 1135 Ser Gln Glu Met Asn Glu Gln Arg Gly Tyr
Asn Pro Asn Glu Thr Ile 1140 1145
1150 Asn Lys Gln Leu Asp Tyr Ser Leu Asp Gln Val Arg Asn Glu Gln
Arg 1155 1160 1165 Cys
Tyr Asn Pro Asn Glu Ile Thr Asn Asp Gln Ser Glu Tyr His Tyr 1170
1175 1180 Ser Gln Glu Met Asn Glu
Gln Arg Cys Tyr Asn Pro Asn Glu Ile Ile1185 1190
1195 1200 Asn Glu His Arg Asn Tyr Val Thr Arg Thr Ile
Asp Phe Phe Pro Asp 1205 1210
1215 Thr Asn Gln Val Arg Ile Asp Ile Gly Glu Thr Glu Gly Thr Phe Lys
1220 1225 1230 Val Glu Ser
Ile Glu Leu Ile Cys Met Lys Ser Gln 1235 1240
24521PRTBacillus thuringiensis 24Met Thr Lys Ser Glu Phe Ile Gln
Asn Ser Cys Arg Lys Met Gln Ser1 5 10
15 Lys Val Arg Val Ile Ile Leu Ser Thr Asn Asp Pro Val
Val Asn Asn 20 25 30
Asn Thr Leu Asp Ile Thr Glu Ile Lys Asp Leu Ala His Leu Ser Gln
35 40 45 Ala Ile Met Leu
Ala Asn Asn Phe Gln Ala Ala Leu Val Pro Thr Ser 50 55
60 Ser Glu Phe Gly Gln Asp Val Leu Arg
Phe Asp Val Asn Gln Gly Ile65 70 75
80 Ser Ile Ala Asn Asn Ile Tyr Pro Lys Ala Val Asp Ile Asn
Tyr Ile 85 90 95
Ser Arg Thr Leu Ser Gln Ser Asn Asn Gln Val Asn Ser Met Ile Asn
100 105 110 Met Val Val Asn Glu
Leu Lys Leu Leu Leu Gly Ile Asn Leu Ala Asp 115
120 125 Ser Val Leu Gln Gln Leu Thr Ser Leu
Val Ala Tyr Thr Phe Thr Asn 130 135
140 Leu Tyr Thr Gln Gln Asn Ser Ala Trp Val Phe Trp Gly
Lys Gln Ala145 150 155
160 Ser Asn Gln Thr Asn Tyr Thr Tyr Asn Ile Val Phe Ala Ile Gln Asn
165 170 175 Ala Gln Thr Gly
Asn Phe Met Lys Ala Ile Pro Met Gly Phe Glu Ile 180
185 190 Ser Ala Tyr Ala Val Lys Glu Gln Val
Leu Phe Phe Thr Ile Gln Asp 195 200
205 Tyr Ala Ser Tyr Ser Val Lys Ile Gln Ala Ile Asn Val Thr
Gln Pro 210 215 220
Leu Ile Asn Ser Ser Tyr Gly Ser Leu Ser Gly Val Tyr Asn Ile Ile225
230 235 240 Thr Ala Leu Asn Asn
Ile Ser Val Ile Thr Met Ser Asn Ser Asp Glu 245
250 255 Asn Val Asn Leu Trp Tyr Asp Asn Asp Asp
Leu Asn Gln Lys Trp Ile 260 265
270 Leu Glu Phe Asn His Asn His Tyr Ala Tyr Ile Ile Arg Asn Leu
Ser 275 280 285 Asn
Arg Ser Leu Val Leu Thr Trp Asp Ser Thr Ser Gly Ser Asn Asn 290
295 300 Val Phe Ala Thr Asn Tyr
Gln Gly Asn Asp Glu Gln Phe Trp Ile Ile305 310
315 320 Gln Asp Thr Asp Asn Asp Tyr Phe Tyr Leu Ser
Asn Met Arg Asp Thr 325 330
335 Gln Tyr Val Leu Glu Ile Ala Gly Ser Val Phe Tyr Asn Gly Thr Asn
340 345 350 Val Ile Val
Asn Lys Lys Thr Ser Ser Leu Asn Gln Lys Phe Ser Ile 355
360 365 Asn Arg Ile Asn Arg Gln Ile Gln
Asn Gly Ile Tyr Asn Ile Thr Thr 370 375
380 Tyr Leu Asn Ala Ser Ser Val Ile Thr Met Ser Thr Asp
Tyr Asn Ile385 390 395
400 Asn Val His Asp Tyr Pro Val Asn Leu Trp Phe Lys Asn Asp Ser Ile
405 410 415 Asn Gln Lys Trp
Ile Phe Glu Phe Asp Ser Asp Lys Ser Ala Tyr Arg 420
425 430 Val Arg Ser Val Ser Asn Pro Ser Leu
Phe Leu Ser Trp Pro Val Ala 435 440
445 Ser Phe Thr Asn Arg Ala Ala Val Thr Pro Asn Pro Arg Asp
Asn Glu 450 455 460
Tyr Phe Trp Phe Leu Gln Ser Ala Gly Leu Gly Thr Phe Tyr Leu Val465
470 475 480 Ser Met Arg Asp Thr
Arg Tyr Val Leu Glu Val Glu Asn Ser Asn Ile 485
490 495 Asp Asn Gly Thr Asn Ile Ile Val Asn Gln
Arg Thr Gly Asn Phe Asn 500 505
510 Gln Arg Phe Tyr Ile Glu Asn Ile Asn 515
520 251992DNAArtificial Sequencesynthetic nucleotide sequence
encoding a toxin protein 25atgcacatca tcgacagcag cagcaatgat
ttctcccaaa gcaacagata tccaagatat 60cctctggcca aggagagcaa ctacaaggac
tggctggcaa gctgtgatga aagcaacctg 120gaccgcctct ccacgccttc ttcagttcaa
gatgctgtgg tgacaagcct caacatcttc 180agctacatct tcggcttcct tgatgctggc
gccacctccg ccggcctcgg catcctcggc 240gtcctcttcg gccagttctg gccatcaaac
aacaatgctg tttgggaaac cttcttgagg 300agcgtggagg agctgattgc aagggagatc
gacatcgtgg agaggaacag gatcatggcg 360cagtttgatg gcctcagaaa tgtgatgagc
aactacaatg gagctctcat cgactgggat 420ggcaacaggg acaacaccgc gctgcaatca
gaggtgagga gcagatttga caatgctgat 480gatgccttcg ccttgaggat tccagagttc
agaatcaagg attttgagat ccagagcctc 540gccgtctatg ctcaagctgc caccctccac
ctgctgctgc tgagagatgc tgtggtgaat 600ggccagctat ggggagttga tcctgtcacc
acccaaagaa gatatgagaa gctggtctgc 660ctctccggcg cctatgctga tcactgcacc
ttcttctaca ggcaagggct ggaggagctg 720agaggaaaag gaaactggac ggccttcaac
aactacagga ggaacatgaa catccaggtg 780ctggatgtca tctccctctt ctcaaactat
gatccaaggc tctatggcaa caacaccaac 840acccagctga caagggagat cttcaccgag
ccgctggcga cgccaggatg gctggacaga 900tacagcaacc cagatcagtt ccagcagatc
gagtacaacc tcaacccttc tccaagcctc 960tcctcaaccc tcctcaacct tgctgctgac
accggcctcg gctactacgg cgccggcgcc 1020gtcacgccgc gccccatcat ccagaggacc
tccatgagga ggctcaacac cggcgccacc 1080gtgcccttca ccaccgcctg gcaaggagct
ccaaatcctc tcatcagcca gcagaagcag 1140ctctcctttg aaggatttga tgttttcaac
atcaacagcg tggtgtcaag agaagtttct 1200tctcaaacaa gcagcctctt cggcgtccag
caagctgtgt tccacaccgt cattgctgga 1260ggaaacatcc ctccaacaat gaagatcatt
gatctccagc caagaggaaa ctacagcacc 1320tccgtcacca gcaacatccc tggaaaaaga
agcgccaccg ccaccgcctc agattacacc 1380caccgcctga gcagcatcac ctccacctcc
gtcggcaccg cctaccgtga caggaccaac 1440atcatggcat atggatggac ccatgtttct
tcagaaaaaa caaacaggat tcttccaaac 1500aggatcaccc agatcccctt cgtcaagggc
atcatcacca gcagcggcac ccatgtgagg 1560agcggccctg atcacactgg aggaagcctg
gtgagcatgg gaggagatgc tcaatttggc 1620atggtggtga caagctccgc gcgccaaaga
tacagggtga ggctgagata tgctgcttca 1680aattctgttg atttccgcct gaggatctcg
ccgctcggcg tggactacaa cttcaccctt 1740cctggaggag gcaccagctt caacccagat
ctccgctaca gcagcttcag atacatcacc 1800ctccccatcg agtttgaaac accaaattca
ttgctgaact tctccttcga cctggacacc 1860ctcaccttga tgaatggcac ctgcttcttc
gacagggtgg agttcctccc cgtcaacagc 1920attgctctgg aatatgaagg aaagcagaag
ctggagaagg ccaagcaagc tgtggacaac 1980ctcttcacca ac
1992261992DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
26atgcacatca tcgacagcag cagcaatgat ttctctcaaa gcaacagata tccaagatat
60cctctggcca aggagagcaa ctacaaggat tggctggcaa gctgtgatga aagcaacctg
120gataggctct ccactccttc ttcagttcaa gatgctgtgg tgacaagcct caacatcttc
180agctacatct ttggcttcct tgatgctgga gcaacctccg ccggccttgg catcctcggc
240gtcctctttg gccagttctg gccatcaaac aacaatgctg tttgggaaac cttcttgagg
300agcgtggagg agctgattgc aagagagatt gacattgtgg agaggaacag gatcatggct
360caatttgatg gcttgagaaa tgtgatgagc aactacaatg gagctctcat tgattgggat
420ggaaacagag acaacaccgc gctgcaatca gaagtgagaa gcagatttga caatgctgat
480gatgcctttg ctctgaggat tccagagttc agaatcaagg attttgagat ccaaagcctc
540gccgtctatg ctcaagctgc aaccctccat ctgctgctgc tgagagatgc tgttgtcaat
600ggccagctat ggggagttga tcctgtcacc acccaaagaa gatatgagaa gctggtttgc
660ttgagcggcg cctatgctga tcattgcacc ttcttctaca ggcaagggct ggaggagctg
720agaggaaaag gaaactggac ggccttcaac aactacagaa gaaacatgaa catccaggtg
780ctggatgtca tctccctctt ctcaaattat gatccaaggc tctatggaaa caacaccaac
840acccagctga caagagagat cttcacagag ccgctggcaa caccaggatg gctggacaga
900tacagcaatc cagatcagtt ccagcagatt gaatacaacc tcaatccttc tccttctctc
960tcttcaacat tgctcaacct tgctgctgac actggccttg gctactatgg cgccggcgcc
1020gtcacgccgc gccccatcat ccaaagaaca agcatgagaa ggctcaacac cggcgccact
1080gttcccttca ccaccgcctg gcaaggagct ccaaatcctc tcatcagcca gcagaagcag
1140ctctcctttg aaggatttga tgttttcaac atcaacagcg tggtttcaag agaagtttct
1200tctcaaacaa gcagcttgtt cggcgtccag caagctgttt tccacaccgt cattgctgga
1260ggaaacattc ctccaacaat gaagatcatt gatcttcaac caagaggaaa ctacagcacc
1320tccgtcacca gcaacattcc tggaaaaaga agcgccaccg ccactgcttc agattacacc
1380caccgcctga gcagcatcac ctccacctct gttggcaccg cctaccgtga cagaacaaac
1440atcatggcat atggatggac acatgtttct tcagaaaaaa caaacaggat tcttccaaac
1500aggatcaccc agatcccctt cgtcaagggc atcatcacca gcagcggcac ccatgtcaga
1560agcggccctg atcacactgg aggaagcctg gtgagcatgg gaggagatgc tcaatttgga
1620atggtggtga caagctccgc gcgccaaaga tacagggtga ggctgagata tgctgcttca
1680aattctgttg atttcagatt gaggatctcg ccgctgggag tggactacaa cttcaccctt
1740cctggaggag gaaccagctt caatccagat ctccgctaca gcagcttcag atacatcacc
1800ctccccattg aatttgaaac accaaattca ttgctgaact tctcctttga tctggacacc
1860ttgaccttga tgaatggaac ctgcttcttc gacagggtgg agttccttcc tgtcaacagc
1920attgctctgg aatatgaagg aaagcagaag ctggagaagg ccaagcaagc tgtggacaac
1980ctcttcacca ac
1992272427DNAArtificial Sequencesynthetic nucleotide sequence encoding a
toxin protein 27atgaacagct accagaacag aaatgaatat gagatcctgg
atgcttctcc aaaccatggc 60aacatctcaa acagatatcc cttcgccaag aacccaaatg
ccatggagaa tgtcaactac 120aagaactggc tgaatgttag agaagatgtt gctccttcct
tcttcggcgg cgcgctgggc 180atcatcgtca acctcttcaa gcaatatgtc agcttcatca
aggctccttc agtttctggc 240ggcgtcggct tcttgaggac catcatcggc ctgatgacaa
acagaaatgt catcaacctc 300accattgatg atgttcaaag gctcatcaac cagagcttgg
acaacatcac cagagatgct 360gccaacacca agttcacctc aatccagaac aactacaacc
agtacctcct caacaggcag 420aactacagga acaggagctt gccaaggaac atcttcgtcc
agagcctcca gaacattgaa 480agggagctga gatcagctct ggacagcacc ttctccctcc
agaacaggga gctgctgctg 540ctgccaaact tcacccagat cgccatgctt catctcaccg
tgctgagaga tgctgtcatc 600ttccaaggaa atgatctcat cgtccccacc atctcagaag
gccccatcaa cccgctgctg 660acaaggccgc caagcaacac ctttgaagaa gctctcctca
cctccatcag gatctacagc 720aactactgcg tccgccaata tgaagttggc ctcaacctgc
tgaggaacag aggaaacacc 780tcaaggaact ggctggactt caatgcctac aggctggaga
tgaccttcaa ggtgctggac 840ttcgtcaccc tcttctccct cttcgacacc gtcaagtacc
ccgtcagcat tgtttcagaa 900acagattctg acagcacctc gccggtggtg taccagctca
gcagggtgat ctacacagat 960cctgttggcg ccatcagatc tgatggaaga ggatggtttg
atcctcctgt tggaacagat 1020cgcgtcacct tcaccagcat tgaaaatgag atccctgctc
caacaacaag ccgccacctc 1080tcagagctca ccatctcctc tgggccgctg ggcttcggcg
tcaacccagc aagaactcac 1140agctggcaag gaaacagaaa tgtgaacatc tcagctccaa
ctgatgtctc cggcgccatc 1200agcaacagga caaggaccat cccagcaagg aacatcttca
gagtgaacag cagggtgtac 1260accttggact ggaggctcta cggcgtctac agagcagagt
tcttccaagg agctcacagc 1320caggtgttct cagaaaatcc tccaactggc attggagctc
aaagcgccaa caacttcaga 1380tttcttcctg gagaaaattc tgaaacgccg acgccgcagg
actacaccca tgtgctgagc 1440agggtggtga atgccaccgt cggcctcacg ccggccaccg
gcaaccagag gaacagcgtg 1500ctgatctttg gatggaccca caagagcttg acatcagaga
acatctacag gatcaatgag 1560atcaccaagg tggccgccgt caacacaaga ggaaacagcg
gcatcagggt gatctcagga 1620cctggcttca ctggaggaga tctggtgagg ctggacccta
atggcagcgt cagctacaac 1680ttcacgccgg ccaaccagca ggcgctgcaa agcaacatca
ggatcaggct gagatatgct 1740tgccaaggaa cagcttcctt gaggatcacc tttggaaatg
cttcaagcca ggtgatctcc 1800ttgatctcca ccaccagcag catcaacaac cttcaatatg
agaacttcca tgtggtgaat 1860gttccaaaca acatcaactt ccagagcgtc ggcacccaga
tcaccatcca gaacatcagc 1920caaaatccaa acatcagctt ggacagcatt gagctcttct
caaacatccc catccctcaa 1980gaacctccct tcaacccggt ggtgccggag ccgcccatca
tctcaggaaa ctaccagatt 2040gtgacgccgc tggacagaag aagcatcatc gacctcaacc
ccaacaacaa tgtcaccctc 2100tggaccaaca acagaagcag caaccagatc tggaacttca
gctatgatca gcagcgcggc 2160gcctacctca tcaggagctt gaggacagga agcctggtgc
tctccatgga ttcaccaagg 2220acaagcaatg tttttggata tccaagcaac tctgatgctt
ctcagttctg gatcttggag 2280ccaaatcaag atggcttcat cttcagatcc ttgagggaca
ggaacctggt gctggatgtt 2340tctggaggaa gagttgatcc tggaacaagg atcatcgtgt
tccccttcac caacagcatc 2400aaccagaggt tcaccctcca gcgcgtc
2427282427DNAArtificial Sequencesynthetic
nucleotide sequence encoding a toxin protein 28atgaacagct accagaacag
aaatgaatat gagatcctgg atgcttctcc aaatcatgga 60aacatctcaa acagatatcc
cttcgccaag aatccaaatg ccatggaaaa tgtcaactac 120aagaactggc tgaatgttag
agaagatgtt gctccttcct tcttcggcgg cgcgctgggc 180atcatcgtca acctcttcaa
gcaatatgtc agcttcatca aggctccttc agtttctgga 240ggcgtcggct tcttgaggac
catcattggg ctgatgacaa acagaaatgt catcaacctc 300accattgatg atgttcaaag
gctcatcaac cagagcttgg acaacatcac aagagatgct 360gcaaacacca agttcacctc
aatccagaac aactacaacc agtacctcct caacaggcag 420aactacagga acaggagctt
gccaaggaac atcttcgtcc aaagcctcca gaacattgaa 480agagagctga gatcagctct
ggacagcacc ttctccctcc agaacaggga gctgctgctg 540ctgccaaact tcacccagat
cgccatgctt catctcaccg tgctgagaga tgctgtcatc 600ttccaaggaa atgatctcat
cgtccccacc atctcagaag gacccatcaa cccgctgctg 660acaaggccgc caagcaacac
ctttgaagaa gctctcctca cctccatcag gatctacagc 720aactactgcg tccgccaata
tgaagttggc ctcaacctgc tgaggaacag aggaaacaca 780tcaaggaact ggctggactt
caatgcctac aggctggaga tgaccttcaa ggtgctggat 840tttgtcaccc tcttctccct
cttcgacacc gtcaagtatc ctgtcagcat tgtttcagaa 900acagattctg acagcacctc
gccggtggtg taccagctct caagggtgat ctacacagat 960cctgttggag ccatcagatc
tgatggaaga ggatggtttg atcctcctgt tggaacagat 1020cgcgtcacct tcaccagcat
tgaaaatgag attcctgctc caacaacaag caggcatctt 1080tcagagctca ccatctcttc
tgggccgctg ggcttcggcg tcaatccagc aagaactcac 1140agctggcaag gaaacagaaa
tgtgaacatc tcagctccaa ctgatgtttc tggcgccatc 1200agcaacagaa caagaaccat
cccagcaagg aacatcttca gagtgaacag cagggtgtac 1260accttggact ggaggctcta
tggcgtctac agagcagagt tcttccaagg agctcacagc 1320caggtgttct cagaaaatcc
tccaacagga attggagctc aaagcgccaa caacttcaga 1380tttcttcctg gagaaaattc
tgaaacaccg acgccgcagg actacaccca tgttctctca 1440agggtggtga atgccaccgt
cggcctcacg ccggccaccg gcaaccaaag aaacagcgtg 1500ctgatctttg gatggaccca
caagagcttg acatcagaaa acatctacag gatcaatgag 1560atcaccaagg tggctgctgt
caacacaaga ggaaacagcg gcatcagggt gatctcagga 1620cctggcttca ctggaggaga
tctggtgagg ctggacccta atggaagcgt cagctacaac 1680ttcacgccgg ccaaccagca
agctctgcaa agcaacatca ggatcaggct gagatatgct 1740tgccaaggaa cagcttcctt
gaggatcacc tttggaaatg cttcaagcca ggtgatctcc 1800ttgatctcca ccaccagcag
catcaacaac cttcaatatg aaaacttcca tgtggtgaat 1860gttccaaaca acatcaactt
ccagagcgtc ggcacccaga tcaccatcca gaacatcagc 1920caaaatccaa acatcagctt
ggacagcatt gagctcttct caaacatccc catccctcaa 1980gaacctccct tcaaccctgt
ggtgccagag cctcccatca tctcaggaaa ctaccagatt 2040gtgacgccgc tggacagaag
aagcatcatc gacctcaacc ccaacaacaa tgtcaccctc 2100tggacaaaca acagaagcag
caaccagatc tggaacttca gctatgatca gcagcgcggc 2160gcctacctca tcaggagctt
gagaactgga agcctggtgc tctccatgga ttcaccaaga 2220acaagcaatg tttttggata
tccaagcaat tctgatgctt ctcagttctg gatcttggag 2280ccaaatcaag atggcttcat
cttcagaagt ttgagggaca ggaacctggt gctggatgtt 2340tctggaggaa gagttgatcc
tggaacaagg atcatcgtct ttcccttcac caacagcatc 2400aaccaaagat tcaccttgca
gcgcgtc 2427292130DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
29atggagagca tcatctgcat gttccgcgtc ctctacatca ggaactacga gcacaccggc
60ggcatcaaga tgaagccata tcaaaatgaa aatgaatatg agatcctgga tgctcttcca
120aagtacagca acatcgtcaa tgtttattca agatatcctc tggccaacaa ccctcaagtt
180cctctccaga acaccagcta caaggactgg ctcaacatgt gccaaaccat cacgccgctc
240tgcaccccca tcgacattga ttcaaagctg gtggccaccg ccataggcat cctcggcgcc
300atcttcaagg ccatgccagg acctggaagc gccgtcggcc tcttcttgaa aaccttctcc
360accatcatcc ccatcttatg gccaaatgac aacaccccca tctggaagga gttcaccaag
420caaggcttgc agctcttccg gccggagctg ggaagagatg ccatcgagat cattggaaat
480gatgttcaat caggcttcaa tgctctcaag gaccacatga atgattttga aacaaaattt
540gagatctggg acaaggacag gacacaaaca aatgccacct acctcatcac cgccttcggc
600gtcgtcaatg gcaagatcat tgatctaaag aaccagttcc tcatcaaccc cgccaaccag
660ccggccttcc tcaacctcta tgctcaaaca gcaaacatcg acctcatcct ctaccaaaga
720ggagctgttt atggagacaa ctgggccaag gccatcaatg acagcagcat ctcacccttc
780aacagcagcc agatcttcta tgacagcttg aaggccaaga tcaaggagta caccaactac
840tgtgctgaaa catacaggaa cagcctcacc atcctcaaga accagccaaa catccaatgg
900gacatctaca acagatacag aagagaagca accctcggcg cgctggatct ggtggcgctc
960ttccccaact atgacatctg caagtacccc atctcaacaa aaacagagct gacaaggaag
1020gtgtacatgc cctccttcta cctccaagct ctccagcaca gcaacatcga ggcgctggag
1080aaccagctca cccatcctcc aagcctcttc acctggctca atgagctcaa cctctacacc
1140atcagggaga acttcaaccc ggcgctccag gtgagcagcc tctcaggcct ccaggccaag
1200tacagataca cccagaacag caccatcctg ccaaatcctc ctgctcaagg catcaccaat
1260ggcaccccca tccccatcat cggcctcaac aacctcttca tctacaagct ctccatgagc
1320cagtaccgcc atccaaatga ttgtgttcca attgctggca tctccgacat gaccttctac
1380aagagcgact acaatggaaa tgcttcagca acacaaacat atcaagctgg aaggaacagc
1440aacaatgtca ttgacacctt catgaatgga cctcaaaatg caagcagcag caacaacatc
1500tccatcaacc aaacaaacca catcctctcc gacatcaaga tgaactatgc aagaagtgga
1560ggagtttatg attttggcta cagcttcgcc tggacccaca cctcagttga tccagacaac
1620ctcatcgtcc ccaacaggat cacccagatc cccgccgtca aggccaactg cctctcctcg
1680ccggcgcgcg tcattgctgg acctggccac actggaggtg atctggtggc gctgctgaat
1740ggaggaacac aagctggaag gatgcaaatt caatgcaaaa ctggcagctt caccggcgcc
1800tcaagaagat atggcatcag gatgagatat gctgcaaata atgccttcac cgtcagcttg
1860agctacaccc tccaaggagg aaacaccatc ggcaccacct tcatcacaga aaggaccttc
1920agcaggccaa acaacatcat cccaacagat ctgaaatatg aggagttcaa gtacaaggag
1980tacaaccaga tcatcaccat gacatcacct caaaacacca tcgtcaccat tgccatccag
2040cagctcaacc ccatcccaaa tgatcagctc atcatcgaca ggatcgagtt ctaccctgtt
2100gatcaaggag tggtggcctg ccccgtcaac
2130302130DNAArtificial Sequencesynthetic nucleotide sequence encoding a
toxin protein 30atggagagca tcatctgcat gttccgcgtc ctctacatca
ggaactatga gcacaccggc 60ggcatcaaga tgaagccata tcaaaatgaa aatgaatatg
agatcctgga tgctcttcca 120aaatacagca acatcgtcaa tgtttattca agatatcctc
tggccaacaa tcctcaagtt 180cctctccaga acaccagcta caaggactgg ctcaacatgt
gccaaaccat cacgccgctc 240tgcaccccca tcgacattga ttcaaagctg gtggccaccg
ccataggcat cctcggcgcc 300atcttcaagg ccatgccagg acctggaagc gccgtcggcc
tcttcttgaa aaccttctcc 360accatcatcc ccatcttatg gccaaatgac aacaccccca
tctggaagga gttcaccaag 420caaggcttgc agctcttccg gccggagctg ggaagagatg
ccatcgagat cattggaaat 480gatgttcaat caggcttcaa tgctctcaag gaccacatga
atgattttga aacaaaattt 540gagatctggg acaaggacag aacacaaaca aatgccacct
acctcatcac cgccttcggc 600gtcgtcaatg gcaagatcat tgatctaaag aaccagttcc
tcatcaaccc cgccaaccag 660ccggccttcc tcaacctcta tgctcaaaca gcaaacatcg
acctcatcct ctaccaaaga 720ggagctgttt atggagacaa ctgggccaag gccatcaatg
acagcagcat ctcacccttc 780aacagcagcc agatcttcta tgacagcttg aaggccaaga
tcaaggagta caccaactac 840tgtgctgaaa catacaggaa cagcctcacc atcttgaaga
accagccaaa catccaatgg 900gacatctaca acagatacag aagagaagca accctcggcg
cgctggatct ggtggcgctc 960ttccccaact atgacatctg caagtacccc atctcaacaa
aaacagagct gacaaggaag 1020gtgtacatgc catccttcta cctccaagct ctccagcaca
gcaacatcga ggcgctggag 1080aaccagctca cccatcctcc ttctctcttc acctggctca
atgagctcaa cctctacacc 1140atcagggaga acttcaaccc tgctctccag gtgagcagcc
tctcaggcct ccaagcaaag 1200tacagataca cccagaacag caccatcctt ccaaatcctc
ctgctcaagg catcaccaat 1260ggaaccccca tccccatcat cggcctcaac aacctcttca
tctacaagct ctccatgagc 1320cagtaccgcc atccaaatga ttgtgttcca attgctggca
tctccgacat gaccttctac 1380aagagcgact acaatggaaa tgcttcagca acacaaacat
atcaagctgg aagaaacagc 1440aacaatgtca ttgacacctt catgaatgga cctcaaaatg
caagcagcag caacaacatc 1500tccatcaacc aaacaaacca catcctctcc gacatcaaga
tgaactatgc aagaagtgga 1560ggagtttatg attttggcta cagcttcgcc tggacccaca
cctcagttga tccagacaac 1620ctcatcgtcc ccaacaggat cacccagatc cctgctgtca
aggccaactg cctctcctcg 1680ccggcgcgcg tcattgctgg acctggccac actggaggtg
atctggtggc gctgctgaat 1740ggaggaacac aagctggaag gatgcaaatt caatgcaaaa
ctggcagctt caccggcgcc 1800tcaagaagat atggcatcag gatgagatat gctgcaaata
atgccttcac cgtcagcttg 1860agctacaccc tccaaggagg aaacaccatc ggcaccacct
tcatcacaga aagaaccttc 1920tcaaggccaa acaacatcat cccaacagat ctgaaatatg
aagagttcaa gtacaaggag 1980tacaaccaga tcatcaccat gacatcacct caaaacacca
tcgtcaccat tgccatccag 2040cagctcaacc ccatcccaaa tgatcagctc atcatcgaca
ggattgagtt ctatcctgtt 2100gatcaaggag tggtggcctg ccctgtcaac
2130312061DNAArtificial Sequencesynthetic
nucleotide sequence encoding a toxin protein 31atgaagccat atcaaaatga
aaatgaatat gagatcctgg atgctcttcc aaagtacagc 60aacatcgtca atgtttattc
aagatatcct ctggccaaca acccccaggt gccgctgcaa 120aacaccagct acaaggactg
gctcaacatg tgccaaacca tcacgccgct ctgcaccccc 180atcgacattg acagcaagct
ggtggccacc gccataggca tcctcggcgc catcttcaag 240gccatgccag gacctggatc
agctgttggc ctcttcttga aaaccttctc caccatcatc 300cccatcctct ggccaaatga
caacaccccc atctggaagg agttcaccaa gcaaggcttg 360cagctcttcc ggccggagct
gggaagagat gccatcgaga tcattggaaa tgatgttcaa 420tcaggcttca atgctctcaa
ggaccacatg aatgattttg aaacaaaatt tgagatctgg 480gacaaggaca ggacacaaac
aaatgccacc tacctcatca ccgccttcgg cgtcgtcaat 540ggcaagatca ttgatctaaa
gaaccagttc ctcatcaacc ccgccaacca gccggccttc 600ctcaacctct atgctcaaac
agcaaacatc gacctcatcc tctaccagcg cggcgccgtc 660tatggagaca actgggccaa
ggccatcaat gattcaagca tctctccctt caacagcagc 720cagatcttct atgacagctt
gaaggccaag atcaaggagt acaccaacta ctgtgctgaa 780acatacagga acagcctcac
catcctaaag aaccagccaa acatccaatg ggacatctac 840aacagataca gaagagaagc
aaccctcggc gcgctggatc tggtggcgct cttccccaac 900tatgacatct gcaagtaccc
catctccacc aagacagagc tgacaaggaa ggtgtacatg 960ccctccttct acctccaagc
tctccagcat tcaaacattg aagctctgga gaaccagctc 1020acccatcctc caagcctctt
cacctggctc aatgagctca acctctacac catcagggag 1080aacttcaacc cggcgctcca
ggtgagcagc ctctcaggcc tccaggccaa gtacagatac 1140acccagaaca gcaccatcct
ccccaacccg ccggcgcaag gcatcaccaa tggcaccccc 1200atccccatca tcggcctcaa
caacctcttc atctacaagc tctccatgtc acagtaccgc 1260catccaaatg attgtgttcc
aattgctggc atctccgaca tgaccttcta caagagcgac 1320tacaatggaa atgcttctgc
cacccaaaca tatcaagctg gaaggaacag caacaatgtc 1380attgacacct tcatgaatgg
acctcaaaat gcttcaagca gcaacaacat ctccatcaac 1440caaacaaacc acatcctctc
cgacatcaag atgaactatg caaggagcgg cggcgtctat 1500gattttggct acagcttcgc
ctggacccac acctcagttg atccagacaa cctcattgtt 1560ccaaacagga tcacccagat
ccccgccgtc aaggccaact gcctctcctc gccggcgcgc 1620gtcattgctg gacctggcca
cactggaggt gatctggtgg cgctgctgaa tggaggaaca 1680caagctggaa ggatgcaaat
tcaatgcaaa actggcagct tcactggagc ttcaagaaga 1740tatggcatca ggatgagata
tgctgcaaat aatgccttca ccgtcagctt gagctacacc 1800ctccaaggag gaaacaccat
cggcaccacc ttcatcacag aaaggacctt cagcaggcca 1860aacaacatca tcccaacaga
tctgaaatat gaggagttca agtacaagga gtacaaccag 1920atcatcacca tgacatcacc
tcaaaacacc atcgtcacca ttgccatcca gcagctcaac 1980cccatcccaa atgatcagct
catcatcgac aggatcgagt tctaccctgt tgatcaagga 2040gtggtggcct gccccgtcaa c
2061322061DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
32atgaagccat atcaaaatga aaatgaatat gagatcctgg atgctcttcc aaaatacagc
60aacatcgtca atgtttattc aagatatcct ctggccaaca accctcaggt gccgctgcaa
120aacaccagct acaaggactg gctcaacatg tgccaaacca tcacgccgct ctgcaccccc
180atcgacattg acagcaagct ggtggccacc gccataggca tcctcggcgc catcttcaag
240gccatgccag gacctggatc agctgttggc ctcttcttga aaaccttctc caccatcatc
300cccatcctct ggccaaatga caacaccccc atctggaagg agttcaccaa gcaaggcttg
360cagctcttcc ggccagagct gggaagagat gccatcgaga tcattggaaa tgatgttcaa
420tcaggcttca atgctctcaa ggatcacatg aatgattttg aaacaaaatt tgagatctgg
480gacaaggaca gaacacaaac aaatgccacc tacctcatca ccgccttcgg cgtcgtcaat
540ggaaagatca ttgatctaaa gaaccagttc ctcatcaacc ccgccaacca gccggccttc
600ctcaacctct atgctcaaac agcaaacatc gacctcatcc tctaccagcg cggcgccgtc
660tatggagaca actgggccaa ggccatcaat gattcaagca tctctccctt caacagcagc
720cagatcttct atgacagctt gaaggccaag atcaaggagt acaccaacta ctgtgctgaa
780acatacagga acagcctcac catcttaaag aaccagccaa acatccaatg ggacatctac
840aacagataca gaagagaagc aaccctcggc gcgctggatc tggtggcgct cttccccaac
900tatgacatct gcaagtaccc catctccacc aagacagagc tgacaaggaa ggtgtacatg
960ccatccttct acctccaagc tctccagcat tcaaacattg aagctctgga gaaccagctc
1020acccatcctc cttctctctt cacctggctc aatgagctca acctctacac catcagggag
1080aacttcaacc ctgctctcca ggtgagcagc ctctcaggcc tccaagcaaa gtacagatac
1140acccagaaca gcaccatcct ccccaacccg ccagctcaag gcatcaccaa tggaaccccc
1200atccccatca tcggcctcaa caacctcttc atctacaagc tctccatgtc acagtaccgc
1260catccaaatg attgtgttcc aattgctggc atctccgaca tgaccttcta caagagcgac
1320tacaatggaa atgcttctgc aacacaaaca tatcaagctg gaagaaacag caacaatgtc
1380attgacacct tcatgaatgg acctcaaaat gcttcaagca gcaacaacat ctccatcaac
1440caaacaaacc acatcctctc cgacatcaag atgaactatg caagaagcgg cggcgtctat
1500gattttggct acagcttcgc ctggacccac acctcagttg atccagacaa cctcattgtt
1560ccaaacagga tcacccagat ccctgctgtc aaggccaact gcctctcctc gccggcgcgc
1620gtcattgctg gacctggcca cactggaggt gatctggtgg cgctgctgaa tggaggaaca
1680caagctggaa ggatgcaaat tcaatgcaaa actggcagct tcactggagc ttcaagaaga
1740tatggcatca ggatgagata tgctgcaaat aatgccttca ccgtcagctt gagctacacc
1800ctccaaggag gaaacaccat cggcaccacc ttcatcacag aaagaacctt ctcaaggcca
1860aacaacatca tcccaacaga tctgaaatat gaagagttca agtacaagga gtacaaccag
1920atcatcacca tgacatcacc tcaaaacacc atcgtcacca ttgccatcca gcagctcaac
1980cccatcccaa atgatcagct catcatcgac aggattgagt tctatcctgt tgatcaagga
2040gtggtggcct gccctgtcaa c
2061332061DNAArtificial Sequencesynthetic nucleotide sequence encoding a
toxin protein 33atgaacagct accagaacaa gaatgaatat gagatcctgg
atgcttctca aaacaacagc 60accatgagca acagatatcc aagatatcct ctggcaaatg
atcctcaagc atcaatgcaa 120aacaccaact acaaggactg gctcaacatg tgtgacagca
acacccagtt tgttggagac 180atctccacct acagctctcc agaagctgct ctctccgtga
gagatgctgt gctcaccggc 240atcaacaccg ccggcaccat cctctcaaac ctcggcgtgc
catttgcttc tcagagcttt 300gggatgattg gtaggatcat cggcatcctc tggccagggc
cagatccatt tgctgctctg 360atggtgctgg tggaggagct catcaaccag aggatcaatg
atgagatcag gaaccatgct 420ctactggagc tcgccggcct caagggcatc atggacctct
acaggacaag atggagagca 480tgggatctga acaaggacaa cccagaaaca agagaagctg
ttcgagctca gtacagaact 540gctgacaact tcttcatcca gaacatgcca aaatttggaa
gagaagatca tggcgtgctg 600ctgctacctg tttatgctca agctgccaac atgcacctca
tcctcctccg tgatgcatat 660gtttttggaa ctggatgggg ccttggacct ggagaggtga
gggacaacta cacaaggctg 720caagagaaga tcagggagta caaggaccac tgcgtcacct
tctacaacca aggcctcaac 780aggttcaaca gatcaaatgc tcaagattgg gtgagcttca
acaggttcag aactgacatg 840accctcaccg tgctggacct cgccatcctc ttccccaact
atgatccaag gatctacccc 900tccgccgtca agacagagct gacaagggag atctacacag
atcctgttgg cttcaccggc 960gtgctgggct caggaggaag aacatatcca tggtacaacc
caaatgacac cagcttcgcc 1020accatggaga actctgcaag aagaaggcct tccttcacca
cctggctgaa caggatcagg 1080atcttcactg gtcacatcgg caacttctct gctgctggaa
atgtttgggg aggccatgag 1140ctctttgaaa gaagcaacaa tggatcagag atcatccaaa
gatttggcaa caccaacacc 1200agctacacgc cggtgaggaa ctgggacttc accaaccaga
acaggacggt gttctccatt 1260gcttcaacag caagggtgct gctggctgga tctgaaggaa
atgctcaccg gccaagccaa 1320tatggagttt caagggtgga catgcacacc gccattggag
gaaacaccag cggcggccag 1380ttcatctatg aagttccaaa tgttcacagc agccagaaca
tcttgagcga gctgccagga 1440gaaaaccagc agcggccaga tgcaaggaac cacagccaca
tcctcagcta catctcaaat 1500tttgatgcaa aaagaggagg aactgttgga aatgtcaggc
tgctgacata tggatggacc 1560cacacctcca tggacaggaa caacaggctg gagagggaca
ggatcactca aattgatgct 1620gtcaagggct ggggcggcgt caccggcagc gtcatccctg
ggccaactgg aggtagcttg 1680gtcaccatcc cttcaaatcc atggagcgtg agcctccgcg
tccaagctcc tcagatccaa 1740acaaactaca ggatcaggct gcgcttcgcc tgcgtctggc
ctggagctca ccacatgtgg 1800gtgacatatg gaggcatcag ccatcctgtg cagctctgca
acaacccttc ttctggaagg 1860ccaagcaaca acctgctgga aagtgatttt ggatatgtgg
tggtgcctgg caccttctca 1920ccaagcatca acccagagat aaggttctcc gccatcagca
atgctcctgt gctggacaag 1980atcgagttca tcccgctgga catctacaat gagcacttcg
tggaggagcg cgccaagacc 2040atcaatgatc tcttcatcaa c
2061342061DNAArtificial Sequencesynthetic
nucleotide sequence encoding a toxin protein 34atgaacagct accaaaacaa
gaatgaatat gagatcctgg atgcttctca aaacaacagc 60accatgagca acagatatcc
aagatatcct cttgcaaatg atcctcaagc atcaatgcaa 120aacaccaact acaaggattg
gctcaacatg tgtgacagca acacccaatt tgttggagac 180atctccacct acagctctcc
agaagctgct ctctctgtga gagatgctgt tctcaccggc 240atcaacaccg ccggcaccat
cctctcaaac ctcggcgtgc catttgcttc tcagagcttt 300gggatgattg gtaggatcat
tggcatcctc tggcctggac cagatccatt tgctgctctg 360atggtgctgg tggaggagct
catcaaccaa aggatcaatg atgagatcag gaaccatgct 420ctactggagc tggctggcct
caagggcatc atggatctct acagaacaag atggagagca 480tgggatctga acaaggacaa
tccagaaaca agagaagctg ttcgagctca gtacagaact 540gctgacaact tcttcatcca
gaacatgcca aaatttggaa gagaagatca tggagtgctg 600ctgctacctg tttatgctca
agctgcaaac atgcacctca tcctcctccg tgatgcatat 660gtttttggaa ctggatgggg
ccttggacct ggagaagtga gggacaacta cacaaggctg 720caagagaaga tcagggagta
caaggatcac tgcgtcacct tctacaacca aggcctcaac 780aggttcaaca gatcaaatgc
tcaagattgg gtgagcttca acaggttcag aactgacatg 840accctcaccg tgctggatct
ggccatcctc ttccccaact atgatccaag gatctacccc 900tccgccgtca agacagagct
gacaagggag atctacacag atcctgttgg cttcaccggc 960gtgctgggat caggaggaag
aacatatcca tggtacaacc caaatgacac cagcttcgcc 1020accatggaga actctgcaag
aagaaggcct tccttcacca cctggctgaa caggatcagg 1080atcttcactg gtcacattgg
aaacttctct gctgctggaa atgtttgggg aggacatgag 1140ctctttgaaa gaagcaacaa
tggatcagag atcatccaaa gatttggaaa caccaacacc 1200agctacacgc cggtgaggaa
ctgggacttc accaaccaaa acaggacggt gttctccatt 1260gcttcaacag caagggtgct
gctggctgga tctgaaggaa atgctcatcg gccttctcaa 1320tatggagttt caagggtgga
catgcacacc gccattggag gaaacaccag cggcggccag 1380ttcatctatg aagttccaaa
tgttcacagc agccaaaaca tcttgagcga gcttcctgga 1440gaaaaccagc agaggccaga
tgcaaggaac cacagccaca tcctcagcta catctcaaat 1500tttgatgcaa aaagaggagg
aactgttgga aatgtcaggc tgctgacata tggatggacc 1560cacacctcca tggacaggaa
caacaggctg gagagggaca ggatcactca aattgatgct 1620gtcaaaggat ggggcggcgt
caccggcagc gtcatccctg gaccaactgg aggtagcttg 1680gtcaccatcc cttcaaatcc
atggagcgtg agcctccgcg tccaagctcc tcagatccaa 1740acaaactaca ggatcaggct
gaggttcgcc tgcgtctggc ctggagctca tcacatgtgg 1800gtgacatatg gaggcatcag
ccatcctgtt cagctctgca acaatccttc ttctggaagg 1860ccaagcaaca acctgctgga
aagtgatttt ggatatgtgg tggttcctgg aaccttctca 1920ccaagcatca atccagaaat
aaggttctcc gccatcagca atgctcctgt gctggacaag 1980attgagttca tccctctgga
catctacaat gagcattttg tggaggagcg cgccaagacc 2040atcaatgatc tcttcatcaa c
2061351872DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
35atgaacagct accagaacaa gaatgaatat gagatcctgg acacctcccc caacaacagc
60accatgagca ccctccatcc aagatatcct ctagcaaaag atccatacaa gccaatgagg
120aacaccaact acaaggaatg gctggccatg tgcgccaaca acaaccaagt tccaattgat
180cctctggaca acacctgggc cggcgtcatg gcggcgctct tcgcctccgc cgccgccatt
240gctgggctga tgtctgctgt tcctgttttc tcagtggtgg ccaccggcac ggcgctcgcc
300gccgcgctca cccccatcct cttcccaagc aatggaccag atgtttcaac tcagctgatg
360agcaacactg aagctctgct gaagagggag ctggacacat atgtccgcgc gcgcgccgac
420agcgagttcc aagctctgga agctcaaagg gagttcttca agagcgcctt cgactactgg
480aagctctacc ccaccaactc aaatgccatt gccaccgtcg ccgcgcgctt ccacaccgtc
540aatggcgcct tcgtcaccgc catgaggctc ttcagaactg ctggatatga agctctgctg
600ctgccagttt atgctcaagc tgcaaggctg cacctcctcc acctccgcga cggcgtcctc
660ttcgccaatg aatggggcct agcaaaagat cctggagatc ttcatgatca agagttcaac
720aaatatgctg ctgaatatgc tgattattgt gaaagcacct acaacacaga gctcaacagg
780atcaagacgg cgccaggaaa aacatggctg gactacaacc agtacaggag gatcatgacc
840attgctgtgc tggacattgc tgccaagttc tcaatcctca acccgcgcct ctacaggctg
900ccgctgcaag aagagatctt gacaaggaag atctacacag atcctgtcaa cttctcacca
960gggccaagca ttgctgatga tgagaacaga tacaccgtgc cgctctcctt ggtgacccag
1020ctggtgaaca gcaggctctt caccaatgtt gcttctgctc aaaatgctgg cttcatcggc
1080aaccagaaca gatacaagaa catcggcgtg ggagatcctg ttgatggccc catcatcggc
1140caatcagttt atgagaaggt ggatgctggc atcccaacaa atgaatgggt gtatgaagtt
1200ggcgtcaatg gcatccaaaa tgattatcca aggaacatcg gcctgaggaa aggatcaacc
1260accgccttca cagatcatct tgctggaagc cagtacaacc tggggccgct gacaagggtg
1320agcatcccca ccaaggacaa tgctcccatc aacaacacca acttcaccca ccgcctctcc
1380gacatcatcc ttcctggcaa caaaggatca tccttcgcct ggacccatgt tgatgttgat
1440ccaacaggaa actacctctc caccaccaag atcaacctca tccccgccac caaggccagc
1500aagattcctc tctccttcta cttgaggaaa ggacctggct tcattggagg agatctggtg
1560aggctgggca gcggctttga atgcagctac aagttcaact tcaagagccc tggaagctca
1620gcaaacttca gaatcaggat cagatatgct ggagctggaa gcggccaagg agctgatggc
1680caggtgtact tcaagctagg aaactacacc tcgccgacga cgccatgggg ccacactgga
1740tttgattatg gaaatgtcaa gtacaaccag ttccgcgtgc tggagctctt tggaacagca
1800gagaacatca ccgacaatga tctgaagatc atcgtctgga caggaagctc tgctcaagat
1860ttcctctcca gg
1872361872DNAArtificial Sequencesynthetic nucleotide sequence encoding a
toxin protein 36atgaacagct accaaaacaa aaatgaatat gagatcttgg
acacctctcc aaacaacagc 60accatgagca ccctccatcc aagatatcct ctagcaaaag
atccatacaa gccaatgagg 120aacaccaact acaaggaatg gctggccatg tgcgccaaca
acaaccaagt tccaattgat 180cctctggaca acacctgggc cggcgtcatg gcggcgctct
tcgcctccgc cgccgccatt 240gctggcttga tgtctgctgt tcctgttttt tcagtggtgg
ccaccggcac ggcgctcgcc 300gccgcgctca cccccatcct cttcccttca aatggaccag
atgtttcaac tcagctgatg 360agcaacactg aagctctgct gaagagggag ctggacacat
atgtccgcgc gcgcgccgac 420agcgagttcc aagctctgga agctcaaaga gagttcttca
agagcgcctt cgactactgg 480aagctctacc caacaaattc aaatgccatt gccaccgtcg
ccgcgcgctt ccacaccgtc 540aatggagcct tcgtcaccgc catgaggctc ttcagaactg
ctggatatga agctctgctg 600cttcctgttt atgctcaagc tgcaaggctg catctcctcc
acctccgaga tggcgtcctc 660tttgcaaatg aatggggcct agcaaaagat cctggagatc
ttcatgatca agagttcaac 720aaatatgctg ctgaatatgc tgattattgt gaaagcacct
acaacacaga gctcaacagg 780atcaagacag ctccaggaaa aacatggctg gactacaacc
agtacagaag gatcatgaca 840attgctgtgc tggacattgc tgcaaagttc tcaatcctca
acccaaggct ctacaggctg 900ccgctgcaag aagaaatctt gacaaggaag atctacacag
atcctgtcaa cttctcacca 960ggaccaagca ttgctgatga tgaaaacaga tacaccgtgc
ctctctcctt ggtgacacag 1020ctggtgaaca gcaggctctt caccaatgtt gcttctgctc
aaaatgctgg cttcattgga 1080aaccaaaaca gatacaagaa catcggcgtt ggagatcctg
ttgatggccc catcattgga 1140caatcagttt atgagaaggt ggatgctggc atcccaacaa
atgaatgggt gtatgaagtt 1200ggtgtcaatg gcatccaaaa tgattatcca agaaacattg
gcttgaggaa aggatcaaca 1260acagccttca cagatcatct tgctggaagc cagtacaacc
ttgggccgct gacaagggtg 1320agcatcccca ccaaggacaa tgctcccatc aacaacacca
acttcaccca ccgcctctct 1380gacatcatcc ttcctggaaa caaaggatca tccttcgcct
ggacacatgt tgatgttgat 1440ccaacaggaa actacctctc caccaccaag atcaacctca
tccctgccac caaggcaagc 1500aagattcctc tctccttcta cttgagaaaa ggacctggct
tcattggagg agatctggtg 1560aggctgggct ctggctttga atgcagctac aagttcaact
tcaagagccc tggaagctca 1620gcaaacttca gaatcaggat cagatatgct ggagctggaa
gcggccaagg agctgatggc 1680caagtttact tcaagctagg aaactacacc tcgccgacga
cgccatgggg ccacactgga 1740tttgattatg gaaatgtcaa gtacaaccag ttcagagtgc
tggagctctt tggaacagca 1800gaaaacatca ccgacaatga tctgaagatc atcgtctgga
caggaagctc tgctcaagat 1860ttcctctcaa ga
187237888DNAArtificial Sequencesynthetic nucleotide
sequence encoding a toxin protein 37atgaagaagc tgatgttctc cttggtggcc
accaccatga gcatgggcct catcctcggc 60agcgcgccgg tgaaggctga tgtcagcaac
aagaacagcg cctaccagga cattgatgaa 120agggtgaaga agatggctca atcagcagca
tggggaggac aagaatacag gaaccacaac 180atcaaggaca tcgagctgaa gggcaacctc
attgatggaa gcatgattga gaacagcgag 240gtgctcaccg tcagcagcga catcctggag
aacaagctgg gtcacaccgt caacatgcca 300tcaacaggat atgaacatga atttgaagaa
acaacaaaca ccaccaacac cagcggctgg 360acatttggct acaactacaa tgcttccttc
tccgtgctga tggtttcagc ttctcagagc 420ttctccgtgg agtacaacat gagcaccagc
aacacccatg agaagaagga gaagaggaag 480ttcactgttc cttccatcga ggttcctgtt
cctgctggca agaagtacaa ggtggaatat 540gtttttgaga aggtgaaggt gtcagggaag
aacaagattg atgccaacct ctatggagat 600gtcacctact actacaacaa ccagccaatg
tcgccgcagc tgctgtacag cgtccaaggc 660ctcgccgccg acaagcaagg cttcgagcag
gtcatcagag attctgctgt tggaaatgac 720agatttggca tcaagacaac tggcattggt
cagttctcaa cagaatttgg aacaaggctg 780acaaggacct tgacagacat caccgacaca
aggaaccccg tcaaggtgga aacaaaaaat 840gttcctgtgg agttcaagac cctctccatc
gacacaaggg tgatcaag 88838888DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
38atgaagaagc tgatgttctc cttggtggcc accaccatga gcatgggcct catcctcggc
60agcgcgccgg tgaaggctga tgtcagcaac aagaacagcg cctaccagga cattgatgaa
120agggtgaaga agatggctca atcagcagca tggggaggac aagaatacag gaaccacaac
180atcaaggaca tcgagctgaa gggcaacctc attgatggaa gcatgattga gaacagcgag
240gtgctcaccg tcagcagcga catcctggag aacaagctgg gtcacaccgt caacatgcca
300tcaacaggat atgaacatga atttgaagaa acaacaaaca ccaccaacac cagcggctgg
360acatttggct acaactacaa tgcttccttc tccgtgctga tggtttcagc ttctcagagc
420ttctccgtgg agtacaacat gagcaccagc aacacccatg agaagaagga gaagaggaag
480ttcactgttc cttccattga agttcctgtt cctgctggca agaagtacaa ggtggaatat
540gtttttgaga aggtgaaggt gtcagggaag aacaagattg atgccaacct ctatggagat
600gtcacctact actacaacaa ccagccaatg tcgccgcagc tgctgtacag cgtccaaggc
660ctcgccgccg acaagcaagg cttcgagcag gtcatcagag attctgctgt tggaaatgac
720agatttggca tcaagacaac tggcattggt cagttctcaa cagaatttgg aacaaggctg
780acaaggacct tgacagacat caccgacaca aggaaccccg tcaaggtgga aacaaaaaat
840gttcctgtgg agttcaagac cctctccatc gacacaaggg tgatcaag
88839924DNAArtificial Sequencesynthetic nucleotide sequence encoding a
toxin protein 39atgaagaaga acaggatgct gctgaaatgg atgtgcggcc
tcaccatcgg catcggcagc 60ttgacaggag gaagcctcaa tgcttttgct gatgaagttt
cagattctct tgctgatgtt 120ggcttcctct atggagacta cctctacaag accaagcagc
atcctcaagg aaccctcccc 180atcacctacc ccatgaggga gatcaacaac taccagatca
tcgacaagag cgtcagccaa 240gttggatcaa cagaatatga agaaggacaa accctctatg
ttgatgatga tgtttttgac 300aacaagacag gaacagatca aaccttcaag accatccagt
ttgagaagga gttctcagaa 360acagcaacaa gcagcaccac ccattctgtt ggcaccagct
tggaggagag cgtgaagttc 420gacttctttg ttggagaagg aagcgccaag ttcaccgtca
actacaactt ctccaagaca 480ggaagcctct ccaccagcaa caagatcaag tacacccttc
cttcacaaag catcaatgtt 540cctgccaaca agaaatatga ggtgatctgc gtgctggaaa
caaagaaggc caaggcaaat 600gttcagttca atgttgatgt tctaggaaat gcaaaatatg
tctacagcaa caacagcccc 660tacacgccaa aatatgagag cggcgccacc atgctcaaga
ccctcaatga gaagaaccca 720actccttctg tcagctggct gggcaaggaa tgggagaaat
gggagtacca tgatggcaag 780gcgcgctaca agaatggatc aggaacagtt tctgctgaat
atggaacaag gatggtgctg 840gtgatcaatg acatcaccaa caacaagaca agaggaagca
aggagattgc aaggattcct 900gtcaccccca tccagaagca gatg
92440924DNAArtificial Sequencesynthetic nucleotide
sequence encoding a toxin protein 40atgaagaaga acaggatgct gctgaaatgg
atgtgcggcc tcaccatcgg catcggcagc 60ttgacaggag gaagcctcaa tgcttttgct
gatgaagttt cagattctct tgctgatgtt 120ggcttcctct atggagacta cctctacaag
accaagcagc atcctcaagg aaccctcccc 180atcacctacc ccatgaggga gatcaacaac
taccagatca tcgacaagag cgtcagccaa 240gttggatcaa cagaatatga agaaggacaa
accctctatg ttgatgatga tgtttttgac 300aacaagacag gaacagatca aaccttcaag
accatccagt ttgagaagga gttctcagaa 360acagcaacaa gcagcaccac ccattctgtt
ggcaccagct tggaggagag cgtgaagttc 420gacttctttg ttggagaagg aagcgccaag
ttcaccgtca actacaactt ctccaagaca 480ggaagcctct ccaccagcaa caagatcaag
tacacccttc cttcacaaag catcaatgtt 540cctgccaaca agaaatatga ggtgatctgc
gtgctggaaa caaagaaggc caaggcaaat 600gttcagttca atgttgatgt tctaggaaat
gcaaaatatg tctacagcaa caacagcccc 660tacacgccaa aatatgagag cggcgccacc
atgctcaaga ccctcaatga gaagaaccca 720actccttctg tcagctggct gggcaaggaa
tgggagaaat gggagtacca tgatggcaag 780gcgcgctaca agaatggatc aggaacagtt
tctgctgaat atggaacaag gatggtgctg 840gtgatcaatg acatcaccaa caacaagaca
agaggaagca aggagattgc aaggattcct 900gtcaccccca tccagaagca gatg
924411995DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
41atgcaaatca tcgacagcag cagcaatgat ttctcccaaa gcaacagata tccaagatat
60cctctggcca aggagagcaa ctacaaggac tggctggcaa gctgtgatga aagcaatgtg
120gacaccctct ccgccaccag caatgtccgc gcctctgttt caagagctct tggcattgtg
180aatcagatcc tcggcttcct cggcctcggc ttcatcggca ccggcctcgg cgtcctctca
240gatctcttca acagcttctg gccaagcaat gacaacagca tctgggagag cttcttgagg
300agcgtggagg agctcatcga ccgccgcgtc agggaggtgg agaggttcag aattgaaagc
360cagttcaccg gcctcagaaa tgtgatgtca aactacaatg gagctcttcg agattggaat
420ggcaacagga acaacctggc gctgcaaagc gaggtgagga gcagatttga caactctgat
480gatgcttttg cttcaaggat gccggagttc agaattgaag gatttgagat ccagagcctc
540gccgtctatg ctcaagctgc caccctccac ctgctgctgc tgagagatgg agtggtgaat
600ggcctccaat ggggcttcga catcgtcacc gtcaacaggc tctatgagaa gctggtctgc
660ctctccggcg cctatgctga tcactgcacc ctcttctaca ggcaagggct ggaggagcta
720aggaacagag gaaactggaa tgccttcaac aactacagga gggacatgac cctccaggtg
780ctggatgtca tctccctctt ccccaactat gatccaaggc tctatgacat caacaccaac
840acccagctga caagggagat ctacacagag ccgctggcca tccctggatg gctcaacagc
900cattcaaatc caacccagtt ccagcagatt gaaaatgatc tcatcagatc tccttctgtg
960ttcagcaacc tggaaactct cttcatggaa gctggcttcg ccttctttca agctggcatt
1020gctcgccaag ctgtgctgag gacaaggaca agcagcctca acatgaacag gaccgccgtc
1080atcgtcacgc catggcaagg agctcctcat ccaaatgttt cacatgagct ccaggtgacc
1140ctccaggaca gaaatgtttt caacatcaac agcgtggtgg gaagggagat ctcctctcaa
1200actggcctcc tcttcggcgt ccagcaggcc accttccact tcgtctgggc tggaggaaat
1260gctgccacca ccacccagtt caaccttcct cccatctcag gccatagcac cagcataaca
1320agcaacatcc ctggcaccaa cagcaccacg ccaacaggaa gcgactacac ccaccgcctc
1380tcctccatca cctccacctc cgtcggcacc tggcagaggg acaggaccaa cataatggca
1440tatggatgga cccatgtttc agctgaaaga acaaacagga tcatccccaa caggatcacc
1500cagatccccg ccgtcaaagg aagcctcttc tccgacaacc ctccaaacac ctcaaggaca
1560agggtggaga atggacctgg tcacactgga ggagggctgg tggtgatgga tggaggaaca
1620tcagtgctcc agatgagggt cacctcctcc gcgcgccaaa gatatgacat gaggctgcgc
1680tatgtggcgc tggcgccggc gacggtggag gtgaggattc cagagctcgg cggccatgtc
1740cgcttccaga tgccaatgac agcaacaggg ctgccggcgc cgctaccata cagccacctc
1800agatatgtgg acatcccgct gagatttgaa actcctcatg gagaaaacac ctggaccttc
1860gagctgcaaa caaccttcgc cgccgtcgcc atcgacaggg tggagttcat ccccgtcaat
1920gcaacagctc tggaatatga aggaaagagg cacctggaga aggccaagaa ggctgttgga
1980gatctcttca tcaac
1995421995DNAArtificial Sequencesynthetic nucleotide sequence encoding a
toxin protein 42atgcaaatca tcgacagcag cagcaatgat ttctctcaaa
gcaacagata tccaagatat 60cctctggcca aggaaagcaa ctacaaggat tggctggcaa
gctgtgatga aagcaatgtt 120gacaccctct ccgccaccag caatgtccgc gcctctgttt
caagagctct tggaattgtg 180aatcagatcc ttggcttcct tggccttggc ttcattggca
ctggcctcgg cgtcctctca 240gatctcttca acagcttctg gccatcaaat gacaacagca
tctgggagag cttcttgagg 300agcgtggagg agctcatcga ccgccgcgtc agagaggtgg
aaaggttcag aattgaaagc 360cagttcactg gcttgagaaa tgtgatgtca aactacaatg
gagctcttcg agattggaat 420ggaaacagaa acaacttggc gctgcaatca gaggtgagaa
gcagatttga caattctgat 480gatgcttttg cttcaaggat gccagagttc agaattgaag
gatttgagat ccaaagcctc 540gccgtctatg ctcaagctgc aaccctccat ctgctgctgc
tgagagatgg agtggtgaat 600ggccttcaat ggggcttcga cattgtcacc gtcaacaggc
tctatgagaa gctggtttgc 660ttgagcggcg cctatgctga tcattgcacc ctcttctaca
ggcaagggct ggaggagcta 720agaaacagag gaaactggaa tgccttcaac aactacagaa
gagacatgac cctccaggtg 780ctggatgtca tctccctctt cccaaactat gatccaaggc
tctatgacat caacaccaac 840acccagctga caagagagat ctacacagag ccgctggcca
tccctggatg gctcaacagc 900cattcaaatc caacacagtt ccagcagatt gaaaatgatc
tcatcagatc tccttctgtt 960ttcagcaacc tggaaacatt gttcatggaa gctggcttcg
ccttctttca agctggaatt 1020gcaaggcaag ctgtgctgag aacaagaaca agcagcttga
acatgaacag gacagctgtc 1080atcgtcactc catggcaagg agctcctcat ccaaatgttt
cacatgagct gcaagtgaca 1140ttgcaagaca gaaatgtttt caacatcaac agcgtggttg
gaagagagat ctcttctcaa 1200actggcctcc tcttcggcgt ccagcaagca accttccact
tcgtctgggc tggaggaaat 1260gctgccacca ccacccagtt caaccttcct cccatctcag
gacatagcac cagcataaca 1320agcaacattc ctggaacaaa cagcacaaca ccaacaggaa
gcgactacac ccaccgcctc 1380tcctccatca cctccacctc tgttggcaca tggcaaagag
acagaacaaa cataatggca 1440tatggatgga cacatgtttc agctgaaaga acaaacagga
tcatccccaa caggatcacc 1500cagatccctg ctgtcaaagg aagcctcttc tctgacaatc
ctccaaacac atcaagaaca 1560agggtggaaa atggacctgg tcacactgga ggagggctgg
tggtgatgga tggaggaaca 1620tcagttcttc agatgagggt cacctcctcc gcgcgccaaa
gatatgacat gaggctgagg 1680tatgttgctc tggcgccggc gacggtggag gtgaggattc
cagagcttgg aggccatgtt 1740aggttccaga tgccaatgac agcaacaggg ctgccggcgc
ctctaccata cagccatctg 1800agatatgttg acattccttt gagatttgaa actcctcatg
gagaaaacac atggacattt 1860gagctgcaaa caaccttcgc cgccgtcgcc atcgacaggg
tggagttcat ccctgtcaat 1920gcaacagctc tggaatatga aggaaaaagg catctggaga
aggccaagaa ggctgttgga 1980gatctcttca tcaac
1995431563DNAArtificial Sequencesynthetic
nucleotide sequence encoding a toxin protein 43atgacaaaga gcgagttcat
ccagaacagc tgccggaaga tgcaaagcaa ggtgcgcgtc 60atcatcctct ccaccaatga
tcctgtggtg aacaacaaca ccttggacat cacagagatc 120aaggatcttg ctcatctctc
ccaggccatc atgctggcca acaacttcca agctgctctg 180gtgccaacaa gctcagaatt
tggccaagat gtgctgagat ttgatgtcaa ccaaggcatc 240agcattgcca acaacatcta
ccccaaggcg gtggacatca actacatcag caggacgctc 300tcccaaagca acaaccaggt
gaacagcatg atcaacatgg tggtgaatga gctgaagctg 360ctgctgggca tcaaccttgc
tgattcagtg ctccagcagc tcaccagctt ggtggcctac 420accttcacca acctctacac
ccagcagaac agcgcctggg tgttctgggg gaagcaagca 480tcaaatcaaa caaactacac
ctacaacatc gtctttgcca tccaaaatgc tcaaactgga 540aacttcatga aggccatccc
catgggcttt gagatctctg catatgctgt caaggagcag 600gtgctcttct tcaccatcca
agattatgct tcatattctg tgaagatcca ggccatcaat 660gtcacccagc cgctcatcaa
cagcagctat ggaagcctct ccggcgtcta caacatcatc 720accgcgctca acaacatctc
cgtcatcacc atgagcaact ctgatgaaaa tgtcaaccta 780tggtatgaca atgatgatct
caaccaaaaa tggatcttgg agttcaacca caaccactac 840gcctacatca tcaggaacct
ctccaacagg agcttggtgc tgacatggga cagcaccagc 900ggcagcaaca atgtttttgc
caccaactac caaggaaatg atgagcagtt ctggatcatc 960caggacaccg acaatgacta
cttctacctc tcaaacatga gggacaccca atatgtgctg 1020gagattgctg gaagcgtgtt
ctacaatgga actaatgtca tcgtcaacaa gaaaacaagc 1080agcctcaacc agaagttctc
catcaacagg atcaacaggc agatccaaaa tggcatctac 1140aacatcacca cctacctgaa
tgcttcttct gtcatcacca tgtcaacaga ctacaacatc 1200aatgttcatg attatcctgt
caacctctgg ttcaagaatg acagcatcaa ccaaaaatgg 1260atcttcgagt ttgattcaga
caagagcgcc tacagggtga ggagcgtcag caacccctcc 1320ctcttcctca gctggccggt
ggcctccttc accaacagag ctgctgtcac cccaaatcca 1380agggacaatg aatacttctg
gttcctccag agcgccggcc tcggcacctt ctacctggtg 1440tcaatgaggg acacaagata
tgtgctggag gtggagaaca gcaacatcga caatggcacc 1500aacatcatcg tcaaccaaag
aactggaaac ttcaaccaga ggttctacat cgagaacatc 1560aac
1563441563DNAArtificial
Sequencesynthetic nucleotide sequence encoding a toxin protein
44atgacaaaga gcgagttcat ccagaacagc tgccggaaga tgcaaagcaa ggtgcgcgtc
60atcatcctct ccaccaatga tcctgtggtg aacaacaaca ccttggacat cacagagatc
120aaggatcttg ctcatctctc ccaggccatc atgctggcca acaacttcca agctgctctg
180gtgccaacaa gctcagaatt tggccaagat gtgctgagat ttgatgtcaa ccaaggcatc
240agcattgcca acaacatcta ccccaaggcg gtggacatca actacatcag caggacgctc
300tcccaaagca acaaccaggt gaacagcatg atcaacatgg tggtgaatga gctgaagctg
360ctgctgggca tcaaccttgc tgattcagtg ctccagcagc tcaccagctt ggtggcctac
420accttcacca acctctacac ccagcagaac agcgcctggg tgttctgggg gaagcaagca
480tcaaatcaaa caaactacac ctacaacatc gtctttgcca tccaaaatgc tcaaactgga
540aacttcatga aggccatccc catgggcttt gagatctctg catatgctgt caaggagcag
600gtgctcttct tcaccatcca agattatgct tcatattctg tgaagatcca ggccatcaat
660gtcacccagc cgctcatcaa cagcagctat ggaagcctct ccggcgtcta caacatcatc
720accgcgctca acaacatctc cgtcatcacc atgagcaact ctgatgaaaa tgtcaaccta
780tggtatgaca atgatgatct caaccaaaaa tggatcttgg agttcaacca caaccactac
840gcctacatca tcaggaacct ctccaacagg agcttggtgc tgacatggga cagcaccagc
900ggcagcaaca atgtttttgc caccaactac caaggaaatg atgagcagtt ctggatcatc
960caggacaccg acaatgacta cttctacctc tcaaacatga gggacaccca atatgtgctg
1020gagattgctg gaagcgtgtt ctacaatgga actaatgtca tcgtcaacaa gaaaacaagc
1080agcctcaacc agaagttctc catcaacagg atcaacaggc agatccaaaa tggcatctac
1140aacatcacca cctacctgaa tgcttcttct gtcatcacca tgtcaacaga ctacaacatc
1200aatgttcatg attatcctgt caacctctgg ttcaagaatg acagcatcaa ccaaaaatgg
1260atcttcgagt ttgattcaga caagagcgcc tacagggtga ggagcgtcag caacccctcc
1320ctcttcctca gctggccggt ggcctccttc accaacagag ctgctgtcac cccaaatcca
1380agggacaatg aatacttctg gttcctccag agcgccggcc tcggcacctt ctacctggtg
1440tcaatgaggg acacaagata tgtgctggag gtggagaaca gcaacatcga caatggcacc
1500aacatcatcg tcaaccaaag aactggaaac ttcaaccaga ggttctacat cgagaacatc
1560aac
1563454PRTArtificial Sequenceendoplasmic reticulum targeting peptide
45Lys Asp Glu Leu1
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