AU2771902A - Plant-optimised genes encoding pesticidal toxins - Google Patents

Description

S&FRef: 507084D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Mycogen Corporation 5501 Oberlin Drive San Diego California 92121 United States of America Guy A. Cardineau, Steven J. Stelman, Kenneth E. Narva Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Plant-Optimised Genes Encoding Pesticidal Toxins The following statement is a full description of this invention, including the best method of performing it known to me/us:so X41 5845c Plant Optimised Genes Encoding Pesticidal Toxins Background of the Invention Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield. reduced crop duality, and increased harvesting costs.

Chemical pesticides have provided an effective method of pest control: however, the public has become concerned about the amount of residual chemicals which might be found in food, ground water, and the environment. Therefore, synthetic chemical pesticides are being Increasingly scrutinised, and correctly so, for their potential toxic environmental consequences.

Synthetic chemical pesticides can poison the soil and underlying aquifers, pollute surface waters as a result of runoff, and destroy non-target life forms. Synthetic chemical control agents have the further disadvantage of presenting public safety hazards when they are applied in areas where pets, farm animals, or children may come into contact with them. They may also provide health S 15 hazards to applicants, especially if the proper application techniques are not followed.

',Regulatory agencies around the world are restricting and/or banning the uses of many pesticides and particularly the synthetic chemical pesticides which are persistent in the environment and enter the food chain. Examples of widely used synthetic chemical pesticides include the S"organochlorines, eg., DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb. and dieldrin; the 20 organophosphates, eg., chlorpyrifos, parathion, malathion, and diazinon; and carbamates.

Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides from the market place could limit economical and effective options for controlling costly pests.

Because of the problems associated with the use of synthetic chemical pesticides, there exists 25 a clear need to limit the use of these agents and a need to identify alternative control agents. The replacement of synthetic chemical pesticides, or combination of these agents with biological pesticides. could reduce the levels of toxic chemicals in the environment.

A biological pesticidal agent that is enjoying increasing popularity is the soil microbe Bacillus thuringiensis The soil microbe Bacillus thuringiensis is a Gram-positive, spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal activity. Some B.t. strains produce, and can be characterised by, parasporal crystalline protein inclusions. These "6-endotoxins," which typically have specific pesticidal activity, are different from exotoxins, which have a non-specific host range. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and are specific in their toxic activity.

Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example. B thuringiensis var.

kurstaki HD-1 produces a crystalline 6-endotoxin which is toxic to the larvae of a number of lepidopteran insects.

The cloning and expression of a B.t. crystal protein gene in Escherichia coli was described in the published literature more than 15 years ago (Schnepf. H.R. Whiteley [1981] Proc. Natl.

LibC/507084D1speci Acad. Sci. USA 78:2893-2897.). US 4 448 885 and 4 467 036 both disclose the expression of B.t.

crystal protein in E. coli. Recombinant DNA-based B.t. products have been produced and approved for use.

Commercial use of B.t. pesticides was originally restricted to a narrow range of lepidopteran (caterpillar) pests. More recently, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of namely israelensis and morrisoni tenebrionis, a.k.a. B.t. have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F.H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R.M. Wilkins. ed., Taylor and Francis, New York and London, 1990, pp. 245-255).

New subspecies of B.t. have now been identified, and genes responsible for active proteins have been isolated and sequenced (H6fte, H.R. Whiteley [1989] Microbiological Reviews 52(2):242-255). H6fte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were cryl (Lepidoptera specific), cryll (Lepidoptera and Diptera specific), crylll (Coleoptera S 15 specific), and crylV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. Payne, L. Kim [1992] Bio/Technology 10:271-275). For example, the designations CryV and CryVI have been proposed for two new groups of nematode-active toxins.

Many Bacillus thuringiensis 5-endotoxin crystal protein molecules are composed of two functional segments. For these proteins, the protease-resistant core toxin is the first segment and corresponds to about the first half of the protein molecule. The three-dimensional structure of a core segment of a CrylllA B.t. 5-endotoxin is known, and it was proposed that all related toxins have that same overall structure (Li, J. Carroll, D.J. Ellar [1991] Nature 353:815-821).

The second half of the molecule is often referred to as the "protoxin segment." The protoxin segment is believed to participate in toxin crystal formation (Arvidson. PE. Dunn, S. Strand. A I.

25 Aronson [1989] Molecular Microbiology 3:1533-1534; Choma. W.K. Surewicz. P.R. Carey. M.

Pozsgay, T. Raynor. H. Kaplan [1990] Eur. J Biochem. 189:523-527) The full 130kDa toxin molecule is typically processed to the resistant core segment by proteases in the insect gut. The protoxin segment may thus convey a partial insect specificity for the toxin by limiting the accessibility of the core to the insect by reducing the protease processing of the toxin molecule (Haider. B.H.

Knowles, D.J. Ellar [1986] Eur. J Biochem. 156:531-540) or by reducing toxin solubility (Aronson. A.I.

E.S. Han, W. McGaughey. D. Johnson [1991] Appl. Environ. Microbiol. 57:981-986).

The 1989 nomenclature and classification scheme of H6fte and Whiteley was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. The number of sequenced Bacillus thuringiensis crystal protein genes currently stands at more than 50. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996] Society for Invertebrate Pathology, 29 h Annual Meeting, Illrd International Colloquium on Bacillus thuringiensis, University of Cordoba, Cordoba, Spain. September 1-6, 1996. abstract). The mnemonic "cry" has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and LibC/507084D1spec

S.

s

S

S

S

5.

S

the parentheses in the tertiary rank have been removed. Many of the original names have been retained, although a number have been reclassified.

With the use of genetic engineering techniques, new approaches for delivering B.t. toxins to agricultural environments are under development, including the use of plants genetically engineered with B t. toxin genes for insect resistance and the use of stabilised, microbial cells as delivery vehicles of B.t. toxins (Gaertner, L. Kim [1988] TIBTECH 6:S4-S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.

Various improvements have been achieved by modifying B.t. toxins and/or their genes. For example, US 5 380 831 and 5 567 862 relate to the production of synthetic insecticidal crystal protein genes having improved expression in plants.

Obstacles to the successful agricultural use of B.t. toxins include the development of resistance to B.t. toxins by insects. In addition, certain insects can be refractory to the effects of B.t. The latter includes insects such as boll weevil and black cutworm as well as adult insects of most species which heretofore have demonstrated no apparent significant sensitivity to B.t. 15 Thus, resistance management strategies in B.t. plant technology have become of great interest, and there remains a great need for new toxin genes. As a result of extensive research and resource investment, other patents have issued for new B.t. isolates, toxins, and genes, and for new uses of B.t. isolates. See Feitelson et al.. supra. for a review. Additional examples include the following: B.t. Isolate, Toxin, Exemplified Pesticidal U.S. Patent No. (unless otherwise indicated) and/or Gene Activity of Toxin PS811, 811A, 811B2 lepidopteran 5 126 133; 5 188 960 CrylAc lepidopteran Adang et al.. GENBANK Acc. No. MI 1068 IC/IA(b) chimeric toxin lepidopteran 5 593 881 IF/IA(b) chimeric toxin lepidopteran 5 527 833 PS158C. 158C2c lepidopteran 5 268 172; 5 723 758 PS31G1, 31Gla lepidopteran W098/00546 However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an 20 empirical, unpredictable art.

There remains a great need for new toxin genes that can be successfully expressed at adequate levels in plants in a manner that will result in the effective control of insects and other pests.

Brief Summary of the Invention The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimised polynucleotide sequences that encode pesticidal toxins (full-length and truncated). Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimised expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transform plants in order to confer pest resistance upon said plants.

In one preferred embodiment, the subject invention provides plant-optimised genes that encode other proteins that are toxic to pests. Preferred embodiments are referred to herein as 1AC1AB-N-PO, LibC/507084D1speci 1AC1AB-PO, 1AC1AB-B-PO, 1AC-T-PO, 1AC-TB-PO, 1AC-TBX-PO, 1C-T-PO, 1C1AB-PO, 158C2c- PO, 158C2c-T-PO, and 31Gla-PO.

The subject invention further provides plant-optimised polynucleotide sequences that encode C-terminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Preferred embodiments of plant-optimised protoxins are designated PT-1AB-PO and PT-1A1B-2-PO.

In addition, the subject invention provides unique amino acids sequences for pesticidal toxins.

These toxins are encoded by the genes designated 1F1AB-PO, 1F-T-PO, 1F-7G-PO, and 1 F-7Z-PO; 1AC1AB-N-PO, 1AC1AB-PO, and 1AC1AB-B-PO, 1C1AB-PO, 158C2c-PO, 158C2c-T-PO, and 31Gla-T-PO. Furthermore. the subject invention provides unique, C-terminal amino acid sequences for protoxin portions (of full-length Bacillus thuringiensis toxins) encoded by the polynucleotide sequences designated PT-1AB-PO and PT-1AB-2-PO.

Brief Description of the Sequences SEQ ID NO.1 is a polynucleotide sequence designated PT-1AB-PO, which is optimised for 15 expression in plants. This gene. which encodes a CrylAb protoxin portion, can be used in conjunction with truncated genes (genes encoding truncated, core toxins) to make full-length toxins. Unless otherwise indicated, the chimeric genes exemplified herein are shown with this polynucleotide sequence (PT- 1AB-PO).

SEQ ID NO.2 is a polynucleotide sequence designated PT-1AB-2-PO, which is optimised for expression in cotton. This polynucleotide sequence is an alternative to PT-1AB-PO (and also encodes a CrylAb protoxin portion) and can also be used in conjunction with truncated genes (genes encoding truncated, core toxins) to make full-length toxins. PT-1AB-2-PO is preferred for use in a host that is transformed with more than one type of endotoxin transgene.

SEQ ID NO.3 is an amino acid sequence of a protoxin portion encoded by the genes 25 designated PT-1AB-PO and PT-1AB-2-PO.

SEQ ID NO.4 is a polynucleotide sequence for a gene designated 1AC1AB-N-PO, which is optimised for expression in plants. This gene encodes a chimeric CrylAc(N-terminal)/CrylAb (protoxin) toxin.

SEQ ID NO.5 is a polynucleotide sequence for a gene designated 1AC1AB-PO, which is optimised for expression in plants. This gene encodes a chimeric CrylAc(N-terminal)CrylAb (protoxin) toxin.

SEQ ID NO.6 is a polynucleotide sequence for a gene designated 1AC1AB-B-PO, which is optimised for expression in plants. This gene encodes a chimeric CrylAc(N-terminal)/CrylAb (protoxin) toxin.

SEQ ID NO.7 is an amino acid sequence of a toxin encoded by the genes designated 1AC1AB- N-PO, 1AC1AB-PO, and 1AC1AB-B-PO.

SEQ ID NO.8 is a polynucleotide sequence for a gene designated 1AC-T-PO, which is optimised for expression in plants. This plant-optimised gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a CrylAc toxin described by Adang et al. in GENBANK (Acc. No. MI 1068).

LibC/507084D1speci SEQ ID NO.9 is a polynucleotide sequence for a gene designated 1AC-TB-PO, which is optimised for expression in plants. This plant-optimised gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a CrvlAc toxin described by Adang et al. in GENBANK (Acc. No. M11068).

SEQ ID NO.10 is an alternative polynucleotide sequence for a gene designated 1AC-TBX-PO, which is optimised for expression in plants. This plant-optimised gene encodes a core toxin, the amino acid sequence of which is the same as that of the truncated form of a CrylAc toxin described by Adang et al. in GENBANK (Acc. No. M11068).

SEQ ID NO.11 is a polynucleotide sequence, optimised for expression in dicots, for a gene designated 1C-T-PO, which encodes the truncated form of a CrylC toxin designated 811B2 in U.S.

Patent No. 5,246,852.

SEQ ID NO.12 is a polynucleotide sequence for a gene designated 1C1AB-PO, which is optimised for expression in plants. This gene encodes a chimeric CrylC(N-terminal)CrylAb(protoxin) toxin.

SEQ ID NO.13 is an amino acid sequence of a toxin encoded by the gene designated 1C1AB-

PO.

SEQ ID NO.14 is a polynucleotide sequence for a gene designated 158C2c-PO.

SEQ ID NO.15 is an amino acid sequence for a full-length toxin encoded by the gene designated 158C2c-PO.

SEQ ID NO.16 is a polynucleotide sequence for a gene designated 158C2c-T-PO.

SEQ ID NO.17 is an amino acid sequence for a truncated toxin encoded by the gene designated 158C2c-T-PO.

SEQ ID NO.18 is a polynucleotide sequence for a gene designated 31Gla-T-PO, which is optimised for expression in maize.

25 SEQ ID NO.19 is an amino acid sequence for a truncated toxin encoded by the gene designated 31G1a-T-PO.

Detailed Disclosure of the Invention The subject invention concerns materials and methods useful in the control of pests and, particularly, plant pests. More specifically, the subject invention provides plant-optimised polynucleotide sequences that encode pesticidal toxins (full-length and truncated). Truncated polynucleotide sequences can be used to produce truncated toxins or for the production of fusion (or chimeric) genes and proteins. The polynucleotide sequences of the subject invention have certain modifications, compared to wild-type sequences, that make them particularly well-suited for optimised expression in plants. Using techniques known to those skilled in the art, the polynucleotide sequences described herein can be used to transform plants in order to confer pest resistance upon said plants.

The subject invention provides plant-optimised genes that encode other proteins that are toxic to pests. Preferred embodiments are referred to herein as 1AC1AB-N-PO, 1AC1AB-PO, 1AC1AB-B- PO, 1AC-T-PO, 1AC-TB-PO, 1AC-TBX-PO, 1C-T-PO, 1C1AB-PO, 158C2c-PO, 158C2c-T-PO, and 31G1a-PO.

LibC/507084D1speci The subject invention further provides plant-optimised polynucleotide sequences that encode C-terminal protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Preferred embodiments of plant-optimised protoxins are designated PT-1AB-PO and PT-1AB-2-PO.

In addition, the subject invention provides unique amino acids sequences for pesticidal toxins.

These toxins are encoded by the genes designated 1F1AB-PO, 1F-T-PO, 1F-7G-PO, and 1F-7Z-PO: 1AC1AB-N-PO, 1AC1AB-PO, and 1AC1AB-B-PO, 1C1AB-PO, 158C2c-PO, 158C2c-T-PO, and 31Gla-T-PO. Furthermore, the subject invention provides unique, C-terminal amino acid sequences for protoxin portions (of full-length Bacillus thuringiensis toxins encoded by the polynucleotide sequences designated PT-1AB-PO and PT-1AB-2-PO.

PT-1AB-PO can be used in preferred embodiments in combination with other truncated genes of the subject invention, such as the 1C-T-PO gene, in order to form other hybrid genes that encode full-length toxins. PT-1AB-2-PO an alternative polynucleotide sequence that encodes a protoxin portion) can also be used with truncated genes (which are smaller than full-length toxin genes, so long as the protein encoded by the truncated gene retains pesticidal activity) to encode chimeric or hybrid toxins. Preferred uses of PT-1AB-2-PC) are described above in the section entitled "Description of the Sequences." It should be apparent to a person skilled in this art that, given the sequences of the genes as set forth herein, the genes of the subject invention can be obtained through several means. In S 20 preferred embodiments, the subject genes may be constructed synthetically by using a gene synthesiser, for example. The specific genes exemplified herein can also be obtained by modifying, according to the teachings of the subject invention, certain wild-type genes (for example, by pointmutation techniques) from certain isolates deposited at a culture depository as discussed below. For example, a wild-type crylF gene can be obtained from B.t. isolate PS811. Likewise, the crylA(b) portions of the hybrid genes of the subject invention can be produced synthetically or can be derived by modifying wild-type genes. CrylA(b) toxins and genes have been described in, for example, H6fte et al. (1986) Eur. J. Biochem. 161:273: Geiser et al. (1986) Gene 48:109; and Haider et al. (1988) Nucleic Acids Res. 16:10927. Clones and additional wild-type isolates are discussed in more detail, above, in the section entitled "Background of the Invention" and in the list, below.

Cultures discussed in this application are available from the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, USA. The strains listed below are disclosed in the patent references as discussed above in the section entitled "Back round of the Invention." Subculture Accession Number Deposit Date B.t. PS811 NRRL B-18484 19 April, 1989 E. coli (NM522) (pMYC603) (811A) NRRL B-18517 30 June, 1989 E. coli (NM522) (pMYC394) (811 B2) NRRL B-i 8500 17 May, 1989 B.t. P5158C2 NRRL B-18872 27 August, 1991 E. coli (NM522) (pMYC2383) (158C2c) NRRL B-21428 11 April, 1995 B.t. PS31G1 NRRL B-21560 18 April,1996 E. coli (NM522) (pMYC2454) (31Gla) NRRL B-21796N 30 Sept, 1997 LibC/507084D1speci 7 It should be understood that the availability of a deposit does not constitute a licence to practice the subject invention in derogation of patent rights granted by governmental action.

Genes and Toxins. The polynucleotides of the subject invention can be used to form complete "genes" to encode proteins or peptides in a desired host cell. For example, as the skilled artisan would readily recognise, the polynucleotides of the subject invention are shown without stop codons. Also, the subject polynucleotides can be appropriately placed under the control of a promoter in a host of interest, as is readily known in the art.

As the skilled artisan would readily recognise, DNA can exist in a double-stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. The "coding strand" is often used in the art to refer to the strand having a series of codons (a codon is three nucleotides that can be read three-at-a-time to yield a particular amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. In order to express a protein in vivo, a strand of DNA is typically translated into a complementary strand of RNA which is used as the template for the protein. As DNA is replicated in a plant (for example) additional, complementary strands of DNA are 15 produced. Thus, the subject invention includes the use of either the exemplified polynucleotides shown in the attached sequence listing or the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to the exemplified DNA are included in the subject invention.

s Certain DNA sequences of the subject invention have been specifically exemplified herein.

These sequences are exemplary of the subject invention. It should be readily apparent that the subject invention includes not only the genes and sequences specifically exemplified herein but also equivalents and variants thereof (such as mutants, fusions, chimerics, truncations, fragments. and smaller genes) that exhibit the same or similar characteristics relating to expressing toxins in plants,.

as compared to those specifically disclosed herein. As used herein, variants" and "equivalents" refer to sequences which have nucleotide (or amino acid) substitutions, deletions (internal and/or terminal), additions, or insertions which do not materially affect the expression of the subject genes, and the resultant pesticidal activity, in plants. Fragments retaining pesticidal activity are also included in this definition. Thus, polynucleotides that are smaller than those specifically exemplified are included in the subject invention, so long as the polynucleotide encodes a pesticidal toxin.

Genes can be modified, and variations of genes may be readily constructed, using standard techniques. For example. techniques for making point mutations are well known in the art. In addition, commercially available exonucleases or endonucleases can be used according to standard procedures, and enzymes such as Ba131 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Useful genes can also be obtained using a variety of restriction enzymes.

It should be noted that equivalent genes will encode toxins that have high amino acid identity or homology with the toxins encoded by the subject genes. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the LibC/507084Dlspeci three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 1 provides a listing of examples of amino acids belonging to each class.

Table 1.

Class of Amino Acid E xamples of Amino Acids Nonpolar Ala, Val, Leu, lie, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic Asp, Glu Basic Lys, Arg, His In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the ability of plants to express the subject DNA sequences or from the biological activity of the toxin.

As used herein, reference to "isolated" polynucleotides and.' or "purified" toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature and would include their use in plants. Thus, reference to "isolated and purified" signifies the involvement of the "hand of man" as described herein.

i "'"Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. In some embodiments of the subject invention, transformed microbial hosts can be used in preliminary steps for preparing precursors. for example, that will i eventually be used to transform, in preferred embodiments, plant cells and plants so that they express the toxins encoded by the genes of the subject invention. Microbes transformed and used in this manner are within the scope of the subject invention. Recombinant microbes may be. for example, S 20 E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise :readily available to the skilled artisan.

S"Thus, in preferred embodiments, expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. When transformed plants are ingested by the pest, the pests will ingest the toxin. The result is a control of the pest. The B.t. toxin gene can be introduced via a suitable vector into a host, preferably a plant host. There are many crops of interest, such as corn, wheat, rice, cotton, soybeans, and sunflowers. The genes of the subject invention are particularly well suited for providing stable maintenance and expression, in the transformed plant, of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.

While the subject invention provides specific embodiments of synthetic genes, other genes that are functionally equivalent to the genes exemplified herein can also be used to transform hosts, preferably plant hosts. Additional guidance for the production of synthetic genes can be found in, for example, U.S. Patent No. 5,380,831.

All of the references cited herein are hereby incorporated by reference.

LibC/507084D1speci Following is an example which illustrates procedures for practicing the invention. This example should not be construed as limiting.

Example 1 Insertion of Toxin Genes Into Plants One aspect of the subject invention is the transformation of plants with the subject polynucleotide sequences encoding insecticidal toxins. The transformed plants are resistant to attack by the target pest. The genes of the subject invention are optimised for use in plants.

Obviously, a promoter region capable of expressing the gene in a plant is needed. Thus, for in planta expression, the DNA of the subject invention is under the control of an appropriate promoter region. Techniques for obtaining in planta expression by using such constructs is known in the art.

Genes encoding pesticidal toxins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example. pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the S 15 sequence encoding the B.t. toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli.

The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis. and other biochemicalmolecular biological methods are generally carried out as methods of analysis. After each 20 manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids.

Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be 25 joined as the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B Alblasserdam. Chapter 5; Fraley et al., Cri:. Rev. Plant Sci. 4:1-46: and An et al. (1985) EMBOJ 4:277-287.

Once the inserted DNA has been integrated in the genome. it is relatively stable there and, as a rule, does not come out again. It normally contains a selection marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.

A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacrerium tumefaciens or Agrobacrerium rhizogenes as transformation agent, fusion, injection. biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The LibC/507084D1speci Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Gener. 163:181-187). The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary S 20 factors. The resulting hybrid individuals have the corresponding phenotypic properties.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

LibC/507084D1speci Page(s) ik\ 2 are claims pages they appear after the sequence listing Sequence Listing <1 10> Cardineau, Guy A.

Stelman, Steven J.

Narva, Kenneth E.

120> Plant-Optimized Genes Encoding Pesticidal Toxins 130> MA-714X02 <140> <141> <150> 60/065,215 <151> 1997-11-12 150> 60/076,445 <151> 1998-03-02 <160> 19 <170> Patentln Ver. 15 <21 0> 1 <21 1> 1641 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. protoxin gene <400>1 gcaacactcg acttcgtcca tccaaccttg 25 gagaaggtca tttcgcggga caaggaggtg tgctatccaa taccagttga 30 aacgccaaac ccaagtccca ggctgtaccg gatggccatg gccctcgcta tgggaaacaa tctcagtatg cgcgttcata gctgctatct aggaatgtca cacgtagatg gcagaagttt gcgtacaaag gacgagctga tgcaatgact ggatacgatg gaggagaagg ggggactaca accgacaagg gagttacttc <210> 2 aggcagagtc atcagattgg ttgagtgcct aacatgctaa tcaacaggca atgatgtgtt catacctgta gaggttacat atgagacagt tcggcaagtg acctgaacga ccaggctagg gagtgaagag acattgtgta ataggctcca gcattcggga ttgaagagtt tcaagaatgg tagaagaaca cacaagaagt aaggatacgg agttcagcaa acactgcgac gagcctatga cttacaccga caccgttacc tttggattga tgatggagga tgacttggaa gctcaagaca ctctgatgag gcgacttagt actagatcgt caaggagaac ccagaagata cgaggacagt caatgtgcct tgcccatcac agacctcggt caatctggag ggctgagaag caaagaagcc agctgatacc agcttacctt agaagggcgc tgacttcaac gaacaatcac tcgtgtctgt agaaggttgc ctgcgtcgag tcaagaggag gagcaactct tggacgtagg agccggctat gattggagaa agagcacaga gatgtgactg ttctgtttgg gatgagcgga ggatggaggg tatgttacgc gatgaatcga caagaccttg gggacgggtt tcacaccact gtgtgggtga tttctagaag aagtggaggg aaagaaagcg aacatagcta cctgaactta atcttcactg aatggcctat cgctctgtcc cctggtcgtg gtcaccatac gaggaagtct tatgagggta tctgtacccg gacaatcctt gtcaccaaag acggaaggaa aggcggtgaa actatcacat atgagaagaa acttgcttca gaagtacgga tcttgggtac aactcaaagc agatctacct cactctggcc tctccttgga tcttcaagat agaaaccact acaagagaga ttgacgctct tgattcatgc gcgtgattcc cattctcctt cctgctggaa ttgttgttcc gctacattct acgagattga acccaaacaa cttacacttc ctgactatgc gcgaatctaa agttagagta cattcattgt tgctctgttc cgatcgcgtt ggagttgtcc agatcccaac catcaccatt ctttgatgag ctacacaaga catcagatac actttcagcc catagacgtt caagactcaa tgttggagaa gaagttggaa gtttgtgaac tgcagacaaa gggtgtcaat gtatgatgcg tgtgaaaggg tgagtgggaa tcgtgttacc gaacaacacc c ac cgt aac t tcgcaatcga atcagcctat cagaggctat c tt tcc agaa tgatagcgtg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1641 LibC/50708401 speci <21 1> 1635 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. protoxin gene <400> 2 *6 ctcgaggctg agcaaccaga cttgtggagt gtgaagcacg ggtatcaaca ggtgacgacg cctacctacc cttaggggat aagcacgaga cctatcggta accgatctca cacgctaggc gctagggtga 20 accaacatcg tacgataggc cacagcatca atcttcgagg gtgatcaaga 25 gatgttgagg gttagccaag aaggagggt t ctcaagttca gactacaccg 30 gatggtgctt aaagcataca tacacccctc aaagtgtgga ct tct catgg agagcgatct tcggtctcaa gt ct tagcga ctaagaggct gacagcttga tgttcaaaga tctatcagaa acattgagga ccgtgaacgt agtgcgctca acgaggatct ttggaaacct agagagcaga tgtacaagga ttcaagcaga gggaggcata agcttgaggg acggagact t agcagaacaa aggttagggt acggtgaggg gtaactgtgt ctacccagga acgagagcaa ctgatggtag ttcctgctgg tcgagatcgg aggag agagagggc t gaccgatgtt cgagttctgc tagcgatgag taggggttgg gaactacgtt gatcgatgag tagccaggat tcctggaacc ccacagccac tggtgtttgg tgagttcctt gaagaagtgg ggctaaggag cactaacatc ccttccagag taggatcttc caacaacggt ccacaggagc ttgccctggt ttgcgttacc ggaggaggag agagtacgag cagcagcgtt gagggacaac ttacgttacc tgagaccgag cagaaggctg accgactacc cttgacgaga aggaaccttc aggggtagca acccttcttg agcaagctca ctggagatct ggtagccttt cacttcagcc gtcatcttca gaggagaagc agagacaaga agcgttgatg gctatgatcc cttagcgtga accgctttca cttagctgct gttctcgtgg aggggttaca atccacgaga gtgtacccta ggaacctaca cctgctgact ccttgcgaga aaggagcttg ggaaccttca tgaacgctct acatcgatag agaaagagct tccaagaccc ccgacatcac gtactttcga aggcttacac atctcatccg ggcctcttag ttgacatcga agatcaagac ctCt tgt tgg gggagaagct ctctcttcgt acgctgctga tccctggagt gcctctacga ggaacgtgaa tgccagagtg tccttagggt tcgagaacaa acaacactgt ccagcaggaa acgctagcgc gcaacagggg agtacttccc tcgtggacag cttcaccagc ggttagcaat tagcgagaag taacttcagg tatccaaggt cgaatgctac caggtatcaa ttacaatgct tgcacctagc tgttggttgc ccaagatggt tgaggctctt tgagtgggag gaacagccag caagagggtt gaacgcagca tgctaggaac gggtcacgtt ggaggctgaa gac tgc t tac cactgatgag tacctgcaac caggggttac atacgaagag ttacggtgac tgagactgac cgttgagctt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1635 <210> 3 <21 1> 547 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 3 Ala Thr Leu Giu Ala Giu Ser Asp Leu Giu Arg Ala Gln Lys Ala Val is 1 5 10 Asn Ala Leu Phe Thr Ser Ser Asn Gin Ile Gly Leu 20 25 Thr Asp Tyr His Ile Asp Arg Val Ser Asn Leu Val Asp Giu Phe Cys Leu Asp Giu Lys Lys Giu Leu Ser 55 His Ala Lys Arg Leu Ser Asp Giu Arg Asn Leu Leu 70 Phe Arg Gly Ile Asn Arg Gin Leu Asp Arg Gly Trp 90 Lys Thr Asp Val Cys Leu Ser Lys Val Lys Gin Asp Pro Arg Giy Ser Thr Tyr Vai Asp Ile Thr Ile Gin Gly Gly Asp Asp 100 105 Thr Leu Leu Gly Thr Phe Asp Giu Cys Phe Lys Glu Asn Tyr Pro Thr Tyr 110 Leu Tyr Gin LibC/50708401 speci 3/23 115 120 125 Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg 130 135 140 Gly Tyr Ile Glu Asp Ser Gin Asp Leu Glu Ile Tyr Leu Ile Arg Tyr 145 150 155 160 Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp 165 170 175 Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His 180 185 190 His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp 195 200 205 Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp Gly His Ala 210 215 220 Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu 225 230 235 240 Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg 245 250 255 Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu 260 265 270 Ser Val Asp Ala Leu Phe Val Asn Ser Gin Tyr Asp Arg Leu Gin Ala 275 280 285 Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser 290 295 300 SIle Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn 25 305 310 315 320 Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser 325 330 335 Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly 340 345 350 30 Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gin Asn 355 360 365 Asn His Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser 370 375 380 Gin Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr 385 390 395 400 Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 405 410 415 Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu 420 425 430 40 Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gin 435 440 445 Glu Glu Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly 450 455 460 Ala Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr 465 470 475 480 Glu Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser 485 490 495 Asn Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr 500 505 510 Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile 515 520 525 Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu 530 535 540 Met Glu Glu 545 <210>4 <211> 3468 <212> DNA <213> Artificial Sequence so <220> LibC/507084D1speci <223> Synthetic B.t. toxin gene 9090 .9 .9 9 0*699e .9 @999 9.

9.9.

9. 9 99 99 *9 9 9* .9 9*9 9 9**9 9 9 9 .9 9.9.

S. 9 09 9.

<400> 4 atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcaactta gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggcaactata ccggattcta ctagacattg tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg accttgtcca tctgtgcttg tacagaaaga ccacctaggc 25 agtaatagca gagtttaaca tttctcttta ctgaattctt ccatcgacat 30 ctcaacgtca tccttggata t ct tccc tag gaccgcttcg gcacaaaagg gtgacggatt tgtctggatg gagcggaatt tggagaggaa gttacgctat 40 gagtcgaaat gacttagaaa acgggttcct catcatttct tgggtgatat ctcgaagaga tggagagaca gaatctgtag atcgcgatga gagctgtctg ttcactgcat ggc ttat cc t tcggtccttg ggtcgtggct accattcatg gaagtatatc gagggtacgt gtaccagctg aatccttgtg acaaaagaat gaaggaacat <21O> atcccaacat tgggtggaga aattcctttt tttggggaat tcaaccaaag gcaaccttta ctgccttaag caattccgct ctgccaacct ttgatgccgc ccgattatgc gagattgggt tcgctctctt cccgggaaat ctcagggcat cgatctacac cacccgttgg cagctccaca gcactctata acgggacaga gcggaacagt aagggtttag gcgttagtat acataattgc atggttctgt ccggcaacaa ctaccagata attggggtaa atctccaatc gtaacatagt aattcattcc cggtgaatgc atcatatcga aaaaaaaaga tacttcaaga gtacggatat tgggtacctt taaaagccta tctatttaat tatggccgct ccttggacat tcaagattaa aaccattagt aacgtgaaaa atgctttatt ttcatgcggc tgattccggg tctccctata gctggaacgt ttgttccgga atatccttcg agatcgagaa caaacaacac acacttctcg attatgcatc aatctaacag tagagtactt tcatcgtgga caacgagtgc acggattgag gtcagagttc ctttggtccc gattgaagag ccagatttac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgttactgca cctgtttact tcgagtatcc attgtccgag tccaaacttt taccatccaa tgatgagtgc tacccgttac tcgctacaat ttcagcccca tgatgttgga gacgcaagat aggagaagca at tggaatgg tgtaaactct agataaacgc tgtcaatgcg tgatgcgaga gaaagggcat atgggaagca tgtcacagcg caatacagac ggtaacgtgt taatcgagga agcctatgaa aggatatggg cccagaaacc cagcgtggaa attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctccgcg agtcgttata tacaacacgg cagttcaggc gactctaggc ccagtcctcg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt ccgatgttct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggttacttcg aatttctccg acgctcgagg tcttccaatc aatttagttg aaagtcaaac agagggatca ggaggcgatg tatccaacgt caattaagag gccaaacacg agtccaatcg tgtacagact ggccatgcaa ctagctcgtg gaaacaaata caatatgata gttcatagca gctatttttg aatgtcatta gtagatgtag gaagtgtcac tacaaggagg gaactgaagt aatgattata tatgacggag gaaaaagcat gattacacac gataaggtat ttacttctta actgcctgag cacctatcga ctggattcgt acgcctttct accaagccat ttcgagagtg tcaatgacat aagttcctct atgtctccgt atgatctgac gtctcgaacg gagagttgac gctacccaat agaacttcga cacacctgat actactggtc ctttcccact tcggtcaggg gcatcaacaa caaatctgcc c tcc a caga a ccatgttccg cttggataca tcccagctgt ctggaggcga ttgaagtgcc cctctgttac cagtaccagc aaagtgccaa gaaccgccgg cagaatctga aaatcgggtt agtgtttatc atgcgaagcg atagacaact acgtattcaa atttatatca ggtatatcga aaacagtaaa gaaaatgtgc taaatgagga gactaggaaa tgaaaagagc ttgtttataa gattacaagc ttcgagaagc aagaattaga aaaatggtga aagaacaaaa aagaagttcg gatatggaga ttagcaactg ctgcgactca cctatgaaag atacagatgg cactaccagc ggattgagat tggaggaa caaccctgag catctcgttg gc ttggac tt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa taggcttatt tgtctgggga actaactgtc ccgtactgtg cggtagcttt ggacatattg agggcatcag ttacgggact cgtgtataga tcaacaattg atccgctgtc caacaacgtt ttcaggcttt tcgtagtgct caaggggaac cttggttagg cattcacttc ccctattcac gacagctaca tgccttcacc agtgataatc tttagaaaga aaaaaccgat tgatgaattt acttagtgat agaccgtggc agagaattac aaaaatagat agatagtcaa tgtgccaggt ccatcattcc cttaggtgta tctagaattt ggagaaaaaa agaggcaaaa ggataccaac ttatctgcct agggcgtatt ttttaataat caaccaccgt tgt ctgt ccg aggttgcgta tgtagaagag agaagaatat caattcttct acgaagagac tggctatgtg cggagaaacg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3468 LibC/507084D1 speci <21 1> 3468 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene

OOOS

0O 0S 0

S

5.55..

0

OS

S

0*

S

S. S

OS

50 S. S 0@

S.

SOS

S 5 <400> atggacaaca gt tgaggtgc tcacttaccc gtcgatatca gagcaactta gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggc aa ctat a ccggattcta ctagacattg 20 tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg 25 accttgtcca tctgtgcttg tacagaaaga ccacctaggc agtaatagca 30 gagtttaaca tttctcttta ctgaattctt ccatcgacat ctcaacgtca tccttggata tcttccctag gaccgcttcg gcacagaagg gtgactgact tgtttggatg gagcggaact tggaggggaa gttacgctct gaatcgaaac gaccttgaga acgggttcac caccacttct tgggtgatct ctagaagaga tggagggaca gaaagcgttg atagctatga gaacttagcg t tcac tgcat ggcctatcct tctgtccttg ggtcgtggct accatacacg gaagtctacc gagggtactt atcccaacat tgggtggaga aattcctttt tttggggaat tcaaccaaag gcaaccttta c tgc ct taag caattccgct ctgccaacct ttgatgccgc ccgattatgc gagattgggt tcgctctctt cccgggaaat ctcagggcat cgatctacac cacccgttgg cagctccaca gcactctata acgggacaga gcggaacagt aagggtttag gcgttagtat acataattgc atggttctgt ccggcaacaa ctaccagata attggggtaa atctccaatc gtaacatagt aattcattcc cggtgaatgc atcacatcga agaagaagga tgcttcaaga gtacggacat tgggtacctt tcaaagccta tctacctcat tctggccact ccttggacat tcaagatcaa aaccacttgt agagagagaa acgctctgtt ttcatgctgc tgattccggg tctccttgta gctggaatgt ttgttcctga acattcttcg agattgagaa caaacaacac acacttctcg caacgagtgc acggat tgag gtcagagttc Ct ttggt CCC gattgaagag ccagatttac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtacaac t ccc aact a c ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgttactgca tctgttcact tcgcgtttcc gttgtccgag tcccaacttt caccattcaa tgatgagtgc cacaagatac cagatacaac ttcagcccca agacgttggc gactcaagat tggagaagcc gttggaatgg tgtgaactct agacaaacgc tgtcaatgct tgatgcgagg gaaagggcac gtgggaagca tgttaccgcg caacaccgac cgtaacttgc caatcgagga attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctccgcg agtcgttata tacaacacgg cagttcaggc gactctaggc ccagtcctcg a tc aggtct C cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt ccgatgttct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggttacttcg aatttctccg acgctcgagg tcgtccaatc aaccttgttg aaggtcaaac cgcgggatca ggaggtgatg tatccaacat cagttgagag gccaaacatg agtcccatcg tgtaccgacc ggccatgcca ctcgctagag gaaacaaaca cagtatgata gttcatagca gctatctttg aatgtcatca gtagatgtag gaagtttcac tacaaagaag gagctgaagt aatgactaca tacgatggag actgcctgag cacctatcga ctggattcgt acgcctttct accaagccat ttcgagagtg tcaatgacat aagttcctct atgtctccgt atgatctgac gtctcgaacg gagagttgac gctacccaat agaacttcga cacacctgat actactggtc ctttcccact tcggtcaggg gcatcaacaa caaatctgcc ctccacagaa ccatgttccg cttggataca tcccagctgt ctggaggcga ttgaagtgcc cctctgttac cagtaccagc aaagtgccaa gaaccgccgg cagagtctga agattgggct agtgcctctc atgctaagcg acaggcaact atgtgttcaa acctgtacca gttacatcga agacagtcaa gcaagtgtgc tgaacgaaga ggctaggcaa tgaagagggc ttgtgtacaa ggctccaagc ttcgggaagc aagagttaga agaatggtga aagaacagaa aagaagt tcg gatacggaga tcagcaactg ctgcgactca cctatgagag caaccctgag catctcgttg gcttggactt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa taggcttatt tgtctgggga actaactgtc ccgtactgtg cggtagcttt ggacatattg agggcatcag ttacgggact cgtgtataga tcaacaattg atccgctgtc caacaacgtt ttcaggcttt tcgtagtgct caaggggaac cttggttagg cattcacttc ccctattcac gacagctaca tgccttcacc agtgataatc cttggaaaga caagacagat tgatgagttc acttagtgat agatcgtgga ggagaactat gaagatagat ggacagtcaa tgtgcctggg ccatcactca cctcggtgtg tctggagttt tgagaagaag agaagccaaa tgataccaac ttaccttcct agggcgcatc cttcaacaat caatcaccgc tgtctgtcct aggttgcgtc cgtcgaggag agaggagtat caactcttct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 050505 00 0 go 00: LibC/50708401 speci gtacccgctg actatgcatc agcctatgag gagaaggctt acaccgatgg acgtagggac 3300 aatccttgcg aatctaacag aggctatggg gactacacac cgttaccagc cggctatgtc 3360 accaaagagt tagagtactt tccagaaacc gacaaggttt ggattgagat tggagaaacg 3420 gaaggaacat tcattgttga tagcgtggag ttacttctga tggaggaa 3468 <210> 6 <21 1> 3468 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 6 atggacaaca atcccaacat gttgaggtgc tgggtggaga tcacttaccc aattcctttt gtcgatatca tttggggaat gagcagttaa ttaaccaaag gaaggcctca gcaaccttta ccgaccaatc ctgccttaag ctgacgaccg caattccgct tacgtgcagg ctgccaacct cggtggggct ttgatgccgc ggcaactata ccgattatgc ccggattcta gagattgggt ctagacattg tcgctctctt tcacaattga cccgggaaat cgaggctcgg ctcagggcat aacagtatca cgatctacac atcatggcat cacccgttgg atgggcaatg cagctccaca accttgtcca gcactctata tctgtgcttg acgggacaga tacagaaaga gcggaacagt ccacctaggc aagggtttag agtaatagca gcgttagtat gagtttaaca acataattgc tttctcttta atggttctgt ctgaattctt ccggcaacaa ccatcgacat ctaccagata ctcaacgtca attggggtaa tccttggata atctccaatc tcttccctag gtaacatagt gaccgcttcg aattcattcc gcacagaagg cggtgaatgc gtgactgact atcacatcga tgtttggatg agaagaagga gagcggaact tgcttcaaga tggaggggaa gtacggacat gttacgctct tgggtacctt gaatcgaaac tcaaagccta gaccttgaga tctacctcat acgggttcac tctggccact caccacttct ccttggacat tgggtgatct tcaagatcaa ctagaagaga aaccacttgt tggagggaca agagagagaa gaaagcgttg acgctctgtt atagctatga ttcatgctgc gaacttagcg tgattccggg ttcactgcat tctccttgta caacgagtgc acggattgag gtcagagttc ctttggtCCC aatagaagaa ccagatttac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gttc t ctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgttactgca tctgttcact tcgcgtttcc gttgtccgag tcccaacttt caccattcaa tgatgagtgc cacaagatac cagatacaac ttcagcccca agacgttggc gactcaagat tggagaagcc gttggaatgg tgtgaactct agacaaacgc tgtcaatgct tgatgcgagg attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctccgcg agtcgttata tacaacacgg cagttcaggc gactctaggc ccagtcctcg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt ccgatgttct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggttacttcg aatttctccg acgctcgagg tcgtccaatc aaccttgttg aaggtcaaac cgcgggatca ggaggtgatg tatccaacat cagttgagag gccaaacatg agtcccatcg tgtaccgacc ggccatgcca ctcgctagag gaaacaaaca cagtatgata gttcatagca gctatctttg aatgtcatca actgcctgag cacctatcga ctggattcgt acgcctttct accaagccat ttcgagagtg tcaatgacat aagttcctct atgtctccgt atgatctgac gtctcgaacg gagagttgac gctacccaat agaacttcga cacacctgat actactggtc ctttcccact tcggtcaggg gcatcaacaa caaatctgcc ctccacagaa ccatgttccg cttggataca tcccagctgt ctggaggcga ttgaagtgcc cctctgttac cagtaccagc aaagtgccaa gaaccgccgg cagagtctga agattgggct agtgcctctc atgctaagcg acaggcaact atgtgttcaa acctgtacca gttacatcga agacagtcaa gcaagtgtgc tgaacgaaga ggctaggcaa tgaagagggc ttgtgtacaa ggctccaagc ttcgggaagc aagagttaga agaatggtga caaccctgag catctcgttg gcttggactt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa taggcttatt tgtctgggga actaactgtc ccgtactgtg cggtagcttt ggacatattg agggcatcag ttacgggact cgtgtataga tcaacaattg atccgctgtc caacaacgtt ttcaggcttt tcgtagtgct caaggggaac cttggttagg cattcacttc ccctattcac gacagctaca tgCCttcacc agtgataatc cttggaaaga caagacagat tgatgagttc acttagtgat agatcgtgga ggagaactat gaagatagat ggacagtcaa tgtgcctggg ccatcactca cctcggtgtg t ctggagt tt tgagaagaag agaagccaaa tgataccaac ttaccttcct agggcgcatc cttcaacaat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 LibC/507084D1 speci ggcctatcct gctggaatgt gaaagggcac tctgtccttg ttgttcctga gtgggaagca ggtcgtggct acattcttcg tgttaccgcg accatacacg agattgagaa caacaccgac gaagtctacc caaacaacac cgtaacttgc gagggtactt acacttctcg caatcgagga gtacccgctg actatgcatc agcctatgag aatccttgcg aatctaacag aggctatggg accaaagagt tagagtactt tccagaaacc gaaggaacat tcattgttga tagcgtggag '<21O> 7 <211> 1156 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 7 7/23 gtagatgtag gaagtttcac tacaaagaag gagctgaagt aatgactaca tacgatggag gagaaggctt gactacacac gacaaggttt ttacttctga aagaacagaa caatcaccgc 2940 aagaagttcg tgtctgtcct 3000 gatacggaga aggttgcgtc 3060 tcagcaactg cgtcgaggag 3120 ctgcgactca agaggagtat 3180 cctatgagag caactcttct 3240 acaccgatgg acgtagggac 3300 cgttaccagc cggctatgtc 3360 ggattgagat tggagaaacg 3420 tggaggaa 3468 Met 1 Ser Tyr Giu Trp Giu Ile Ser Glu Ile 145 Tyr Val Tyr Arg Asp 225 Leu Ile Leu Arg Ile 305 Ile Asp Asn Thr Phe 50 Gly Gin Ser Phe Met 130 Pro Val1 Phe As n Trp 210 Trp Asp Arg Giu Ser 290 Tyr Met Asn Pro Pro Val1 Ile Leu Arg Arg 115 Arg Leu Gin Giy Asp 195 Tyr Val1 Ile Thr Asn 275 Ile Thr Ala Pro 5 Val1 Asp Gly Gly Asn Glu Trp Gin Al a Al a 165 Arg Thr Thr Tyr Ala 245 Ser Asp Ser Al a Pro 325 As n Giu Ile Al a Pro 70 Gin Gly Giu Phe Val 150 Asn Trp Arg Gly Asn 230 Leu Gin Gly Pro His 310 Val1 Asn Leu Leu 40 Phe Gin Ile Ser Asp 120 Asp As n His Phe Ile 200 Giu Phe Pro Thr Phe 280 Leu Gly Phe Cys 10 Gly Leu Leu Asp Giu 90 Leu Thr Asn Gin Ser 170 Al a Asn Val1 Arg Tyr 250 Giu Gly Asp Tyr Gly 330 Pro Arg Gin Leu Phe Ala Gin Pro Ala 140 Pro Leu Thr Thr Gly 220 Leu Ser Tyr Ala Leu 300 Trp Glu Tyr Ile Phe Val1 Leu Arg Ile Ala 125 Leu Leu Arg Ile Asp 205 Pro Thr Arg Thr Gin 285 Asn Ser Phe Asn Giu Leu Asp Val1 Asn Tyr 110 Leu Thr Leu Asp Asn 190 Tyr Asp Leu Arg Asn 270 Gly Ser Gly Thr Cys Thr Leu Ile Gin Gin Ala Arg Thr Ser Val 175 Ser Ala Ser Thr Tyr 255 Pro Ile Ile His Phe 335 Leu Gly Ser Ile Ile Ala Giu Giu Ala Val 160 Ser Arg Val Arg Val 240 Pro Val1 Giu Thr Gin 320 Pro LibC/507084D1 speci Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg Ile Val Ala 340 345 350 Gin Leu Gly Gin Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gin Gin Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gin 405 410 415 Asn Asn Asn Val Pro Pro Arg Gin Gly Phe Ser His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 Ile Ile Ala Ser Asp Ser Ile Thr Gin Ile Pro Ala Val Lys Gly Asn 465 470 475 480 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 485 490 495 Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gin Asn Arg Gly 500 505 510 Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg 515 520 525 S 25 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn 530 535 540 Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr 545 550 555 560 Ser Leu Asp Asn Leu Gin Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala 565 570 575 Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe 580 585 590 Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 595 600 605 Thr Ala Thr Leu Glu Ala Glu Ser Asp Leu Glu Arg Ala Gin Lys Ala 610 615 620 Val Asn Ala Leu Phe Thr Ser Ser Asn Gin Ile Gly Leu Lys Thr Asp 625 630 635 640 Val Thr Asp Tyr His Ile Asp Arg Val Ser Asn Leu Val Glu Cys Leu 40 645 650 655 Ser Asp Glu Phe Cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val 660 665 670 Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro 675 680 685 Asn Phe Arg Gly Ile Asn Arg Gin Leu Asp Arg Gly Trp Arg Gly Ser 690 695 700 Thr Asp Ile Thr Ile Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr 705 710 715 720 Val Thr Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr 725 730 735 Gin Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu 740 745 750 Arg Gly Tyr Ile Glu Asp Ser Gin Asp Leu Glu Ile Tyr Leu Ile Arg 755 760 765 Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu 770 775 780 Trp Pro Leu Ser Ala Pro Ser Pro Ile Gly Lys Cys Ala His His Ser 785 790 795 800 His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu 805 810 815 Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp Gly His 820 825 830 Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly LibC/507084Dspeci 845 Trp Giu Ala 850 Arq Giu Ala Arg Val Lys 855 Giu Ala Giu Lys Arg Asp Lys Lys Leu Giu Thr Asn Ile Lys Giu Ala 865 Giu Ser Val Asp Ala 885 Ile Phe Val Asn Tyr Asp Arg Leu Gin 895 Ala Asp Thr Ser Ile Arg 915 Asn Ala Ala Asn 900 Giu Ala Met Ile His 905 Glu Ala Asp Lys Arg Val His 910 Pro Gly Val Ala Tyr Leu Leu Ser Val Ile 925 Phe Ile Phe Glu Glu 935 Asn Glu Gly Arg Ile 940 Gly Thr Ala Phe Ser 945 Gly Tyr Asp Ala Val Ile Lys Asp Phe Asn Leu Ser Cys Trp 965 Ser Val Lys Gly Asp Val Giu Giu Gin 975 Asn Asn His Arg 980 Val Leu Val Val 985 Gly Glu Trp Glu oo .o :.o:o o o 0. 0.

o o Ser Gin Thr Ala 1010 Ile Giu 1025 Giu Val Gin Giu Gly Ala Tyr Glu Giu Val Arg Val Cys Pro 995 1000 Tyr Lys Giu Gly Tyr Gly 1015 Asn Asn Thr Asp Giu Leu 1030 Tyr Pro Asn Asn Thr Val 1045 Glu Tyr Giu Gly Thr Tyr 1060 Tvr Glu SerAsn Ser Ser Glu Gly Cys Val Thr 1020 Lys Phe Ser Asn Cys 1035 Thr Cys Asn Asp Tyr 1050 Thr Ser Arg Asn Arg 1065 Val Pro Ala Asp Tyr 1085 Glv Arg Arg Asp Asn Arg Gly Tyr Ile 1005 Ala Giu Val 990 Leu Arg Val Ile His Glu Val Glu Glu 1040 Thr Ala Thr 1055 Gly Tyr Asp 1070 Ala Ser Ala Pro Cys Giu .075 Glu Lys Ala Tyr Thr 1080 Asp 1090 1095 Ser Asn Arg Gly Tyr Gly Asp 1105 1110 Thr Lys Glu Leu Giu Tyr Phe 1125 Ile Gly Glu Thr Giu Gly Thr 1140 40 Leu Met Giu Giu 1155 <210> 8 <21 1> 1839 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 8 1100 Tyr Thr Pro Leu 1115 Pro Glu Thr Asp 1130 Phe Ile Val Asp 1145 Pro Ala Gly Tyr Val 1120 Lys Val Trp Ile Giu 1135 Ser Val Glu Leu Leu 1150 atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcaactta gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggcaactata atcccaacat tgggtggaga aattcctttt tttggggaat tcaaccaaag gcaaccttta ctgccttaag caattccgct ctgccaacct ttgatgccgc ccgattatgc caacgagtgc acggattgag gtcagagttc ct ttggt CCC gattgaagag ccagatttac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctccgcg agtcgttata tacaacacgg actgcctgag cacctatcga ctggattcgt acgcctttct accaagccat ttcgagagtg tcaatgacat aagt tcct ct atgtctccgt atgatctgac gtctcgaacg caaccctgag catctcgttg gcttggactt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa taggcttatt tgtctgggga LibC/507084D1 speci ccggattcta ctagaoattg tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg accttgtcca to tgtgc ttg tacagaaaga cc acc tagg c agtaatagca gagtttaaca tttctcttta ctgaattctt ocatogacat ctcaacgtca tccttggata tcttccctag gaccgcttog <210> 9 gagattgggt tcgctctctt cccgggaaat o toagggo at cgatctacac cacccgttgg cagctccaca gcactctata acgggacaga gcggaacagt aagggtttag gcgttagtat acataattgc atggttctgt ccggcaacaa ctaccagata attggggtaa atctccaatc gtaacatagt aattcattcc oaggtacaac tcocaactac ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgc~tt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga ogttactgca 10/23 cagttcaggc gactotaggo ccagtcotcg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt ccgatgttct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggt tact tcg aatt t ct ccg acgctcgag gagagttgac gctacooaat agaacttcga cacacctgat actactggtc ctttcocact tcggtoaggg goatcaaoaa caaatctgcc ctccaoagaa ccatgttoog cttggataoa tcccagctgt ctggaggoga ttgaagtgco octotgttac c agt ac cago aaagtgcoaa gaaccgccgg aotaaotgto Oogtaotgtg oggtagcttt ggaoatattg agggcatoag ttacgggaot ogtgtataga tcaaoaattg atco go tgt 0 oaaoaaogtt ttcaggottt tcgtagtgot caaggggaac ottggttagg cattoaottc coctattoac gacagotaca tgc otto ac agtgataatc 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1839 <21 1> 1839 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 9 atggaoaaca gttgaggtgc tcacttaoc gtcgatatca gagcagttaa gaaggcctoa ccgaccaatc ctgacgacog tacgtgoagg cggtggggot ggcaaotata coggattota ctagacattg tcacaattga cgaggotcgg aacagtatca atcatggcat atgggoaatg accttgtcca tctgtgcttg tacagaaaga ccacctaggc agtaatagca gagtttaaca tttctottta otgaattctt ocatogacat otcaaogtca toot tggat a tot to cotag gacogottog <210> atcccaaoat tgggtggaga aattoctttt tttggggaat ttaaccaaag gcaaocttta ctgocttaag caattccgot ctgcoaaoot ttgatgcogc ccgattatgo gagattgggt tcgctctott ccogggaaat ctoagggoat cgatotaoao caoccgttgg cagctcoaoa goactotata aogggaoaga goggaaoagt aagggtttag gogttagtat aoataattgo atggt totgt ooggcaacaa otaccagata attggggtaa atotccaato gtaacatagt aattcattcc caaogagtgc acggattgag gtoagagtto otttggtCCo aatagaagaa ocagatotac agaggagatg ottogcogtt gcacttgtcg aactatcaat tgttcgctgg oaggtacaao toccaactao otacacaaac agagagaago ogatgcgcac gt tototgga aoaacgtatt taggagacot atttgcotat tgatagottg ooatcgcctt catoagagot atoogatagc oatttcagga oatocagaat tcgtgttogt ttoctocato tagogattto aggtgttaga cgttactgca attoottaca actggttaca gtgcooggtg tctcaatggg ttcgotagga gcagaatctt cgcattoaat cagaattacc gtgctcogcg agtcgttata t ac aacac gg cagttcaggc gactotaggo ccagtcctog atcaggtcto cgcggttatt ccagaattoa gttgctcaao ttcaaoatog ggaaootcot gatgagatcc agcoatgtgt ccgatgttct attactoaga ccaggattca agagggtata gtaaggtatg ttttocaata ggttacttog aatttotcog acgctcgag aotgootgag cacotatoga ctggattogt acgctttot acoaagooat ttogagagtg tcaatgaoat aagttootot atgtcoto cgt atgatotgac gtctcgaacg gagagttgac gotacooaat agaacttoga oaoacotgat actaotggtc otttoccaot tcggtcaggg gcatoaaoaa oaaatctgoc otcoacagaa ocatgttccg cttggataoa tcccagctgt otggaggoga ttgaagtgoo cototgttac cagtaooago aaagtgocaa gaaccgccgg caacootgag catctcgttg got tgga t t tgtacagata ctoaaggtta ggaagcagac gaaoagcgcg tttatocgtg gttcggaoaa taggcttatt tgtotgggga aotaaotgto oogtaotgtg cggtagcttt ggaoatattg agggcatoag ttaogggaot Cgtgtataga tcaacaattg atoogctgtc caaoaacgtt ttcaggcttt tcgtagtgct caaggggaac cttggttagg cattoaottc coctattoac gacagotaca tgcttcacc agtgataatO 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1839 LibC/5O7OB4D1 speci 11/23 <21 1> 1839 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcagttaa gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggcaactata ccggattcta ctagacattg tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg accttgtcca tctgtgcttg tacagaaaga ccacctaggc agtaatagca gagtttaaca tttctcttta ctgaattctt ccatcgacat ctcaacgtca tccttggata tcttccctag gaccgcttcg <210> 11 atcccaacat tgggtggaga aattcctttt tttggggaat ttaaccaaag gcaaccttta ctgccttaag caattccgct ctgccaacct ttgatgccgc ccgattatgc gagattgggt t cgctct Ct t cccgggaaat ctcagggcat cgatctacac cacccgttgg cagctccaca gcactctata acgggacaga gcggaacagt aagggtttag gcgttagtat acataattgc atggttctgt ccggcaacaa ctaccagata attggggtaa atctccaatc gtaacatagt aattcattcc caacgagtgc acggattgag gtcagagttc Ct ttggt CCC aatagaagaa ccagatctac agaggagatg Ct tcgc cgt t gcacttgtcg aactatcaat tgttcgctgg caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gt t ct Ctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgttactgca attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctccgcg agtcgttata tacaacacgg cagttcaggc gactctaggc ccagtcctgg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt ccgatgttct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggttacttcg aatttctccg acgctcgag actgcctgag cacctatcga ctggattcgt acgcctttct accaagccat ttcgagagtg tcaatgacat aagttcctCt atgtctccgt atgatctgac gtctcgaaCg gagagt tgac gctacccaat agaacttcga cacacctgat actactggtc ctttcccact tcggtcaggg gcatcaacaa caaatctgcc ctccacagaa ccatgttccg cttggataca tcccagctgt ctggaggcga ttgaagtgcc cctctgttac cagtaccagc aaagtgccaa gaaccgccgg caaccctgag catctcgttg gcttggactt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa t aggc ttat t tgtctgggga actaactgtc ccgtactgtg cggtagcttt ggacatattg agggcatcag ttacgggact cgtgtataga tcaacaattg atccgctgtc caacaacgtt ttcaggcttt tcgtagtgct caaggggaac cttggttagg cattcacttc ccctattcac gacagctaca tgccttcacc agtgataatc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1839 <21 1> 1860 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 11 atggaggaga gtacttcttg cttgttcagt gactttgtgt cagttgatca ggattgggaa aacaatccag gaaagggaca gctcaagctg tggggattga gagtatgctg acgtatcaag acaatcagaa atggagaacg ttcttgtctc ggggcattgt atgagaggat acaacttcaa caacccgtac ttccttcgtt ccaatctgca caactatcaa atcactgtgc attggatcac tcagtgcata gatctcaact caactttgtt tggcccatct agctgagttt catctatgtg gagagtcatt tcgaatctcg tcttgcgatt tgtcaatgag caacacctac ctacaatcga ccctacaact ggtaactcta ccagggggtg caatgggatg gctaggaatg gaagcct tc a gatcgctttc ggctttgaag ctaagagatt aactacaaca aatcgtggtc ttgaggaggg gcttgagcaa gcattgacat gc t ttct tgt cctttcttgt ctgccattgc aagaatggga ggatacttga tgccgttgct ctgtgatctt gactcatcag tcaacaactt atctcacatt tcctgaagag ctcactgtca tggactcata acagattgaa caatctggaa agaagatCCC tgggc tact t ctccgtgtat tggagaaaga acacattgat accgaagtct gactgtcttg 120 180 240 300 360 420 480 540 600 660 720 LibC/50708401 speci gacattgctg caactaacaa gttgctcagt tttgacattc tactggggtg atctatggta ttcaggactt ttcaacttac cgtggaagag cctcgcgaag ccgttcctga accattgatc ggcac tt ctg tttggtgact ctaaggtttc tccacaggag ggggagaact agagccaatc agtagcggtg <210> 12 ctttctttcc gggaagtgta tacctacttt tcaacaatct gacatcgtgt gggaggcgaa tgtccaatcc gtggtgttga gtcaagttga gctacagtca caactggtgt cagagaggat tgatcaccgg ttgtatctct gttacgcttc ttggaggcca tgacctctag cagacatcat aactgtacat aaactatgac cactgatcca caatgtgatg taccatcttc gatctctagc tcaggagcct tact ttgcga aggagtagag ttcgttgact tcgtctctgt tgtcttctcc caatcagata tccaggattc tcaagtcaac cagtagagat agttagtgtc aac ct t tcgc tgggatcagt agacaagatt 12/23 aacagaagat ctcdtcaact gaaagctcag actgattggt ttgataggtg ccaagatcct ttgttacaac ttctcaacac gagttaccgc cacgcaacct tggactcatc cctcttgtga acaggagggg atcaactcac gcacgtgtga aacatgcctc tacaccgact gaacaacctc gagatcattc atcccattca tcaatccaca ccatcaggaa tcagtgttgg gaggtaacat tcactttcaa aaccatggcc ccaccaacag ctgaggacaa ttgttcaaag gtagcgcaac aaggcttcag acattcttcg ccatcacaca tagtactcac ttcagaagac tcagcaatcc tctttggtgc tagctgatgc accagttggt gttacaatct tccacacttg acgcaacttc cacatctcct tggacccgtc tgctccacca cttcacgtat ctcagttcca gtctggaaca tcttaccaac agtttggggg acgcaacacc aagataccgt aggagctgca tatggagata cttctcattc aggttccatc aacactcgag 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 <21 1 >3489 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 12 atggaggaga gtacttcttg cttgttcagt gactttgtgt cagttgatca ggattgggaa aacaatccag gaaagggaca gctcaagctg tggggattga gagtatgctg acgtatcaag gacattgctg caactaacaa gttgctcagt tttgacattc tactggggtg at c tatggt a ttcaggactt ttcaacttac cgtggaagag cctcgcgaag ccgttcctga accattgatc ggcacttctg tttggtgact c taaggt tt c tccacaggag ggggagaact agagccaatc agtagcggtg gcagagtctg cagattgggc acaatcagaa atggagaacg ttcttgtctc ggggcattgt atgagaggat acaacttcaa caacccgtac ttccttcgtt ccaatctgca caactatcaa atcactgtgc attggatcac ctttctttcc gggaagtgta tacctacttt tcaacaatct gacatcgtgt gggaggcgaa tgtccaatcc gtggtgttga gtcaagttga gctacagtca caactggtgt cagagaggat tgatcaccgg ttgtatctct gt tacgc tt c ttggaggcca tgacctctag cagacatcat aactgtacat acttggaaag tcaagacaga tcagtgcata gatctcaact caactttgtt tggc ccat ct agctgagttt catctatgtg gagagtcatt tcgaatctcg tcttgcgatt tgtcaatgag caacacctac ctacaatcga aaactatgac cactgatcca caatgtgatg taccatcttc gatctctagc tcaggagcct tactttgcga aggagt agag ttcgttgact tcgtctctgt tgtcttctcc caatcagata tccaggattc tcaagtcaac cagtagagat agttagtgtc aacctttcgc tgggatcagt agacaagatt agcacagaag tgtgactgac ccctacaact ggtaactcta ccagggggtg caatgggatg gctaggaatg gaagccttca gat cgct tt c ggctttgaag ctaagagatt aactacaaca aatcgtggtc ttgaggaggg aacagaagat ctcatcaact gaaagctcag actgattggt ttgataggtg ccaagatcct ttgttacaac ttctcaacac gagttaccgc cacgcaacct tggactcatc cctcttgtga acaggagggg atcaactcac gcacgtgtga aacatgcctc tacaccgact gaacaacctc gagatcattc gcggtgaatg tatcacatcg gcttgagcaa gcattgacat gctttcttgt cctttcttgt ctgccattgc aagaatggga ggatacttga tgccgttgct ctgtgatctt gactcatcag tcaacaactt atctcacatt atcccattca tcaatccaca ccatcaggaa tcagtgttgg gaggtaacat tcactttcaa aaccatggcc ccaccaacag ctgaggacaa ttgttcaaag gtagcgcaac aaggcttcag acattcttcg ccatcacaca tagtactcac ttcagaagac tcagcaatcc tctttggtgc tagctgatgc ctctgttcac at cgcgt t tc tcctgaagag ctcactgtca tggactcata acagattgaa caatctggaa agaagatccc tgggc tact t ctccgtgtat tggagaaaga acacattgat accgaagtct gactgtcttg accagttggt gttacaatct tccacacttg acgcaacttc cacatctcct tggacccgtc tgctccacca cttcacgtat ct cag t tcc a gtctggaaca tcttaccaac agtttggggg acgcaacacc aagataccgt aggagctgca tatggagata cttctcattc aggttccatc aacactcgag ttcgtccaat caaccttgtt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 LibC/50708401 speci 13/23 gagtgcctct catgctaagc aacaggcaac gatgtgttca tacctgtacc ggttacatcg gagacagtca ggcaagtgtg ctgaacgaag aggctaggca gtgaagaggg attgtgtaca aggctccaag attcgggaag gaagagttag aagaatggtg gaagaacaga caagaagttc ggatacggag ttcagcaact actgcgactc gcctatgaga tacaccgatg ccgttaccag tggattgaga atggaggaa <210> 13 <211> 1163 ctgatgagtt gacttagtga tagatcgtgg aggagaacta agaagataga aggacagtca atgtgcctgg cccatcactc acctcggtgt atctggagtt ctgagaagaa aagaagccaa ctgataccaa c t tac ct tcc aagggcgcat acttcaacaa acaatcaccg gtgtctgtcc aaggttgcgt gcgtcgagga aagaggagta gcaactcttc gacgtaggga ccggctatgt ttggagaaac ctgtttggat tgagcggaac atggagggga tgt tacgct c tgaatcgaaa agaccttgag gacgggttca acaccacttc gtgggtgatc tctagaagag gtggagggac agaaagcgtt catagctatg tgaacttagc cttcactgca tggcctatcc ctctgtcctt tggtcgtggc caccatacac ggaagtctac tgagggtact tgtacccgct caatccttgc caccaaagag ggaaggaaca gagaagaagg ttgcttcaag agtacggaca ttgggtacct ctcaaagcct atctacctca ctctggccac tccttggaca ttcaagatca aaaccacttg aagagagaga gacgctctgt attcatgctg gtgattccgg ttctccttgt tgctggaatg gttgttcctg tacattcttc gagattgaga ccaaacaaca tacacttctc gactatgcat gaatctaaca ttagagtact ttcattgttg agttgtccga atcccaactt tcaccattca ttgatgagtg acacaagata tcagatacaa tt t cagc ccc tagacgttgg agactcaaga ttggagaagc agttggaatg ttgtgaactc cagacaaacg gtgtcaatgc atgatgcgag tgaaagggca agtgggaagc gtgttaccgc acaacaccga ccgtaacttg gcaatcgagg cagcctatga gaggctatgg ttccagaaac atagcgtgga gaaggtcaaa tcgcgggatc aggaggtgat ctatccaaca ccagttgaga cgccaaacat aagtcccatc ctgtaccgac tggccatgcc cc tcgc taga ggaaacaaac tcagtatgat cgttcatagc tgc tat ctt t gaatgtcatc cgtagatgta agaagtttca gtacaaagaa cgagctgaag caatgactac atacgatgga ggagaaggct ggactacaca cgacaaggtt gttacttctg 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3489 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 13 Met Giu Giu Asn Asn 1 5 Asn Pro Giu Giu Val Gin Asn Gin Cys Ile 10 Giu Pro Tyr Asn Cys Leu Ser Leu Leu Asp Arg Ile Ser 20 Asp Ser Ser Ile Phe Val Pro Ile Ser Leu Ser Leu Val Gin Phe Thr Gly Asn Val Ser Asn Phe Val Trp Gly Gly Gly Vai Gly Leu Glv Ile Ile Leu Vai Gly Pro Ser Arg Trp Asp Aia Val Gin Ile Giu Leu Ile Asn Ile Ala Giu Phe Phe Arg Asn Ala Ala Ile Ala Asn Leu Phe Lys Giu 115 Val Ile Asp Giu 100 Trp Leu Gly Asn Asn Ile Tyr Giu Giu Asp Pro 120 Leu Asn Pro Ala Vai Giu Ala 110 Arg Thr Arg Arg Asp Ile Arg Phe Arg Asp Giy Leu 130 Pro Ser Leu 140 Leu Phe Arg Ile Ser 150 Leu Phe Giu Val Leu Ser Val Tyr 160 Gin Ala Ala His Leu Ala Ile 170 Ile Arg Asp Ser Val Ile 175 Phe Gly Giu Asn Arg Leu 195 Arg 180 Ile Gly Leu Thr Asn Val Asn Giu Asn Tyr 190 Cys Ala Asn Arg His Ile Asp 200 Tyr Ala Asp LibC/50708401 speci 14/23 Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gin Asp 210 215 220 Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val Leu 225 230 235 240 Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro Ile 245 250 255 Gin Pro Val Gly Gin Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu Ile 260 265 270 Asn Phe Asn Pro Gin Leu Gin Ser Val Ala Gin Leu Pro Thr Phe Asn 275 280 285 Val Met Glu Ser Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile Leu 290 295 300 Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn Phe 305 310 315 320 Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly Asn 325 330 335 Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gin Glu Pro Pro Arg 340 345 350 Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro Thr 355 360 365 Leu Arg Leu Leu Gin Gin Pro Trp Pro Ala Pro Pro Phe Asn Leu Arg 370 375 380 Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr Tyr 385 390 395 400 S 25 Arg Gly Arg Gly Gin Val Asp Ser Leu Thr Glu Leu Pro Pro Glu Asp 405 410 415 Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His Ala 420 425 430 Thr Phe Val Gin Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val Val 435 440 445 Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp Pro 450 455 460 Glu Arg Ile Asn Gin Ile Pro Leu Val Lys Gly Phe Arg Val Trp Gly 465 470 475 480 Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu A 485 490 495 Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gin Val Asn Ile Asn 500 505 510 Ser Pro Ile Thr Gin Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser Ser 40 515 520 525 Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly Val 530 535 540 Gly Gly Gin Val Ser Val Asn Met Pro Leu Gin Lys Thr Met Glu Ile 545 550 555 560 Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn 565 570 575 Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gin 580 585 590 Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp 595 600 605 Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Leu Glu Ala Glu Ser Asp 610 615 620 Leu Glu Arg Ala Gin Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn 625 630 635 640 Gin Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Arg Val 645 650 655 Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys 660 665 670 Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu 675 680 685 Arg Asn Leu Leu Gin Asp Pro Asn Phe Arg Gly Ile Asn Arg Gin Leu 690 695 700 Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gin Gly Gly Asp LibC/507084Dlspeci 15/23 705 710 715 720 Asp Val Phe Lys Giu Asn Tyr Val Thr Leu Leu Gly Thr Phe Asp Glu 725 730 735 Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys Ile Asp Glu Ser Lys Leu Lys 740 745 750 Ala Tyr Thr Arg Tyr Gin Leu Arg Gly Tyr Ile Glu Asp Ser Gin Asp 755 760 765 Leu Giu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Giu Thr Val Asn 770 775 780 Val Pro Gly Thr Giy Ser Leu Trp Pro Leu Ser Ala Pro Ser Pro Ile 785 790 795 800 Giy Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp Val 805 810 815 Gly Cys Thr Asp Leu Asn Giu Asp Leu Gly Val Trp Val Ile Phe Lys 820 825 830 Ile Lys Thr Gin Asp Gly His Ala Arg Leu Gly Asn Leu Giu Phe Leu 835 840 845 Giu Giu Lys Pro Leu Val Gly Giu Ala Leu Ala Arg Val Lys Arg Ala 850 855 860 Giu Lys Lys Trp Arg Asp Lys Arg Giu Lys Leu Giu Trp Giu Thr Asn 865 870 875 880 Ile Vai Tyr Lys Giu Ala Lys Giu Ser Vai Asp Ala Leu Phe Val Asn *885 890 895 Ser Gin Tyr Asp Arg Leu Gin Ala Asp Thr Asn Ilie Aia Met Ile His 25900 905 910 '~Ala Aia Asp Lys Arg Val His Ser Ile Arg Giu Ala Tyr Leu Pro Glu 915 920 925 Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Giu Giu Leu Giu *930 935 940 Gly Arg Ile Phe Thr Aia Phe Ser Leu Tyr Asp Ala Arg Asn Vai Ile 945 950 955 960 Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly *to965 970 975 His Val Asp Val Giu Glu Gin Asn Asn His Arg Ser Val Leu Val Val 980 985 990 Pro Giu Trp Giu Ala Giu Val Ser Gin Giu Val Arg Val Cys Pro Gly 995 1000 1005 Arg Giy Tyr Ile Leu Arg Val Thr Ala Tyr Lys Giu Gly Tyr Gly Glu 1010 lois 1020 Gly Cys Val Thr Ilie His Giu Ile Giu Asn Asn Thr Asp Giu Leu Lys *1025 1030 1035 1040 Phe Ser Asn Cys Val Glu Giu Giu Val Tyr Pro Asn Asn Thr Val Thr 1045 1050 1055 Cys Asn Asp Tyr Thr Ala Thr Gin Giu Giu Tyr Giu Gly Thr Tyr Thr 1060 1065 1070 Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Giu Ser Asn Ser Ser Val 1075 1080 1085 Pro Ala Asp Tyr Ala Ser Ala Tyr Giu Giu Lys Ala Tyr Thr Asp Gly 1090 1095 1100 Arg Arg Asp Asn Pro Cys Giu Ser Asn Arg Gly Tyr Gly Asp Tyr Thr 1105 1110 1115 1120 Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Giu 1125 1130 1135 Thr Asp Lys Val Trp Ile Giu Ilie Gly Giu Thr Giu Gly Thr Phe Ile 1140 1145 1150 Val Asp Ser Val Giu Leu Leu Leu Met Giu Giu 1155 1160 <210O> 14 <21 1> 3558 <212> DNA <213> Artificial Sequence LibC/507084D1 speci 16/23 <220> <223> Synthetic B.t. toxin gene <400> 14 atgacttcta tctaaccatt attgctgagg aacattgctg ttctactctt cttgagcatg cttgctagac tggcttgaga cttgagcttg cttctcatgg ttgttcggat gttgagaaga aaccttagag actcttggag atgaacactt gctccatctg atcgaggctg ttctctgttc cttgagtcta tctatcaacc gctggaatca tggagaaacc gttggtaccc aactacgagt agagctccag gattctatca 30 gtttctggac ctttctatgg tacgctgctt caaggattcc gctgagttcc aacaacgctg actctcgagg tcgtccaatc aaccttgttg aaggtcaaac cgcgggatca ggaggtgatg tatccaacat cagttgagag gccaaacatg agtcccatcg tgtaccgacc ggccatgcca ctcgctagag gaaacaaaca cagtatgata gttcatagca gctatctttg aatgtcatca gtagatgtag gaagtttcac tacaaagaag gagctgaagt aatgactaca tacgatggag gagaaggctt gactacacac gacaaggttt acagaaagaa ctgctcagat gaaacaacat gaagaatcct tccttgttgg ttgagcagtt ttcaaggact acagagatga acttcttgaa tgtacgctca ctgagttcgg ctagagagta gaactaacgc ttcttgatct ctgctcaact gattcgcttc cagtgatcag tttctagatg gaactatcag cagttactct acattcttct cattgaactc agttgttcga cttactctca tgtactcttg ctcagattcc caggattcac gattgaactt ctcaaactat catctactat cagttggaat gaagacaaac cagagtctga agattgggct agtgcctctc atgctaagcg acaggcaact atgtgttcaa acctgtacca gttacatcga agacagtcaa gcaagtgtgc tgaacgaaga ggctaggcaa tgaagagggc ttgtgtacaa ggctccaagc ttcgggaagc aagagttaga agaatggtga aagaacagaa aagaagttcg gatacggaga t cagc aac tg ctgcgactca cctatgagag acaccgatgg cgttaccagc ggattgagat cgagaacgag gaacctttct tgatccattc tggagttctt agagctttgg gattcgtcaa tggaaactct tgctagaact cgctatgcca agctgctaac acttacttct ctctgactac tgagtcttgg tgttgccttg tactagagag tactaactgg accaccacat gtctaacact aggat ctct t tcagttcact tactactcca tcttagaggt ttctgagact tagactttct gactcataga acttgtgaag tggaggagac caacaacact ggttcttaga gtctgctaac ctctgcttct tttccacttc cttggaaaga caagacagat tgatgagttc acttagtgat agatcgtgga ggagaactat gaagatagat ggacagtcaa tgtgcctggg ccatcactca cctcggtgtg tctggagttt tgagaagaag agaagccaaa tgataccaac ttaccttcct agggcgcatc cttcaacaat caatcaccgc tgtctgtcct aggttgcgtc cgtcgaggag agaggagtat caactcttct acgtagggac cggctatgtc tggagaaacg atcatcaacg actgatgcta gtttctgctt ggagt tcc at cctaggggaa caagttactg ttcagagctt agatctgtgt ttgttcgcta cttcatcttc caagagattc tgcgctaggt cttagataca ttcccatctt atctacactg ttcaacaaca cttcttgact cagtacatga tctacttcta tctagagatg gtgaacggag tccttgttgt gagcttccac aacattcgtt tctgctgata tctttcaact atcatcagaa tctcttcaaa gttactgttg gagtctctta ggatctcaaa gacaagattg gcacagaagg gtgactgact tgtttggatg gagcggaact tggaggggaa gttacgctct gaatcgaaac gaccttgaga acgggttcac caccacttct tgggtgatct ctagaagaga tggagggaca gaaagcgttg atagctatga gaacttagcg ttcactgcat ggc ctat cc t tctgtccttg ggtcgtggct accatacacg gaagtctacc gagggtactt gtacccgctg aatccttgcg accaaagagt gaaggaacat ctctttctat gaatcgagga ctactgttca tcgctggaca gagatccttg agaacactag accaacaatc tgtacactca tcagaaacca ttcttcttag aaagatacta ggtacaacac accagttcag acgatactag atccaatcgg acgctccatc tcccagagca actactgggt ctcatggaaa tgtacagaac ttccttgggc acaccattgg cagagactac tgatctctgg gaactaacac tgaactctgg ctaacgtgaa gatacagagt gaggatctac cttctcaatc c tgc tggaat agttcattcc cggtgaatgc atcacatcga agaagaagga tgcttcaaga gtacggacat tgggtacctt tcaaagccta tctacctcat tctggccact ccttggacat tcaagatcaa aac cact tgt agagagagaa acgctctgtt ttcatgctgc tgattccggg tctccttgta gctggaatgt ttgttcctga acattcttcg agattgagaa caaacaacac acacttctcg actatgcatc aatctaacag tagagtactt tcattgttga cccagctgtt ttctctttgc aactggaatc gattgcttct ggagatcttc agatactgct tcttgaggat gtacattgct agaggttcca agatgctagc cgagagacaa tggattgaac aagagatctt agtgtaccct aagaactaac tttctctgct acttactatc tggacataga cactaacact tgagtctttc tagattcaac atacactgga tgagagacca aaacactctt catctcttct aacttctgtt cggatctgtt tagagttaga tactttcgat tttcagattc ctctatctct aatcactgct tctgttcact tcgcgtttcc gttgtccgag tcccaacttt caccattcaa tgatgagtgc cacaagatac cagatacaac ttcagcccca agacgttggc gactcaagat tggagaagcc gttggaatgg tgtgaactct agacaaacgc tgtcaatgct tgatgcgagg gaaagggcac gtgggaagca tgttaccgcg caacaccgac cgtaacttgc caatcgagga agcctatgag aggctatggg tccagaaacc tagcgtggag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 LibCI5O7O84D1 speci 17/23 ttacttctga tggaggaa <210> <211> 1186 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 3558 Met 1 Ile Ala Pro Arg Phe Trp Thr Asn Arg 145 Leu Gin Leu Thr Arg 225 Asn Arg Ser Arg Phe 305 Ile Gin Met Ser Val Thr Ser Pro Ala Arg Ile Phe Val Ile Leu Tyr Ser Glu Ile Glu Asn 115 Ser Phe 130 Asp Asp Glu Leu Glu Val Leu Leu 195 Ser Gin 210 Glu Tyr Leu Arg Arg Asp Tyr Asp 275 Glu Ile 290 Ala Ser Glu Ala Leu Thr Asn Tyr 355 Leu Ser 370 Thr Leu Asn Arg 5 Val Ser Glu Asp Ser Ala Gly Val Phe Leu 85 Phe Leu 100 Thr Arg Arg Ala Ala Arg Asp Phe 165 Pro Leu 180 Leu Arg Glu Ile Ser Asp Gly Thr 245 Leu Thr 260 Thr Arg Tyr Thr Thr Asn Ala Val 325 Ile Phe 340 Trp Val Thr Ser Gin Phe Lys Asn Ser Ser Leu 70 Val Glu Asp Tyr Thr 150 Leu Leu Asp Gin Tyr 230 Asn Leu Val Asp Trp 310 Ile Ser Gly Thr Thr Asn His Leu Thr 55 Gly Gly His Thr Gin 135 Arg Asn Met Ala Arg 215 Cys Ala Gly Tyr Pro 295 Phe Arg Val His His 375 Ser Glu Asn Ser Ala 25 Cys Ile 40 Val Gin Val Pro Glu Leu Val Glu 105 Ala Leu 120 Gin Ser Ser Val Ala Met Val Tyr 185 Ser Leu 200 Tyr Tyr Ala Arg Glu Ser Val Leu 265 Pro Met 280 Ile Gly Asn Asn Pro Pro Leu Ser 345 Arg Leu 360 Gly Asn Arg Asp Glu 10 Gin Ala Thr Phe Trp 90 Gin Ala Leu Leu Pro 170 Ala Phe Glu Trp Trp 250 Asp Asn Arg Asn His 330 Arg Glu Thr Val Ile I Met A Glu G Gly I Ala G 75 Pro A Leu I Arg L Glu A 1 Tyr T 155 Leu P Gin A Gly S Arg G 2 Tyr A 235 Leu A Leu V Thr S Thr A 3 Ala P 315 Leu L Trp S Ser A Asn TI 3 Tyr A 395

I

l e

E

s 4 e 1 2 s r a e s 0 r e h 8 r r Le Asn sn Leu Ly Asn Le Asn Ly Gin rg Gly e Arg !u Gin 125 sp Trp L0 ir Gin ie Ala a Ala ir Glu 205 n Val !0 n Thr -g Tyr 1I Ala r Ala 285 n Ala i0 o Ser u Asp r Asn g Thr 365 Ir Ser 0 g Thr Ala Ser Asn Ile Ile Arg Gin 110 Gly Leu Tyr Ile Asn 190 Phe Glu Gly Asn Leu 270 Gin Pro Phe Phe Thr 350 Ile Ile Glu Leu Ser Thr Asp Ile Asp Ala Gly Ala Ser Asp Pro Gin Val Leu Gly Glu Asn Ile Ala 160 Arg Asn 175 Leu His Gly Leu Lys Thr Leu Asn 240 Gin Phe 255 Phe Pro Leu Thr Ser Gly Ser Ala 320 Pro Glu 335 Gin Tyr Arg Gly Asn Pro Ser Phe 400 385 390 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp LibC/507084D1speci 18/23 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gin Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ser Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ser Ser Asp Ser Ile Thr Gin Ile Pro Leu Val Lys Ser Phe 500 505 510 Asn Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr Gly 515 520 525 Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val Leu Ser Met Gly 530 535 540 Leu Asn Phe Asn Asn Thr Ser Leu Gin Arg Tyr Arg Val Arg Val Arg 545 550 555 560 Tyr Ala Ala Ser Gin Thr Met Val Leu Arg Val Thr Val Gly Gly Ser 565 570 575 Thr Thr Phe Asp Gin Gly Phe Pro Ser Thr Met Ser Ala Asn Glu Ser 580 585 590 Leu Thr Ser Gin Ser Phe Arg Phe Ala Glu Phe Pro Val Gly Ile Ser 595 600 605 Ala Ser Gly Ser Gin Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala Gly 610 615 620 Arg Gin Thr Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr Ala 625 630 635 640 Thr Leu Glu Ala Glu Ser Asp Leu Glu Arg Ala Gin Lys Ala Val Asn 645 650 655 S. Ala Leu Phe Thr Ser Ser Asn Gin Ile Gly Leu Lys Thr Asp Val Thr 660 665 670 Asp Tyr His Ile Asp Arg Val Ser Asn Leu Val Glu Cys Leu Ser Asp 675 680 685 Glu Phe Cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His 690 695 700 Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gin Asp Pro Asn Phe 705 710 715 720 Arg Gly Ile Asn Arg Gin Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp 725 730 735 Ile Thr Ile Gin Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr 740 745 750 Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gin Lys 755 760 765 Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gin Leu Arg Gly 770 775 780 Tyr Ile Glu Asp Ser Gin Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn 785 790 795 800 Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro 805 810 815 Leu Ser Ala Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His His 820 825 830 Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu 835 840 845 Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gin Asp Gly His Ala Arg 850 855 860 Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala 865 870 875 880 Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu 885 890 895 Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser LibC/507084D1speci 19/23 Val Asp Ala 915 Thr Asn Ile 900 Leu Ala Phe Val Asn Ser 920 Met Ile His Ala 905 Gin Ala Tyr Asp Arg Leu 925 Asp Lys Arg Val 910 Gin Ala Asp His Ser Ile 930 Arg Giu 945 Ala Ile 935 Glu 940 Pro Ala Tyr Leu Phe Giu Giu 965 Leu Ser Val Ile 955 Phe Giy Vai Asn Al a 960 Giu Giy Arg Thr Ala Phe Ser Leu 975 Tyr Asp Ser Cys His Arg 1010 Giu Val 1025 Tyr Lys Asn Asn Ala Arg Asn 980 Val Ile Lys Asn 985 Asp Phe Asn Trp Asn Val Lys Gly His Vai 995 1000 Ser Val Leu Val Vai Pro Giu 1015 Arg Val Cys Pro Gly Arg Gly 1030 Giu Gly Tyr Gly Giu Gly Cys Asp Val Glu Glu 1005 Glu Asn Gly Leu 990 Gin Asn Asn Val Ser Gin Trp Giu Tyr Ile 1035 Val Thr Al a 1020 Leu Arg Val Thr Ala 1040 Glu Ile His Giu Ile Thr 1045 1050 Asp Giu Leu Lys Phe Ser Asn Cys Val Giu 1060 1065 Tyr Pro Asn Asn Thr Val Thr Cys Asn 1075 1080 Glu Tyr Giu Giy Thr Tyr Thr Ser Arg Asp Tyr Thr Ala 1085 Asn Arg Gly Tyr 1055 Giu Giu Val L070 Thr Gin Giu Asp Gly Ala 1090 1095 1100 Tyr Glu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Giu 1105 1110 1115 1120 Giu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Giu Ser Asn 1125 1130 1135 Arg Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys 1140 1145 1150 Giu Leu Giu Tyr Phe Pro Giu Thr Asp Lys Val Trp Ile Giu Ile Gly 1155 1160 1165 Giu Thr Giu Gly Thr Phe Ile Val Asp Ser Val Giu Leu Leu Leu Met 1170 1175 1180 Giu Giu 1185 <210> 16 <21 1> 1929 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 16 atgacttcta tctaaccatt attgctgagg aacattgctg ttctactctt cttgagcatg cttgctagac tggcttgaga cttgagcttg cttctcatgg ttgttcggat gttgagaaga aaccttagag actcttggag atgaacactt acagaaagaa ctgctcagat gaaacaacat gaagaatcct t cc ttgt tgg ttgagcagtt ttcaaggact acagagatga acttcttgaa tgtacgctca ctgagttcgg ctagagagta gaactaacgc ttcttgatct ctgctcaact cgagaacgag gaac ctt t ct tgatccattc tggagttctt agagctttgg gattcgtcaa tggaaactct tgctagaact cgctatgcca agctgctaac acttacttct ctctgactac tgagtcttgg tgttgccttg tactagagag atcatcaacg actgatgcta gtttctgctt ggagttccat cctaggggaa caagttactg t t cagagct t agatctgtgt ttgttcgcta cttcatcttc caagagattc tgcgctaggt cttagataca ttcccatctt atctacactg ctctttctat gaatcgagga ctactgttca tcgctggaca gagatccttg agaacactag accaacaatc tgtacactca tcagaaacca ttcttcttag aaagatacta ggtacaacac accagttcag acgatactag atccaatcgg cccagctgtt t t ct ct ttgc aactggaatc gattgcttct ggagatcttc agatactgct tcttgaggat gtacattgct agaggttcca agatgctagc cgagagacaa tggattgaac aagagatctt agtgtaccct aagaactaac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 LibC/507084D1 speci gctccatctg atcgaggctg ttctctgttc cttgagtcta tctatoaacc gctggaatca tggagaaaoo gttggtaccc aactacgagt agagctocag gattctatca gtttctggac ctttotatgg tacgctgctt caaggattcc gotgagttcc aacaacgctg actctcgag <210O> 17 <21 1 >643 gattcgcttc oagtgatcag tttctagatg gaactatcag cagt taotc t acattcttct oattgaactc agttgttoga cttactctca tgtactcttg ctcagattoo caggattoac gattgaactt ctcaaactat catctactat cagttggaat gaagacaaac tactaactgg acoaccacat gtctaacaot aggatct ott tcagttcact tactactcca tot tagaggt ttctgagact t agao t ttot gaotoataga aottgtgaag tggaggagao oaaoaaoaot ggttottaga gtotgotaao ototgottot tttccacttc 20/23 ttoaaoaaoa ottottgaot oagtaoatga totaottota totagagatg gtgaaoggag toottgttgt gagottooao aaoattogtt totgotgata tot tto aao t at oatcoagaa totottoaaa gttaotgttg gagtototta ggatotoaaa gacaagattg acgotooato tcooagagoa actaotgggt otoatggaaa tgtaoagaao ttoottgggo acaooattgg oagagaotao tgatototgg gaaotaaoao tgaaototgg otaaogtgaa gataoagagt gaggatotao ottotoaato otgotggaat agt toat too tttototgot aottaotato tggaoataga oaotaaoaot tgagtottto tagattoaao ataoaotgga tgagagaooa aaaoaotott oatotottot aaottotgtt oggatotgtt tagagttaga taotttogat t tto agat to ototatotot aatcactgct 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1929 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <220> <223> Toxin encoded by synthetic B.t. gene <400> 17 Met Thr Ser Asn Arg 1 5 Ile Pro Ala Val Ser Lys Asn Giu Asn Giu 10 Gin Ile Ile Asn Ala Leu Ser Asn His Ser Met Asn Leu Glu Ala Arg Ile Pro Phe Val Asp Ser Leu Cys Vali Ala Giu Gly Ser Thr Asp Asn Ile Asp Ile Ala Gly Ser Ala Ser Gin Thr Giy Arq Ile Ile Gly Leu Gly Vai Phe Leu Val Val Pro Phe Gin Ile Ala Tyr Ser Phe Gly Giu Leu Trp Gin Arg Giy Arg Asp Pro Trp Giu Ile Thr Giu Asn 115 Asn Ser Phe Phe 100 Thr Giu His Val Leu Ile Arg Arg Asp Thr Ala 120 Gin Ala Arg Leu Gin Gin Gin Val 110 Gly Leu Gly Leu Giu Asn Arg Ala Tyr Ser Leu Giu 130 Arg Asp Asp 140 Thr Asp Ala Arg 145 Leu Thr 150 Leu Ser Val Leu Tyr 155 Leu Gin Tyr Ile Giu Leu Asp Asn Ala Met Pro 170 Ala Phe Ala Ile Arg Asn 175 Gin Giu Val Leu Leu Leu 195 Thr Ser Gin Pro 180 Leu Leu Met Val Tyr 185 Leu Gin Ala Ala Arg Asp Ala Ser 200 Tyr Phe Gly Ser Asn Leu His 190 Phe Gly Leu Giu Lys Thr Giu Ile Gin Tyr Giu Arg 210 Arq Giu Gin 220 Asn Tyr Ser Asp 225 Asn Tyr 230 Asn Ala Arg Trp Tyr 235 Leu Thr Gly Leu Leu Arg Gly Thr Ala Giu Ser Trp Arg Tyr Asn Gin LibC/507084D1 speci 21/23 245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Val Tyr Pro Met Asn Thr Ser Ala Gin Leu Thr 275 280 285 Arg Giu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Giu Ala Ala Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro Giu 325 330 335 Gin Leu Thr Ile Phe Ser Val Leu Ser Arg Trp Ser Asn Thr Gin Tyr 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Giu Ser Arg Thr Ile Arg Gly 355 360 365 Ser Leu Ser Thr Ser Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gin Phe Thr Ser Arg Asp Val Tyr Arg Thr Giu Ser Phe 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Vai Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gin Leu Phe Asp Ser 435 440 445 *Giu Thr Giu Leu Pro Pro Giu Thr Thr Giu Arg Pro Asn Tyr Giu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ser Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Tr485 490 495 h Ile Ser Ser Asp Ser Ile Thr Gin Ile Pro Leu Val Lys Ser Phe 500 505 510 Asn Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr Gly 515 520 525 Gly Asp Ile Ile Arg Thr Asn Val Asn Giy Ser Val Leu Ser Met Giy *530 535 540 Leu Asn Phe Asn Asn Thr Ser Leu Gin Arg Tyr Arg Val Arg Val Arg 545 550 555 560 Tyr Ala Ala Ser Gin Thr Met Val Leu Arg Val Thr Val Gly Gly Ser ***565 570 575 Thr Thr Phe Asp Gin Gly Phe Pro Ser Thr Met Ser Ala Asn Giu Ser 580 585 590 Leu Thr Ser Gin Ser Phe Arg Phe Ala Giu Phe Pro Val Gly Ile Ser 595 600 605 Ala Ser Gly Ser Gin Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala Gly 610 615 620 Arg Gin Thr Phe His Phe Asp Lys Ile Giu Phe Ile Pro Ile Thr Ala 625 630 635 640 Thr Leu Giu <210O> 18 <21 1> 1965 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 18 atgaaccgca acaacccgaa cgagtacgag atcatcgacg ccccgtactg cggctgcccg tccgacgacg acgtgcgcta cccgctcgcc tccgacccga acgccgcctt ccagaacatg 120 LibC/507084D1 speci aactacaagg aacctctcca ct cggc t tcc aaccagctct gagctcatcg ggcctccacg aacggcgctc acccgcatgc accgtgtacg ggcgcccgct cgcacccgca cgcggcacca atggcgatgg gccaacccgc aaccagggca aacgccttca aaccgctaca cagtcccagc aacacccgcc ttcggcaacc gtgatgtccg gagctcccgc aacaccaccc cgggaggacg gtcaaggcct ggggacatcc gccccgctca gtgaacctct aacggctccc tcccagtccg tacgtggaca <210> 19 <21 1> 655 agtacctcca tcaacccgcg tgggcgtgcc ggccgaccaa accagaagat actactacga gcgccaacct cgtcgttcgg cccaggccgc ggggcctcca tctacaccaa acaccgagtc acctcgtggc agctcacccg tctgccgccg tcaggccgcc ccgccccgac acgccaacaa tcttcaacac tctacgccaa agatcaccaa tggagaacaa agggcggccc tcgacttcac ccgagatcgg tccgccgcac cccagcagta tcgtgaacaa tcacctacga acaccaccct agctcgagat gacctacgac cgacgtcctc gttcgccggc cgacaacgcc ctccgcccag ggagtacctc cgtgacccag gacggggcca caacctccac gcagggccag ccactgcgtg ctggctcaac CCtCttCCCg cgagatctac ctggggcaac gcacctcttc caccaactcc cccgaccacc caccaacggc cctctacggc cgccgccaac caacttcaac actcgcgacg caacaccatc cggcggcacg cgacggcggc ccgcatccgc Ct Ccgc tgc c gtccttcaac ccgcctcaac cgtgccgatc 22/23 ggcgactaca cagaccggca cagctcgtga gtgtgggagg gtggtgcgca gcggcgctgg aggttcgaga gggagccaga CtCCtCCtCC atcaacctct gagacctaca taccaccgct ttctacaacg accgacccga aacccgtaca gagcgcctca ttcctcgact tacgagacct gccagggcca gtgtCCtCCC acctgccgcc CtCCtCtCCC ctggggttcg accgccgacc acggtcgtca gctgtgggqa ctccgctacg ggcttcaccc accctggagg atcttcccgt aacccgaccc cc ggC t ccct tcaacatcgt ccttctacac cgttcatggc acgccctcga aggagtggct acctccacac gggacgccgt tcaaggacgc acttcaacgc accgcggcct tccgcaggga tgcgccagta tcgtgtacaa acaccttctc accgcctcac actggtccgg cctacggcca tcgactccag tcaacatctt aggacctcac acgtgacctt tcccgaccta gcatcacgca aggggccggg ccatccgcgc cctccaccac tcccgtccac tgacgcacac ccatcagcgg gcgag catcaacccg ggggcgcatc CttCCtCCtC ccagatcgag cgacctcacc ggagaggccg cgccttcgtg cgcgc tCCt C cgagatctac ccagcaggag ggaggacgtg gatgaccctc cccgaacggc cccgccggcc cgagctggag catctcccgc ccacaccctg gatcacctcc ggcgcgcaat cccgaccggc caccaccgag CCtCCgCttC cgtgtggacc gc tcc cgtgg cttcaccggg caacgtgaac ctccttcgtg gatggcccag catccgcttc ccaggaggtg 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1965 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 19 Met Asn 1 Cys Gly Arg Asn Asn Pro Asn Giu Tyr Glu 10 Arg Ile Ile Asp Ala Pro Tyr Cys Pro Ser Asp Asp Asp Tyr Pro Leu Pro Asn Ala Tyr Asp Gly Ala Phe Gin Asn Met Ser Tyr Lys Giu Ala Ser Asp Leu Gin Thr Leu Ser Ile Asp Tyr Thr Leu Ile Asn Asn Pro Pro Ile Arg Asp Val Leu Leu Val1 Thr Gly Ile Val Gly Arg Giy Phe Leu Pro Phe Ala Gly Thr Leu Val Thr Phe Tyr Thr Phe Leu Leu 100 Met Gin Leu Trp Asn Asp Asn Giu Ala Phe 115 Ala Gin Val Ala Gin Ile Giu 120 Leu Leu Ile Asp Ala Val Trp 110 Lys Ile Ser Leu His Asp Val Arg Asn Asp Asp Leu 130 Tyr Thr 140 Trp Tyr 145 Asn Giu Giu Tyr Leu 150 Asn Ala Leu Glu Glu Leu Giu Arg Pro 160 His Gly Ala Arg Ala Leu Val Thr Gin Phe Giu Asn Leu LibC/50708401 speci 23/23 165 170 175 Thr Ala Phe Val Thr Arg Met Pro Ser Phe Gly Thr Gly Pro Gly Ser 180 185 190 Gin Arg Asp Ala Val Ala Leu Leu Thr Val Tyr Ala Gin Ala Ala Asn 195 200 205 Leu His Leu Leu Leu Leu Lys Asp Ala Glu Ile Tyr Gly Ala Arg Trp 210 215 220 Gly Leu Gin Gin Gly Gin Ile Asn Leu Tyr Phe Asn Ala Gin Gin Glu 225 230 235 240 Arg Thr Arg Ile Tyr Thr Asn His Cys Val Glu Thr Tyr Asn Arg Gly 245 250 255 Leu Glu Asp Val Arg Gly Thr Asn Thr Glu Ser Trp Leu Asn Tyr His 260 265 270 Arg Phe Arg Arg Glu Met Thr Leu Met Ala Met Asp Leu Val Ala Leu 275 280 285 Phe Pro Phe Tyr Asn Val Arg Gin Tyr Pro Asn Gly Ala Asn Pro Gin 290 295 300 Leu Thr Arg Glu Ile Tyr Thr Asp Pro Ile Val Tyr Asn Pro Pro Ala 305 310 315 320 Asn Gin Gly Ile Cys Arg Arg Trp Gly Asn Asn Pro Tyr Asn Thr Phe 325 330 335 Ser Glu Leu Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe Glu Arg 340 345 350 I Leu Asn Arg Leu Thr Ile Ser Arg Asn Arg Tyr Thr Ala Pro Thr Thr 355 360 365 Asn Ser Phe Leu Asp Tyr Trp Ser Gly His Thr Leu Gin Ser Gin His 370 375 380 Ala Asn Asn Pro Thr Thr Tyr Glu Thr Ser Tyr Gly Gin Ile Thr Ser .385 390 395 400 Asn Thr Arg Leu Phe Asn Thr Thr Asn Gly Ala Arg Ala Ile Asp Ser 405 410 415 Arg Ala Arg Asn Phe Gly Asn Leu Tyr Ala Asn Leu Tyr Gly Val Ser 420 425 430 Ser Leu Asn Ile Phe Pro Thr Gly Val Met Ser Glu Ile Thr Asn Ala 435 440 445 Ala Asn Thr Cys Arg Gin Asp Leu Thr Thr Thr Glu Glu Leu Pro Leu 450 455 460 Glu Asn Asn Asn Phe Asn Leu Leu Ser His Val Thr Phe Leu Arg Phe 465 470 475 480 40 Asn Thr Thr Gin Gly Gly Pro Leu Ala Thr Leu Gly Phe Val Pro Thr 485 490 495 Tyr Val Trp Thr Arg Glu Asp Val Asp Phe Thr Asn Thr Ile Thr Ala 500 505 510 Asp Arg Ile Thr Gin Leu Pro Trp Val Lys Ala Ser Glu Ile Gly Gly 515 520 525 Gly Thr Thr Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu 530 535 540 Arg Arg Thr Asp Gly Gly Ala Val Gly Thr Ile Arg Ala Asn Val Asn 545 550 555 560 Ala Pro Leu Thr Gin Gin Tyr Arg Ile Arg Leu Arg Tyr Ala Ser Thr 565 570 575 Thr Ser Phe Val Val Asn Leu Phe Val Asn Asn Ser Ala Ala Gly Phe 580 585 590 Thr Leu Pro Ser Thr Met Ala Gin Asn Gly Ser Leu Thr Tyr Glu Ser 595 600 605 Phe Asn Thr Leu Glu Val Thr His Thr Ile Arg Phe Ser Gin Ser Asp 610 615 620 Thr Thr Leu Arg Leu Asn Ile Phe Pro Ser Ile Ser Gly Gin Glu Val 625 630 635 640 Tyr Val Asp Lys Leu Glu Ile Val Pro Ile Asn Pro Thr Arg Glu 645 650 655 LibC/507084D1speci

Claims (11)

1. A polynucleotide sequence optimised for expression in a plant, wherein said sequence encodes a pesticidal toxin selected from the group consisting of CrylAc, CrylC, 158C2c and 31G1a.

2. The polynucleotide sequence according to claim 1, wherein said polynucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO 7, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17 and SEQ ID NO 19.

3. A polynucleotide sequence optimised for expression in a plant, wherein said sequence encodes a pesticidal toxin comprising a CrylAb protoxin portion.

4. The polynucleotide sequence according to claim 3, wherein said CrylAb protoxin portion comprises an amino acid sequence shown in SEQ ID NO. 3. The polynucleotide sequence according to claim 1, wherein said polynucleotide sequence is shown in a sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 17 and SEQ ID NO. 18. S 1 6. The polynucleotide sequence according to claim 3, wherein said polynucleotide Ssequence comprises a sequence selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2. A pesticidal toxin comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17 and SEQ ID NO. 20 19.

8. A transformed host cell comprising a polynucleotide sequence optimised for expression in a plant, wherein said sequence encodes a pesticidal toxin selected from the group consisting of CrylAc, CryiC, 158C2c, 31G1a, and toxins comprising a CrylAb protoxin portion.

9. The transformed host cell according to claim 8, wherein said polynucleotide sequence 25 encodes a pesticidal toxin comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17 and SEQ ID NO. 19. The transformed host cell according to claim 8, wherein said cell comprises a polynucleotide sequence shown in a sequence selected from the group consisting of SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 18.

11. A DNA construct comprising a promoter region capable of expression in a plant cell and a polynucleotide sequence optimised for expression in a plant, wherein said polynucleotide sequence is under the control of said promoter region. and wherein said polynucleotide sequence encodes a pesticidal toxin selected from the group consisting of CrylAc, CrylC, 158C2c, 31G1a, and toxins comprising a CrylAb protoxin portion.

12. The DNA construct according to claim 11, wherein said polynucleotide sequence encodes a pesticidal toxin comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17 and SEQ ID NO. 19.

13. The DNA construct according to claim 11, wherein said construct comprises a polynucleotide sequence shown in a sequence selected from the group consisting of SEQ ID NO.1, LibC/507084D1speci 12 SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 18.

14. A method for controlling a pest, wherein said method comprises contacting said pest with a toxin produced by a transformed host, wherein said transformed host comprises a polynucleotide sequence optimised for expression in a plant, and wherein said polynucleotide sequence encodes a pesticidal toxin selected from the group consisting of CrylAc, CryiC, 158C2c and 31Gla, and toxins comprising a CrylAb protoxin portion. The method according to claim 14, wherein said polynucleotide comprises a sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17 and SEQ ID NO. 19.

16. The method according to claim 14, wherein said polynucleotide comprises a sequence shown in a sequence selected from the group consisting SEQ ID NO.1, SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 18. 15 Dated 27 March 2002 SI MYCOGEN CORPORATION Patent Attorneys for the Applicant/Nominated Person SPRUSON&FERGUSON LibC/507084D1speci