AU774176B2 - Plant-optimised genes encoding pesticidal toxins - Google Patents

Description

I

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:- 5845c 1 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 1o 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 Is are applied in areas where pets, farm animals, or children may come into contact with them.

They may also provide health 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 organochlorines, DDT, mirex, kepone, lindane, aldrin, chlordane, aldicarb, and dieldrin; the organophosphates, eg., chlorpyrifos, parathion, malathion, and diazinon; and carbamates.

Stringent new restrictions on the use of pesticides and the elimination of some 25 effective pesticides from the market place could limit economical and effective options for *o controlling costly pests.

Because of the problems associated with the use of synthetic chemical pesticides, ,°there exists 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.

4e.° A biological pesticidal agent that is enjoying increasing popularity is the soil microbe oBacillus thuringiensis The soil microbe Bacillus thuringiensis is a Grampositive, spore-forming bacterium. Most strains of B.t. do not exhibit pesticidal activity.

:35 Some B.t. strains produce, and can be characterised by, parasporal crystalline protein inclusions. These "8-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.

LibC/507084D speci 2 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 5-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. Acad. Sci. USA 78:2893-2897.). U.S. Patent No. 4,448,885 and U.S.

Patent No. 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 endotoxin 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), cryllI (Coleoptera specific), and crylV (Dipteraspecific). 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 25 designations CryV and CryVI have been proposed for two new groups of nematode-active Stoxins.

Many Bacillus thuringiensis 8-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 threedimensional structure of a core segment of a CryIIIA B.t. 6-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, P.E.

35 Dunn, S. Strand, A.I. Aronson [1989] Molecular Microbiology 3:1533-1534; Choma, C.T., 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 LibC/507084D1spec 3 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, o0 2 9 th Annual Meeting, III rd 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 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, U.S. Patent Nos. 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 25 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. 8-endotoxins.

i 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: **o LibC/507084D1speci B.t. Isolate, Toxin, Exemplified Pesticidal U.S. Patent No. (unless otherwise indicated) and/or Gene Activity of Toxin PS81I, 81IA, 81IB2 lepidopteran 5,126,133; 5,188,960 CrylAc lepidopteran Adang et al.. GENBANK Acc. No. M11068 IC/IA(b) chimeric lepidopteran 5,593,881 toxin IF/IA(b) chimeric lepidopteran 5,527,833 toxin PS158C, 158C2c lepidopteran 5,268,172; 5,723,758 PS31G1, 31Gla lepidopteranI W098/00546 However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an 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 plantoptimised 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 S 15 known to those skilled in the art, the polynucleotide sequences described herein can be used S" to transform plants in order to confer pest resistance upon said plants.

Herein disclosed are plant-optimised genes that encode other proteins that are toxic to Spests. Such genes may include those 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- 20 PO, 158C2c-T-PO, and 31Gla-PO.

Also herein disclosed are plant-optimised polynucleotide sequences that encode Cterminal, protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Plant-optimised protoxins described herein include those designated PT-1AB-PO and PT-1A1B-2-PO.

25 Also herein disclosed are 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, 1CIAB-PO, 158C2c-PO, 158C2c- T-PO, and 31Gl a-T-PO. Also herein disclosed are unique, C-terminal amino acid sequences LibC/507084D1speci for protoxin portions (of full-length Bacillus thuringiensis toxins) encoded by the polynucleotide sequences designated PT-1AB-PO and PT-1AB-2-PO.

According to a first embodiment of the invention, there is provided polynucleotide optimised for expression in a plant and which encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, and wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.

6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. According to another embodiment of the invention, there is provided a DNA construct comprising a promoter region capable of expression in a plant cell and a polynucleotide optimised for expression in a plant, wherein said polynucleotide is under the control of said promoter region, and wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, and wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO.

6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. According to another embodiment of the invention, there is provided a method for transforming a host cell to a phenotype capable of expressing a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, said method comprising introducing into said cell a polynucleotide or DNA construct according to the invention.

According to another embodiment of the invention, there is provided a transformed host cell comprising a polynucleotide optimised for expression in a plant, wherein said sequence encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin and wherein said sequence is selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10. Preferably the cell is capable of expressing the pesticidal toxin, more preferably the cell is a plant cell, and even more preferably a cotton or maize cell.

According to another embodiment of the invention, there is provided a transformed plant regenerated from a transformed cell according to the invention.

According to another embodiment of the invention, there is provided a transformed plant comprising a plurality of transformed cells according to the invention.

30 Pesticidal compositions comprising transformed cells or transformed plantsaccording to the invention are also provided.

According to another embodiment of the invention, there is provided a method for producing a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, said method comprising culturing a transformed cell or transformed plant according to the invention under 35 conditions promoting expression of the pesticidal toxin and optionally isolating the pesticidal toxin. CrylAc or chimeric CrylAc/CrylAB pesticidal toxins produced by this tmethod, and pesticidal compositions comprising these thus produced toxins are also provided.

According to another embodiment of the invention, there is provided a method for i* 40 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 optimised for expression in a plant, and wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin and wherein said polynucleotide comprises a LibC/507084DIspeci 6 nucleotide 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, and SEQ ID NO. According to another embodiment of the invention, there is provided a method for controlling a pest, wherein said method comprises contacting said pest with a CrylAc or chimeric Cry 1Ac/Cry 1AB pesticidal toxin produced by a method according to the invention or a pesticidal composition of the invention.

Brief Description of the Sequences SEQ ID NO.1 is a polynucleotide sequence designated PT-1AB-PO, which is optimised for expression in plants. This gene, which encodes a CrylAb protoxin portion, o1 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 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 (Nterminal)/ 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 (Nterminal)/ Cry 1Ab (protoxin) toxin.

30 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 S 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 35 described by Adang et al. in GENBANK (Acc. No. M11068).

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 Cry 1 Ac toxin described by Adang et al. in GENBANK (Acc. No. M1 1068).

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 LibC/507084D1spec 7 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 81IB2 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 0o 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 15822c-T-PO.

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

SEQ ID NO.19 is an amino acid sequence for a truncated toxin encoded by the gene designated 31Gla-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 plantoptimised polynucleotide sequences that encode pesticidal toxins (full-length and 25 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 S. make them particularly well-suited for optimised expression in plants. Using techniques .o known to those skilled in the art, the polynucleotide sequences described herein can be used 30 to transform plants in order to confer pest resistance upon said plants.

Herein disclosed are plant-optimised genes that encode other proteins that are toxic to pests. Such genes may include those referred to herein as 1ACIAB-N-PO, 1ACIAB-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.

35 Also herein disclosed are plant-optimised polynucleotide sequences that encode Ci. terminal protoxin portions that can be used with genes encoding truncated, core toxins to produce full-length toxins. Plant-optimised protoxins described herein include those designated PT-1AB-PO and PT-1AB-2-PO.

Also herein disclosed are 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- LibC/507084D1speci 8 PO: 1AC1AB-N-PO, 1AC1AB-PO, and 1AC1AB-B-PO, 1CIAB-PO, 158C2c-PO, 158C2c- T-PO, and 31Gla-T-PO. Also herein disclosed are 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 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 fulllength 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 0o 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 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 point-mutation 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 PS81I. 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.

25 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 "Background of the Invention." Subculture Accession Number Deposit Date B.t. PS81I NRRL B-18484 19 April, 1989 E. coli (NM522) (pMYC1603) (811A) NRRL B-18517 30 June, 1989 E. coli (NM522) (pMYC394) (81IB2) NRRL B-18500 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,199 6 E. coli (NM522) (pMYC2454) (31Gla) NRRL B-21796N 30 Sept, 1997 5.4 4',c

S

a

S

*u S

S

5 .5*5 LibC/507084D1speci 9 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 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.

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 30 resultant pesticidal activity, in plants. Fragments retaining pesticidal activity are also i 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 35 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 Bal31 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.

LibC/507084D1speci 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 threedimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions lo 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 Examples of Amino Acids Nonpolar Ala, Val, Leu, lie, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gin Acidic I 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 20 would be found in nature and would include their use in plants. Thus, reference to "isolated o and purified" signifies the involvement of the "hand of man" as described herein.

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 :0o 25 preparing precursors. for example, that will 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, 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.

Thus, in preferred embodiments, expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. When LibC/507084D1speci 11 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 lo 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.

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 25 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 sequence encoding the B.t. toxin can be inserted 30 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 biochemical-molecular biological methods are generally carried out as methods of analysis. After each 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 LibC/507084Dlspei 12 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 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 Alblasserdam. Chapter 5; Fraley et al., Crit.

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, to 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 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.

S 25 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. Genet. 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 30 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 S: 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 35 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 LibC/507084D1spec 13 other hereditary 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.

e** Sf 5 LibC/507084D1spec EDITORIAL NOTE APPLICATION NUMBER 27719/02 The following Sequence Listing pages 1 to 23 are part of the description. The claims pages follow on pages 14 to 16.

1/23 Sequence Listing 11 0> Cardineau, Guy A.

Stelman, Steven J.

Narva, Kenneth E.

120> Plant-Optimized Genes Encoding Pesticidal Toxins 130> MA-714XC2 <140> <141> <1 50> 60/065,215 <151> 1997-11-12 150> 60/076,445 <151> 1998-03-02 <160> 19 <170> Patentln Ver. <21 0> 1 <21 1> 1641 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. protoxin gene

S.

S

S. S S

S

C*

<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 <21 0> 2 aggcagagtc atcagattgg ttgagtgcct aacatgctaa tcaacaggca atgatgtgtt catacctgta gaggttacat atgagacagt tcggcaagtg acctgaacga ccaggctagg gagtgaagag acattgtgta ataggctcca gcattcggga ttgaagagtt tcaagaatgg tagaagaaca cacaagaagt aaggatacgg agt t cagc aa 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 t tt c tagaag aagtggaggg aaagaaagcg aacatagcta cc tgaac tt a atcttcactg aatggcctat cgc t ctgt cc cctggtcgtg gtcaccatac gaggaagtct tatgagggta tctgtacccg gacaatcctt gtcaccaaag acggaaggaa aggcggtgaa actatcacat atgagaagaa act tgct t ca 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 agat ccc aac catcaccatt ctttgatgag ctacacaaga catcagatac actttcagcc catagacgtt caagactcaa tgttggagaa gaagttggaa gtttgtgaac tgcagacaaa gggtgtcaat gtatgatgcg tgtgaaaggg tgagtgggaa tcgtgttacc gaacaacacc caccgtaact tcgcaatcga atcagcctat cagaggctat ctttccagaa 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

S

S

*5S* S S. *5

S

S S. S S <400> 2 ctcgaggctg agcaaccaga cttgtggagt gtgaagcacg ggtatcaaca ggtgacgacg cctacctacc cttaggggat aagcacgaga cc tat cggt a accgatctca cacgctaggc gctagggtga 20 accaacatcg tacgataggc cacagcatca atcttcgagg gtgatcaaga 25 gatgttgagg gttagccaag aaggagggt t ctcaagttca gactacaccg 30 gatggtgctt aaagcataca tacacccctc aaagtgtgga ct tc tcatgg agagcgatct tcggtctcaa gtcttagcga ctaagaggct gacagcttga tgttcaaaga tctatcagaa acattgagga ccgtgaacgt agtgcgctca acgaggatct ttggaaacct agagagcaga tgtacaagga ttcaagcaga gggaggcata agcttgaggg acggagactt agcagaacaa aggttagggt acggtgaggg gtaactgtgt ctacccagga acgagagcaa ctgatggtag t tcc tgc tgg tcgagatcgg aggag agagagggct gaccgatgtt cgagttctgc tagcgatgag taggggttgg gaactacgtt gatcgatgag tagccaggat tcctggaacc ccacagccac tggtgtttgg tgagtt oct t 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 gt cat ott ca gaggagaagc agagacaaga agcgttgatg gctatgatcc cttagcgtga accgctttca cttagctgct gttctcgtgg aggggttaca atccacgaga gtgtacccta ggaacctaca cctgctgact ccttgcgaga aaggagcttg ggaaccttca tgaacgctct acatcgatag agaaagagct tccaagaoccc ccgacatcac gtactttcga aggcttacac atctcatccg ggcctcttag ttgacatcga agatcaagac St ct tgt tgg gggagaagct ctctcttcgt acgctgctga tccctggagt gcctctacga ggaacgtgaa tgccagagtg t oct tagggt tcgagaacaa acaacactgt ccagcaggaa acgctagcgc gcaacagggg agtacttoccc tcgtggacag cttcaccagc ggttagcaat tagcgagaag taacttcagg tatccaaggt cgaatgctac caggtatcaa ttacaatgct tgcacctagc tgttggttgc ccaagatggt tgaggct ott tgagtgggag gaacagccag caagagggtt gaacgcagca tgctaggaac gggtcacgtt ggaggctgaa gactgcttac 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 Glu Ala Glu Ser Asp Leu Glu Arg Ala Gin Lys 1 5 10 Asn Ala Leu Phe Thr Ser Ser Asn Gin Ile Gly Leu Lys Thr 20 25 Thr Asp Tyr His Ile Asp Arg Val Ser Asn Leu Val Giu Cys 40 Asp Giu Phe Cys Leu Asp Giu Lys Lys Giu Leu Ser Giu Lys 55 His Ala Lys Arg Leu Ser Asp Giu Arg Asn Leu Leu Gin Asp 70 75 Phe Arg Gly Ile Asn Arg Gin Leu Asp Arg Gly Trp Arg Gly 90 Asp Ile Thr Ile Gin Gly Gly Asp Asp Val Phe Lys Giu Asn 100 105 110 Thr Leu Leu Gly Thr Phe Asp Giu Cys Tyr Pro Thr Tyr Leu Ala Vai Asp Val Leu Ser Val Lys Pro Asn Ser Thr Tyr Val Tyr Gin LibC/50708401 speci 0 0.

0 *.0.0 115 Lys Ile Asp 130 Giy Tyr Ile 145 Asn Ala Lys Pro Leu Ser His Phe Ser 195 Leu Gly Val 210 Arg Leu Gly 225 Ala Leu Ala Glu Lys Leu Ser Val Asp 275 Asp Thr Asn 290 Ile Arg Giu 305 Ala Ala Ile Leu Tyr Asp 30 Leu Ser Cys 355 Asn His Arg 370 Gin Giu Val 35 385 Ala Tyr Lys Giu Asn Asn Val Tyr Pro 435 Giu Glu Tyr 450 Ala Tyr Glu 465 Giu Giu Lys Asn Arg Gly Lys Giu Leu 515 Gly Giu Thr 530 Met Glu Giu 545 <210> 4 <21 1 >3468 Giu Giu His Ala 180 Leu Trp, As n Arg Giu 260 Ala Ile Aia Phe Ala 340 Trp Ser Arg Giu Thr 420 Asn Giu Ser Ala Tyr 500 Giu Giu Ser Asp Giu 165 Pro Asp Val Leu Val1 245 Trp Leu Ala Tyr Giu 325 Arg As n Val1 Val1 Gly 405 Asp Asn Gly Asn Tyr 485 Gly Tyr Lys Ser 150 Thr Ser Ile Ile Glu 230 Lys Giu Phe Met Leu 310 Giu Asn Val Leu Cys 390 Tyr Giu Thr Thr Ser 470 Thr Asp Phe Leu 135 Gin Val Pro Asp Phe 215 Phe Arg Thr Vai Ile 295 Pro Leu Val1 Lys Vai 375 Pro Gly Leu Val1 Tyr 455 Ser Asp Tyr Pro Lys Asp Asn Ile Val 200 Lys Leu Ala Asn Asn 280 His Giu Giu Ile Gly 360 Val1 Gly Giu Lys Thr 440 Thr Vai Gly Thr Glu 520 Ala Leu Vai Gly 185 Gly Ile Giu Glu Ile 265 Ser Ala Leu Gly Lys 345 His Pro Arg Gly Phe 425 Cys Ser Pro Arg Pro 505 Thr Tyr Giu Pro 170 Lys Cys Lys Giu Lys 250 Val Gin Aia Ser Arg 330 Asn Val1 Giu Giy Cys 410 Ser Asn Arg Ala Arg 490 Leu Asp Thr Ile 155 Gly Cys Thr Thr Lys 235 Lys Tyr Tyr Asp Vai 315 Ile Gly Asp Trp Tyr 395 Val1 As n Asp Asn Asp 475 Asp Pro Lys Arg 140 Tyr Thr Ala Asp Gin 220 Pro Trp Lys Asp Lys 300 Ile Phe Asp Val1 Giu 380 Ile Thr Cys Tyr Arg 460 Tyr Asn Ala Val Tyr Leu Gly His Leu 205 Asp Leu Arg Glu Arg 285 Arg Pro Thr Phe Giu 365 Ala Leu Ile Val1 Thr 445 Gly Ala Pro Gly Trp 525 Gin Ile Ser His 190 Asn Giy Val1 Asp Aia 270 Leu Val1 Giy Ala Asn 350 Giu Giu Arg His Giu 430 Ala Tyr Ser Cys Tyr 510 Ile Leu Arg Leu 175 Ser Giu His Gly Lys 255 Lys Gin His Vai Phe 335 Asn Gin Val1 Val1 Giu 415 Glu Thr Asp Ala Giu 495 Val1 Glu Gly Thr Phe Ile Val Asp Ser Val Giu Leu Leu Leu <212> DNA <213> Artificial Sequence <220> LibC/50708401 speci <223> Synthetic BAt. toxin gene we$* 4, *00 0.0 <400> 4 atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcaactta gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggcaactata ccggattcta ctagacattg tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg accttgtcca tctgtgcttg tacagaaaga ccacctaggc agtaatagca gagtttaaca tttctcttta ctgaattctt ccatcgacat 30 ctcaacgtca tccttggata tcttccctag gaccgcttcg gcacaaaagg gtgacggatt tgtctggatg gagcggaatt tggagaggaa gttacgctat 40 gagtcgaaat gacttagaaa acgggttcct catcatttct tgggtgatat ctcgaagaga tggagagaca gaatctgtag atcgcgatga gagctgtctg ttcactgcat ggcttatcct tcggtccttg ggtcgtggct accattcatg gaagtatatc gagggtacgt gtaccagctg aatccttgtg acaaaagaat gaaggaacat <210> 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 t tgt t ccgga atatccttcg agatcgagaa caaacaacac acacttctcg attatgcatc aatctaacag tagagtactt tcatcgtgga caacgagtgc acggattgag gtcagagttc c t ttggt ccc 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 t cgc tac aat ttcagcccca tgatgttgga gacgcaagat aggagaagca attggaatgg 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 agt cc aat cg 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 ct t tccc act tcggtcaggg gcatcaacaa caaatctgcc ctccacagaa 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 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 tttagaaaga aaaaaccgat tgatgaattt acttagtgat agaccgtggc agagaattac aaaaatagat agatagtcaa tgtgccaggt ccatcattcc cttaggtgta tctagaattt ggagaaaaaa agaggcaaaa ggataccaac ttatctgcct agggcgtatt ttttaataat caaccaccgt tgtctgtccg 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 S. S 0* LibC/507084D1 speci <21 1> 3468 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene 006B 00 0e 0 0 @00000 0 00 0 00 0000 0 00 0 00 *0 OS 0 @0 *0 0000

S

0000 <400> atggacaaca atcccaacat gttgaggtgc tgggtggaga tcacttaccc aattcctttt gtcgatatca tttggggaat gagcaactta tcaaccaaag gaaggcctca gcaaccttta ccgaccaatc ctgccttaag ctgacgaccg caattccgct tacgtgcagg ctgccaacct cggtggggct ttgatgccgc ggcaactata ccgattatgc ccggattcta gagattgggt ctagacattg tcgctctctt 20 tcacaattga cccgggaaat cgaggctcgg ctcagggcat aacagtatca cgatctacac atcatggcat cacocgttgg atgggcaatg cagctcoaca 25 accttgtcca gcactctata tctgtgcttg acgggacaga tacagaaaga gcggaacagt ccacctaggc aagggtttag agtaatagca gcgttagtat 30 gagtttaaca acataattgc tttctcttta atggttctgt ctgaattctt ccggcaacaa ccatcgacat otaccagata ctcaacgtca attggggtaa tccttggata atctccaatc tcttccctag gtaacatagt gacogottog aattcattco gcacagaagg cggtgaatgc gtgactgact atcacatcga tgtttggatg agaagaagga gagcggaact tgcttcaaga tggaggggaa gtacggacat gttacgctct tgggtacctt gaatcgaaac tcaaagccta gaccttgaga tctacctcat acgggttcac tctggocact caccacttct ccttggacat tgggtgatct tcaagatcaa ctagaagaga aaccacttgt tggagggaca agagagagaa gaaagcgttg aogctctgtt atagctatga ttcatgctgo gaacttagcg tgattccggg ttcactgcat tctccttgta ggcotatcct gotggaatgt tctgtccttg ttgttcctga ggtcgtggct acattcttcg accatacacg agattgagaa gaagtctacc caaacaacac gagggtaott acacttotcg oaacgagtgc acggattgag gtcagagttc ott tggt CCC gattgaagag ccagatttac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtaoaac t ccc aac tac ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgt tac tgc a tctgttcact t cgogt ttoo gttgtccgag tcccaacttt caccattcaa tgatgagtgc cacaagatac cagatacaac t toag cOccc agacgttggc gactcaagat tggagaagcc gttggaatgg tgtgaactct agacaaacgc tgtcaatgct tgatgcgagg gaaagggcac gtgggaagca tgttaccgcg caacaccgac cgtaacttgc caatcgagga attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctocgcg agtcgttata t a caac acgg cagttcaggc gactctaggc ccagtcctcg at caggt cto 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 ttgaagtgco cctctgttac cagtaccagc aaagtgccaa gaaccgccgg cagagtctga agattgggct agtgcctctc atgctaagcg acaggcaact atgtgttcaa acctgtacca gttacatcga agacagtcaa gcaagtgtgc tgaacgaaga ggctaggcaa tgaagagggc ttgtgtacaa ggCtccaagc ttcgggaagc aagagttaga agaatggtga aagaacagaa aagaagttcg gatacggaga tcagcaactg ctgcgactca cctatgagag caaccctgag catctcgttg gcttggactt tgtacagata otcaaggtta ggaagoagac gaacagcgcg tttatocgtg 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 agatogtgga ggagaactat gaagatagat ggacagtcaa tgtgcctggg ccatcactca cctcggtgtg to tggagtt t 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 00 0000 0 00 0 0 00 0 00 LibC/507084D1 speci 6/23 gtaccogctg actatgcatc agoctatgag gagaaggctt acaccgatgg acgtagggac 3300 aatccttgcg aatctaaoag aggctatggg gactacacac cgttaccagc cggctatgtc 3360 aocaaagagt tagagtaott tcoagaaaco 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 CC C <400> 6 atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcagttaa gaaggcctca ccgaccaatc ctgacgacog tacgtgcagg cggtggggct ggcaactata ccggattcta ctagacattg toaoaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg aocttgtcca totgtgcttg tacagaaaga ccacctaggc agtaatagca gagtttaaca tttctcttta ctgaattctt coatogacat ctcaacgtca tccttggata tcttccctag gaccgcttcg gcacagaagg gtgactgact tgtttggatg gagcggaact tggaggggaa gttacgctct gaatcgaaac gaccttgaga acgggttcac caccacttct tgggtgatct ctagaagaga tggagggaca gaaagcgttg atagctatga gaacttagcg ttcactgcat atcccaacat tgggtggaga aattcctttt tttggggaat ttaaccaaag gcaaccttta ctgccttaag caattcogct ctgccaacct t tgatgcogo ccgattatgc gagattgggt tcgctctctt cccgggaaat ctcagggcat cgatctacac cacccgttgg cagctccaca gcactctata acgggacaga gcggaacagt aagggtttag gcgttagtat acataattgo atggttctgt ccggcaacaa otacoagata attggggtaa atotccaatc gtaacatagt aattcattoc cggtgaatgc atcaoatcga agaagaagga tgcttcaaga gtacggacat tgggtacctt toaaagccta tctacotcat tctggccact ccttggacat tcaagatcaa aaccacttgt agagagagaa acgctctgtt ttcatgctgc tgattccggg tctccttgta caacgagtgc acggattgag gt cagagt to CtttggtCCC aatagaagaa ccagatttac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gt tot ctgga aoaaogtatt taggagaoot atttgootat tgatagottg ooatogoott oatoagagot atoogatago oatttoagga oatooagaat togtgttogt ttootooato tagogattto aggtgttaga ogttaotgoa totgttoaot togogtttoo gttgtoogag toooaattt oaooattoaa tgatgagtgo oaoaagatao oagataoaao ttoagooooa agaogttggc gaotoaagat tggagaagoo gttggaatgg tgtgaaotot agaoaaaogo tgtcaatgot tgatgogagg attoottaoa aotggttaoa gtgoooggtg totoaatggg ttogotagga goagaatott ogoattoaat oagaattaoo gtgotoogog agtogttata taoaaoaogg oagttoaggo gaototaggo ooagtootog atoaggtoto ogoggttatt ooagaattoa gttgotoaao ttoaaoatog ggaaootoot gatgagatoo agooatgtgt oogatgttot attaotoaga ooaggattoa agagggtata gtaaggtatg ttttooaata ggttaottog aatttotocg aogotogagg togtooaato aao 0t tgt tg aaggtoaaao ogogggatoa ggaggtgatg tatooaaoat oagttgagag gooaaaoatg agtoooatog tgtaoogaoo ggcoatgcca otogotagag gaaacaaaca oagtatgata gttcatagca gotatctttg aatgtcatca aotgootgag oaootatoga otggattogt aogotttot aooaagooat ttogagagtg toaatgaoat aagt tootcot atgtotoogt atgatotgao gtotogaaog gagagttgao gotaoooaat agaaottoga oaoaootgat aotaotggto otttcooaot toggtoaggg goatoaaoaa oaaatotgoo otooaoagaa ooatgttoog ottggataoa toooagotgt otggaggoga ttgaagtgoo oototgttao oagtaooago aaagtgooaa gaaoogoogg oagagtotga agattgggct agtgoototo atgotaagog aoaggoaaot atgtgttoaa aootgtaooa gttaoatoga agaoagtoaa goaagtgtgo tgaaogaaga ggotaggoaa tgaagagggo ttgtgtaoaa ggotooaago ttogggaago aagagttaga agaatggtga oaaoootgag oatotogttg gottggactt tgtaoagata otoaaggtta ggaagoagao gaaoagogog tttatoogtg gttoggaoaa taggttatt tgtotgggga aotaaotgto oogtaotgtg oggt ago t tt ggaoatattg agggoatoag ttaogggaot ogtgtataga toaaoaattg atoogotgto oaaoaaogtt t toaggo tt t togtagtgot oaaggggaao ottggttagg oattoaotto oootattoao gaoagotaoa tgoottoaoo agtgataato ottggaaaga oaagaoagat tgatgagttc aottagtgat agatogtgga ggagaaotat gaagatagat ggaoagtoaa tgtgootggg ccatcactca ootoggtgtg totggagttt tgagaagaag agaagooaaa tgataooaao ttaoottoot agggogoato 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/507084D1speci ggcctatcct tctgtccttg ggtcgtggct accatacacg gaagtctacc gagggtactt gtacccgctg aatccttgcg accaaagagt gaaggaacat <210> 7 gctggaatgt ttgttcctga acattcttcg agattgagaa caaacaacac acacttctcg actatgcatc aatctaacag tagagtactt tcattgttga gaaagggcac gtgggaagca tgttaccgcg caacaccgac cgtaacttgc caatcgagga agcctatgag aggctatggg tccagaaacc tagcgtggag gtagatgtag gaagtttcac tacaaagaag gagctgaagt aatgactaca tacgatggag gagaaggctt gactacacac gacaaggttt ttacttctga aagaacagaa aagaagttcg gatacggaga tcagcaactg ctgcgactca cctatgagag acaccgatgg cgttaccagc ggattgagat tggaggaa caatcaccgc tgtctgtcct aggttgcgtc cgtcgaggag agaggagtat caactcttct acgtagggac cggctatgtc tggagaaacg 2940 3000 3060 3120 3180 3240 3300 3360 3420 3468 <211> 1156 <212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400 Met 1 Ser 7 Asp Asn Asn Asn Pro Glu Thr Pro Ile Pro 5 Val1 Asn Ile Asn Glu Ile Pro Tyr Asn Cys Leu Glu Val Leu Gly 25 Ser Giu Arg Ile Tyr Asp Ile Ser Giu Phe Val Leu 40 Phe Leu Thr Gin Phe Val Giu Thr Gly Leu Leu Ser Asp Ile Ile Pro Gly Ala Trp Gly Gly Ser Val Leu Gly Ile Phe Gly Gin Trp Asp Giu Aia Phe Leu Val Gin Gin Leu Ile Asn Giu Arg Ile Giu Aia Arg Asn Gin Ala Ile Ser Arg Gly Leu Ser Asn 105 Pro Tyr Gin Ile Ser Phe Arg 115 Giu Met Arg Trp Giu Ala Thr Asn Pro Ala 125 Leu Tyr Ala Giu 110 Leu Arg Giu Thr Thr Ala Ile Gin Phe 130 Ile Pro Asn 135 Gin Met Asn Ser Leu Phe Ala 145 Tyr Val 150 Asn Asn Tyr Gin Val 155 Val1 Leu Leu Ser Val 160 Val Gin Ala Ala 165 Arg Leu His Leu Ser 170 Ala Leu Arg Asp Val Ser 175 Val Phe Giy Gin 180 Leu Trp Gly Phe Ala Thr Ile Tyr Asn Asp 195 Arg Trp Tyr Thr Arg Leu Ile 200 Glu Asn Tyr Thr Asn Ser Arg 190 Tyr Ala Val Asp Ser Arg Asn Thr Gly Arg Val Trp 210 Asp Trp Gly 220 Leu Val Arg Tyr Asn 230 Leu Phe Arg Arg Glu 235 Asp Thr Leu Thr Val1 240 Asp Ile Val Ala 245 Ser Phe Pro Asn Tyr 250 Giu Ser Arg Arg Tyr Pro 255 Ile Arg Thr Leu Giu Asn 275 Arg Ser Ile Val1 260 Phe Gin Leu Thr Ile Tyr Thr Asp Gly Ser Phe 280 Leu Gly Ser Ala Gin 285 Asn Asn Pro Val 270 Gly Ile Giu Ser Ile Thr Arg Ser Pro 290 Ile Tyr His 295 Arg Met Asp Ile Thr Asp Ala Gly Tyr Tyr 305 Ile Tyr 315 Pro Ser Gly His Gin 320 Pro Met Ala Ser Pro 325 Gly Phe Ser Gly 330 Giu Phe Thr Phe 335 LibC/507084D1 speci Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gin Gin Arg Ile Val Ala :'.000 00: Gin Arg Giy 385 Tyr Asn Val1 Arg Ile 465 Phe Asp Tyr Val Trp 545 Ser 30 Asn Ser Thr Val 625 Val1 Ser Lys Asn Thr 705 Val1 Gin Arg Tyr Trp 785 His Asp Gly 355 Phe Glu Lys Asn Met 435 Pro Ala Phe Val1 Giu 515 Val Asn Asp Phe Thr 595 Thr Ala Asp Giu Ala 675 Arg Ile Leu Ile Tyr 755 Ala Leu Phe Gly 340 Gin Asn Phe Ser Val1 420 Phe Met Ser Asn Arg 500 Val Arg Ser Asn Thr 580 Ala Leu Leu Tyr Phe 660 Lys Gly Thr Leu Asp 740 Ile Lys Ser Ser Val Gly Ile Ala Gly 405 Pro Arg Phe Asp Gly 485 Leu Pro Tyr Ser Leu 565 Ser Gly Giu Phe His 645 Cys Arg Ile Ile Gly 725 Giu Giu His Ala Leu 805 Trp Val Gly Tyr 390 Thr Pro Ser Ser Ser 470 Ser As n Ile Al a Ile 550 Gin Ser Val Ala Thr 630 Ile Leu Leu Asn Gin 710 Thr Ser Asp Giu Pro 790 Asp Val1 Tyr Ile 375 Gly Val1 Arg Gly Trp 455 Ile Val1 Ser His Ser 535 Phe Ser Leu Ile Giu 615 Ser Asp Asp Ser Arg 695 Gly Phe Lys Ser Thr 775 Ser Ile Ile Arg 360 Asn Thr Asp Gin Phe 440 Ile Thr Ile Ser Phe 520 Val Ser Ser Gly Ile 600 Ser Ser Arg Giu Asp 680 Gin Gly Asp Leu Gin 760 Val Pro Asp Phe 345 Thr Asn Ser Ser Gly 425 Ser His Gin Ser Gly 505 Pro Thr Asn Asp Asn 585 Asp Asp Asn Val1 Lys 665 Glu Leu Asp Giu Lys 745 Asp Asn Ile Val Lys Leu Gin Ser Leu 410 Phe Asn Arg Ile Gly 490 Asn Ser Pro Thr Phe 570 Ile Arg Leu Gin Ser 650 Lys Arg Asp Asp Cys 730 Ala Leu Val Gly Gly 810 Ile Ser Gin Asn 395 Asp Ser Ser Ser Pro 475 Pro Asn Thr Ile Val1 555 Gly Val1 Phe Glu Ile 635 Asn Giu Asn Arg Val1 715 Tyr Tyr Glu Pro Lys 795 Cys Lys Ser Leu 380 Leu Glu His Ser Ala 460 Ala Gly Ile Ser His 540 Pro Tyr Gly Giu Arg 620 Gly Leu Leu Leu Gly 700 Phe Pro Thr Ile Gly 780 Cys Thr Thr Thr 365 Ser Pro Ile Arg Val 445 Giu Val1 Phe Gin Thr 525 Leu Ala Phe Val Phe 605 Ala Leu Val1 Ser Leu 685 Trp Lys Thr Arg Tyr 765 Thr Ala Asp Gin 350 Leu Val1 Ser Pro Leu 430 Ser Phe Lys Thr Asn 510 Arg Asn Thr Giu Arg 590 Ile Gin Lys Giu Glu 670 Gin Arg Giu Tyr Tyr 750 Leu Gly His Leu Asp Tyr Leu Aia Pro 415 Ser Ile Asn Gly Gly 495 Arg Tyr Val1 Ala Ser 575 Asn Pro Lys Thr Cys 655 Lys Asp Gly Asn Leu 735 Gin Ile Ser His Asn 815 Gly Arg Asp Val1 400 Gin His Ile Asn Asn 480 Giy Gly Arg Asn Thr 560 Ala Phe Val1 Ala Asp 640 Leu Val Pro Ser Tyr 720 Tyr Leu Arg Leu Ser 800 Giu His 820 825 830 Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Giu Lys Pro Leu Val Gly LibC/50708401 speci 835 Leu 845 Trp Glu Ala 850 Arq Giu Ala Arg Val Lys 855 Giu Ala Glu Lys Lys 860 Tyr Arg Asp Lys Lys Leu Giu 865 Glu Trp 870 Leu Thr Asn Ile Lys Giu Ala Lys 880 Ser Val Asp Ala 885 Ile Phe Val Asn Tyr Asp Arg Leu Gin 895 Ala Asp Thr Ser Ilie Arg 915 Asn Ala Ala Asn 900 Glu Ala Met Ile His 905 Glu Ala Asp Lys Ala Tyr Leu Leu Ser Val Ile 925 Phe Arg Vai His 910 Pro Gly Val Thr Ala Phe Ile Phe Giu Giu 935 Asn Giu Gly Arg Ile 940 Gly Ser 945 Gly Tyr Asp Ala Arg 950 Asn Val Ile Lys Asn 955 Val1 Asp Phe Asn Leu Ser Cys Val Lys Gly Asp Val Giu Giu Gin 975 Asn Asn His Ser Gin Giu 995 Thr Ala Tyr Arg 980 Val Leu Val Val 985 Giu Trp Giu a.

1010 Ile Giu 1025 Giu Val Val Arg Val Cys Pro 1000 Lys Giu Gly Tyr Gly 1015 Asn Thr Asp Giu Leu 1030 Pro Asn Asn Thr Val Gly Arg Gly Tyr Ile 1005 Giu Gly Cys Val Thr 1020 Lys Phe Ser Asn Cys 1035 Thr Cys Asn Asp Tyr Ala Giu Val 990 Leu Arg Val Ile His Giu Val Giu Giu 1040 Thr Ala Thr Asn 1045 Giu Tyr Giu 1050 Thr Ser 1055 Asn Arg Gly Tyr Asp Gin Giu Gly Thr Tyr Arg 1060 1065 Gly Ala Tyr Giu SerAsn Ser Ser Val 1070 Ala Ser Ala Pro Ala Asp Tyr 1075 1080 Tyr Giu Giu Lys Ala Tyr Thr Asp 1090 1095 Ser Asn Arg Gly Tyr Gly Asp Tyr 1105 1110 Thr Lys Giu Leu Giu Tyr Phe Pro 1125 Ile Gly Giu Thr Giu Gly Thr Phe 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 Gly Arg Arg Thr Pro Leu 1115 Giu Thr Asp 1130 Ile Val Asp 1145 Asp 1100 Pro L085 Asn Pro Cys Giu Ala Gly Tyr Val 1120 Val Trp Ile Giu Lys 1135 Ser Val Giu Leu Leu 1150 atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcaactta gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggcaactata atcccaacat tgggtggaga aat tcct tt t tttggggaat tcaaccaaag gcaaccttta ctgccttaag caattccgct ctgccaacct ttgatgccgc ccgattatgc caacgagtgc acggattgag gtcagagttc ctttggtccc 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 aagttcctct atgtctccgt atgatctgac gtctcgaacg caaccctgag catctcgttg gcttggactt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa taggcttatt tgtctgggga 120 180 240 300 360 420 480 540 600 660 LibCI5O7OB4D1 speci ccggattcta ctagacattg tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg accttgtcca tctgtgcttg tacagaaaga ccacctaggc agtaatagca gagtttaaca tttctcttta ctgaattctt ccatcgacat ctcaacgtca tccttggata t ctt ccc tag gaccgcttcg <210> 9 gagattgggt tcgctctctt cccgggaaat ctcagggcat cgatctacac cacccgttgg cagctccaca gcactctata acgggacaga gcggaacagt aagggtttag gcgttagtat acataattgc atggttctgt ccggcaacaa ctaccagata attggggtaa atctccaatc gtaacatagt aattcattcc caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgttactgca 10/23 cagttcaggc gactctaggc ccagtcctcg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt c cgatgtt ct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggt tact tcg aatttctccg acgctcgag gagagttgac gctacccaat agaacttcga cacacctgat actactggtc ctttcccact tcggtcaggg gcatcaacaa caaatctgcc ctccacagaa ccatgttccg cttggataca tcccagctgt ctggaggcga ttgaagtgcc cctctgttac cagtaccagc aaagtgccaa gaaccgccgg actaactgtc ccgtactgtg cggtagcttt ggacatattg agggcatcag ttacgggact cgtgtataga tcaacaattg atccgctgtc caacaacgtt ttcaggcttt tcgtagtgct caaggggaac cttggttagg cattcacttc ccctattcac gacagctaca tgccttcacc agtgataatc 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1839 .0 <21 1> 1839 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene <400> 9 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 t c ttccc tag gaccgcttcg <210> atcccaacat tgggtggaga aattcctttt t ttggggaat ttaaccaaag 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 caacgagtgc acggattgag gtcagagttc ctttggtccc aatagaagaa ccagatctac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc cat t tcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgttaga cgttactgca attccttaca ac tggt tac a gtgcccggtg tctcaatggg ttcgctagga gc agaat ct t cgcattcaat c agaat tac c gtgctccgcg agtcgttata tacaacacgg cagttcaggc gactctaggc ccagtcctcg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacctcct gatgagatcc agccatgtgt ccgatgttct attactcaga ccaggattca agagggtata gtaaggtatg ttttccaata ggttacttcg aatttctccg acgctcgag actgcctgag cacctatcga ctggattcgt acgc ctt tc t 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 caaccctgag catctcgttg gcttggactt tgtacagata ctcaaggtta ggaagcagac gaacagcgcg tttatccgtg gttcggacaa taggcttatt tgtctgggga act aac tgt c 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 LibC/507084D1 speci 11/23 <21 1> 1839 <212> DNA <213> Artificial Sequence <220> <223> Synthetic B.t. toxin gene a <400> atggacaaca gttgaggtgc tcacttaccc gtcgatatca gagcagttaa gaaggcctca ccgaccaatc ctgacgaccg tacgtgcagg cggtggggct ggcaactata ccggattcta ctagacattg 20 tcacaattga cgaggctcgg aacagtatca atcatggcat atgggcaatg accttgtcca tctgtgcttg tacagaaaga ccacctaggc agtaatagca gagtttaaca tttctcttta ctgaattctt ccatcgacat ctcaacgtca tccttggata t ct tccc tag gaccgcttcg <210> 11 atcccaacat tgggtggaga aattcctttt tttggggaat ttaaccaaag 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 caacgagtgc acggattgag gtcagagttc ctttggtccc aatagaagaa ccagatctac agaggagatg cttcgccgtt gcacttgtcg aactatcaat tgttcgctgg caggtacaac tcccaactac ctacacaaac agagagaagc cgatgcgcac gttctctgga acaacgtatt taggagacct atttgcctat tgatagcttg ccatcgcctt catcagagct atccgatagc catttcagga catccagaat tcgtgttcgt ttcctccatc tagcgatttc aggtgt taga cgttactgca attccttaca actggttaca gtgcccggtg tctcaatggg ttcgctagga gcagaatctt cgcattcaat cagaattacc gtgctccgcg agtcgttata tacaacacgg cagttcaggc gactctaggc ccagtcctgg atcaggtctc cgcggttatt ccagaattca gttgctcaac ttcaacatcg ggaacct cc t 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 c aaat ctgc c ctccacagaa ccatgttccg cttggataca tcccagctgt ctggaggcga ttgaagtgcc cctctgttac cagtaccagc aaagtgccaa gaaccgccgg 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 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 gc tc aagc tg tggggat tga gagtatgctg acgtatcaag acaatcagaa atggagaacg ttcttgtctc ggggcattgt atgagaggat acaacttcaa caacccgtac t tcc tt cgt t ccaatctgca caactatcaa atcactgtgc attggatcac tcagtgcata gatctcaact c aac t ttgt t tggcccatct agctgagttt catctatgtg gagagtcatt tcgaatctcg tcttgcgatt tgtcaatgag caacacctac ctacaatcga ccctacaact ggtaac tc ta ccagggggtg caatgggatg gctaggaatg gaagccttca gatcgctttc ggctttgaag ctaagagatt aactacaaca aatcgtggtc t tgaggaggg gcttgagcaa gcattgacat gctttcttgt cctttcttgt ctgccattgc aagaatggga ggatacttga tgccgttgct ctgtgatctt gactcatcag tcaacaactt atctcacatt tcctgaagag ctcactgtca tggactcata acagattgaa caatctggaa agaagatccc tgggctactt ctccgtgtat tggagaaaga acacattgat accgaagtct gac tgt c ttg LibC/507084D1 speci 12/23 ctttctttcc aaactatgac aacagaagat atcccattca accagttggt 780 gacattgctg caactaacaa gttgctcagt tttgacattc tactggggtg atctatggta ttcaggactt ttcaacttac cgtggaagag cctcgcgaag ccgttcctga accattgatc ggcact t ctg tttggtgact ctaaggtttc tccacaggag ggggagaact agagccaatc agtagcggtg <210> 12 gggaagtgta tacctacttt tcaacaatct gacatcgtgt gggaggcgaa tgtccaatcc gtggtgttga gtcaagttga gctacagtca caactggtgt cagagaggat tgatcaccgg ttgtatctct gttacgcttc t tggaggcca tgacctctag cagacatcat aactgtacat cactgatcca caatgtgatg taccatcttc gatctctagc tcaggagcct tactttgcga aggagtagag ttcgttgact tcgtctctgt tgtcttctcc caatcagata tccaggattc tcaagtcaac cagtagagat agttagtgtc aacctttcgc tgggatcagt agacaagatt ctcAtcaact gaaagctcag actgattggt ttgataggtg ccaagatcct ttgttacaac ttctcaacac gagttaccgc cacgcaacct tggactcatc cctcttgtga acaggagggg atcaactcac gcacgtgtga aacatgcctc tacaccgact gaacaacctc gagatcattc tcaatccaca ccatcaggaa tcagtgttgg gaggtaacat tcactttcaa aaccatggcc ccaccaacag ctgaggacaa ttgttcaaag gtagcgcaac aaggcttcag acattcttcg ccatcacaca tagtactcac ttcagaagac tcagcaatcc tctttggtgc tagctgatgc gttacaatct tccacacttg acgcaacttc cacatctcct tggacccgtc tgctccacca cttcacgtat ctcagttcca gtctggaaca tcttaccaac agtttggggg acgcaacacc aagataccgt aggagctgca tatggagata cttctcattc aggttccatc aacactcgag 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 c agt tgat ca ggattgggaa aacaatccag gaaagggaca gctcaagctg tggggattga gagtatgctg acgtatcaag gacattgctg caactaacaa gttgctcagt tttgacattc tactggggtg atctatggta ttcaggactt ttcaacttac cgtggaagag cctcgcgaag ccgttcctga accattgatc ggcacttctg tttggtgact ctaaggtttc tccacaggag ggggagaact agagccaatc agtagcggtg gcagagtctg cagattgggc acaatcagaa atggagaacg ttcttgtctc ggggcattgt atgagaggat acaacttcaa caacccgtac t tcc t tcgt t ccaatctgca caactatcaa atcactgtgc attggatcac ctttctttcc gggaagtgta tacctacttt tcaacaatct gacatcgtgt gggaggcgaa tgtccaatcc gtggtgttga gtcaagttga gctacagtca caactggtgt cagagaggat tgatcaccgg ttgtatctct gttacgcttc ttggaggcca tgacctctag cagacatcat aactgtacat acttggaaag tcaagacaga tcagtgcata gatctcaact caactttgtt tggcccatct agctgagttt catctatgtg gagagtcatt tcgaatctcg t c ttgcgat t tgtcaatgag caacacctac ctacaatcga aaactatgac cactgatcca caatgtgatg taccatcttc gatctctagc tcaggagcct tact ttgcga aggagt agag ttcgttgact tcgtctctgt tgtcttctcc caatcagata tccaggattc tcaagtcaac cagtagagat agttagtgtc aacct t tcgc tgggatcagt agacaagatt agcacagaag tgtgactgac ccctacaact ggtaactcta ccagggggtg caatgggatg gctaggaatg gaagccttca gatcgctttc ggctttgaag ctaagagatt aactacaaca aatcgtggtc ttgaggaggg aacagaagat ctcatcaact gaaagctcag actgattggt ttgataggtg ccaagatcct ttgttacaac ttctcaacac gagttaccgc cacgcaacct tggactcatc cctcttgtga acaggagggg at caac t cac 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 tcagcaatc tctttggtgc tagctgatgc ctctgttcac atcgcgtttc tcctgaagag ctcactgtca tggactcata acagattgaa caatctggaa agaagatccc tgggctactt ctccgtgtat tggagaaaga acacattgat accgaagtct gactgtcttg accagttggt gttacaatct tccacacttg acgcaacttc cacatctcct tggacccgtc tgctccacca cttcacgtat ctcagttcca 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 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 <21 1> 1163 <212> PRT ctgatgagtt gacttagtga tagatcgtgg aggagaacta agaagataga aggacagtca atgtgcctgg cccatcactc acctcggtgt atctggagtt ctgagaagaa aagaagccaa ctgataccaa cttaccttcc aagggcgcat acttcaacaa acaatcaccg gtgtctgtcc aaggttgcgt gcgtcgagga aagaggagta gcaactcttc gacgtaggga ccggctatgt t tggagaaac ctgtttggat tgagcggaac atggagggga tgttacgctc tgaatcgaaa agaccttgag gacgggttca acaccacttc gtgggtgatc tctagaagag gtggagggac agaaagcgtt catagctatg tgaacttagc cttcactgca tggcctatcc ctctgtcctt tggtcgtggc caccatacac ggaagtctac tgagggtact tgtacccgct caatccttgc caccaaagag ggaaggaaca 13/23 gagaagaagg ttgcttcaag agtacggaca ttgggtacct ctcaaagcct atctacctca ctctggccac t cc ttggaca ttcaagatca aaaccacttg aagagagaga gacgctctgt attcatgctg gtgattccgg ttctccttgt tgctggaatg gttgttcctg tacattcttc gagattgaga ccaaacaaca tacacttctc gactatgcat gaatctaaca ttagagtact ttcattgttg agttgtccga atcccaactt tcaccattca ttgatgagtg acacaagata tcagatacaa tttcagcccc 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 cctcgctaga ggaaacaaac tcagtatgat cgttcatagc tgctatcttt gaatgtcatc cgtagatgta agaagtttca gtacaaagaa cgagctgaag caatgactac atacgatgga ggagaaggc t 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 <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 13 Met Glu Glu Asn Asn 1 5 Asn Pro Glu Glu Val Gin Asn Gin Cys Ile 10 Glu Pro Tyr Asn Cys Leu Ser Leu Leu Asp 20 Asp Gly 25 Leu Arg Ile Ser Thr Gly Asn Vai Ser Asn Ser Ser Ile Phe Val Pro Ile Ser Leu Ser 40 Leu Val Gin Phe Leu Asp Gly Gly Gly Vai Giy Leu Phe Val Trp Giv Ile Val Giy Pro Gin Ser 70 Arg Trp Asp Aia Val Gin Ile Giu Leu Ile Asn Ile Ala Giu Phe Phe Arg Asn Ala Ala Ile Ala Asn Leu Glu 100 Trp Leu Gly Asn Asn Ile Tyr Phe Lys Giu 115 Val Ile Asp Giu Giu Asp Pro 120 Leu Asn Pro Ala Val Giu Ala 110 Arg Thr Arg Arg Asp Ile Arg Phe Arg Asp Gly Leu 130 Pro Ser Leu 140 Leu Phe Arg Ile 145 Ala Ser 150 Leu Phe Giu Val Pro 155 Leu Leu Ser Val Tyr 160 Gin Ala Ala His Leu Ala Ile 170 Ile Arg Asp Ser Val Ile 175 Phe Gly Glu 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 His 205 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 g A485 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 S530 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/507084D1speci tee' 0* ~q

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Asp Val Cys Tyr Ala Tyr Leu Giu 770 Vai Pro 785 Gly Lys Gly Cys Ile Lys Giu Giu 850 Giu Lys 865 Ile Vai Ser Gin 25 Ala Ala Leu Ser 930 Gly Arg 945 Lys Asn His Val Pro Giu Arg Gly 1010 40 Gly Cys 1025 Phe Ser Cys Asn 15/23 710 715 Lys Giu Asn Tyr Vai Thr Leu Leu Gly Thr 725 730 Thr Tyr Leu Tyr Gin Lys Ile Asp Giu Ser 740 745 7Arg Tyr Gin Leu Arg Giy Tyr Ile Giu Asp 760 765 Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu 775 780 rThr Gly Ser Leu Trp Pro Leu Ser Aia Pro 790 795 Ala His His Ser His His Phe Ser Leu Asp 805 810 7 Asp Leu Asn Giu Asp Leu Gly Val Trp Val 820 825 7Gin Asp Gly His Ala Arg Leu Gly Asn Leu 840 845 Pro Leu Vai Gly Giu Ala Leu Ala Arg Val 855 860 Trp Arg Asp Lys Arg Giu Lys Leu Giu Trp 870 875 7 Lys Giu Ala Lys Giu Ser Val Asp Ala Leu 885 890 7Asp Arg Leu Gin Ala Asp Thr Asn Ile Ala 900 905 Lys Arg Val His Ser Ile Arg Giu Ala Tyr 920 925 Sle Pro Gly Val Asn Ala Ala Ile Phe Giu 935 940 *Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg 950 955 Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn 965 970 Val Giu Glu Gin Asn Asn His Arg Ser Val 980 985 Giu Ala Giu Val Ser Gin Giu Val Arg Val 1000 1005 7 Ile Leu Arg Val Thr Ala Tyr Lys Giu Gly 1015 1020 Thr Ilie His Giu Ile Giu Asn Asn Thr Asp 1030 1035 iCys Val Giu Giu Giu Val Tyr Pro Asn Asn 1045 1050 Tyr Thr Ala Thr Gin Giu Giu Tyr Giu Gly 1060 1065 Arg Gly Tyr Asp Gly Ala Tyr Giu Ser Asn 1080 1085 Tyr Ala Ser Ala Tyr Giu Giu Lys Ala Tyr 1095 1100 Asn Pro Cys Giu Ser Asn Arg Giy Tyr Gly 1110 1115 Ala Gly Tyr Val Thr Lys Giu Leu Giu Tyr 1125 1130 Val Trp Ile Giu Ile Gly Giu Thr Giu Gly 1140 1145 720 Phe Asp Giu 735 Lys Leu Lys 750 Ser Gin Asp Thr Val Asn Ser Pro Ile 800 Ile Asp Val 815 Ile Phe Lys 830 Giu Phe Leu Lys Arg Ala Giu Thr Asn 880 Phe Val Asn 895 Met Ile His 910 Leu Pro Giu Giu Leu Giu Asn Val Ile 960 Val Lys Gly 975 Leu Val Val 990 Cys Pro Gly Tyr Gly Giu Giu Leu Lys 1040 Thr Val Thr 1055 Thr Tyr Thr 1070 Ser Ser Val Thr Asp Gly Asp Tyr Thr 1120 Phe Pro Giu 1135 Thr Phe Ile 1150 S. *S V S S5*#

S

S. S S C S 55 Ser Arg Asn 1075 Pro Ala Asp 1090 Arg Arg Asp 1105 Pro Leu Prc Thr Asp Lys Val Asp Ser Val Giu Leu Leu Leu Met Giu Giu 1155 <210O> 14 <21 1> 3558 <212> DNA <213> Artificial Sequence 1160 LibC/50708401 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 20 atcgaggctg ttctctgttc cttgagtcta tctatcaacc gctggaatca tggagaaacc gttggtaccc aactacgagt agagctccag gattctatca 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 gagc tgaagt 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 cagt tggaat gaagacaaac cagagtctga agattgggct agtgcctctc atgctaagcg acaggcaact atgtgttcaa acctgtacca gttacatcga agacagtcaa gcaagtgtgc tgaacgaaga ggctaggcaa tgaagagggc ttgtgtacaa ggctccaagc ttcgggaagc aagagttaga agaatggtga aagaacagaa aagaagttcg gatacggaga tcagcaactg ctgcgactca cctatgagag acaccgatgg cgttaccagc ggattgagat cgagaacgag gaacctttct tgatccattc tggagttctt agagctttgg gat tcgt caa 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 ct Ctgc tt ct 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 ggagttccat cctaggggaa caagttactg ttcagagctt agatctgtgt ttgttcgcta cttcatcttc caagagattc tgcgctaggt cttagataca ttcccatctt atctacactg ttcaacaaca ct tct tgac t 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 ggcctatcct tctgtccttg ggtcgtggct accatacacg gaagtctacc gagggtactt gtacccgctg aatccttgcg accaaagagt gaaggaacat ctctttctat gaatcgagga ctactgttca tcgctggaca gagatccttg agaacactag accaacaatc tgtacactca tcagaaacca t tctt ct tag aaagatacta ggtacaacac accagttcag acgatactag atccaatcgg acgctccatc tcccagagca actactgggt ctcatggaaa tgtacagaac ttccttgggc acaccattgg cagagactac tgatctctgg gaactaacac tgaactctgg ctaacgtgaa gatacagagt gaggatctac ct t ctCaat C ctgctggaat agt tcat tcc cggtgaatgc atcacatcga agaagaagga tgcttcaaga gtacggacat tgggtacctt tcaaagccta tctacctcat tctggccact ccttggacat tcaagatcaa aaccacttgt 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 tgagtct tt C tagattcaac atacactgga tgagagacca aaacactctt catctcttct aacttctgtt cggatctgtt tagagttaga tact t tcgat tttcagattc ctctatctct aatcactgct tctgttcact tcgcgtttcc gttgtccgag t ccc aac tt t 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 LibC/5O7O84D1 speci 17/23 ttacttctga tggaggaa <210O> <211> 1186 <212> PRI <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 3558 0. 0.

Met Thr 1.

Ile Pro Ala Arg Pro Phe Arg Ile Phe Tyr Trp Giu Thr Giu Asn Ser 130 Arg Asp 145 Leu Giu 30 Gin Giu Leu Leu Thr Ser 21.0 Arg Giu 225 Asn Leu Arg Arg Ser Tyr Arg Giu 290 Phe Ala 305 Ile Glu Gin Leu Met Asn Ser Leu 370 Vai Thr 385 Ser Aia Ile Vali Leu Ser Ile As n 115 Phe Asp Leu Val Leu 195 Gin Tyr Arg Asp Asp 275 Ile Ser Aia Thr Tyr 355 Ser Leu Asn Vali Giu Ser Gly Phe Phe 2.00 Thr Arg Aia Asp Pro 180 Leu Giu Ser Giy Leu 260 Thr Tyr Thr Ala Ile 340 Trp Thr Gin Arg 5 Ser Asp Ala Val Leu 85 Leu Arg Ala Arg Phe 165 Leu Arg Ile Asp Thr 245 Thr Arg Thr Asn Val2 325 Phe Val1 Ser Phe Lys Asn Ser Ser Leu 70 Val Giu Asp Tyr Thr i50 Leu Leu Asp Gin Tyr 230 Asn Leu Val2 Asp Trp, 310 Ile Ser Gly Thr Thr 390 Asn His Leu Thr 55 Gly Gly His Thr Gin 135 Arg Asn Met Ala Arg 22.5 Cys Al a Gly Tyr Pro 295 Phe Arg Val2 His His 375 Ser Glu Ser Cys 40 Val Val Giu Val Ala 120 Gin Ser Ala Val Ser 200 Tyr Ala Giu Val Pro 280 Ile Asn Pro Leu Arg 360 Gly Arg Asn Al a 25 Ile Gin Pro Leu Giu 105 Leu Ser Val Met Tyr 2.85 Leu Tyr Arg Ser Leu 265 Met Gly Asn Pro Ser 345 Leu Asn Asp Glu 2.0 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 Giu Thr Val Ile Ile Met Asn Giu Gly Gly Ile Ala Gly 75 Pro Arg Leu Ile Arg Leu Giu Asp 2.40 Tyr Thr 155 Leu Phe Gin Ala Giy Ser Arg Gin 220 Tyr Asn 235 Leu Arg Leu Val Thr Ser Thr Asn 300 Ala Pro 315 Leu Leu Trp Ser Ser Arg Asn Thr 380 Tyr Arg Asn Ala Leu Ser Asn Asn Asn Ile Gin Ile Giy Arg Arg Gin 110 Gin Gly 2.25 Trp Leu Gin Tyr Ala Ile Ala Asn 190 Giu Phe 205 Val Giu Thr Gly Tyr Asn Ala Leu 270 Ala Gin 285 Ala Pro Ser Phe Asp Phe Asn Thr 350 Thr Ile 365 Ser Ile Thr Giu Leu Thr Ile Ala Al a Asp Gin Leu Giu Ile Arg 2.75 Leu Gly Lys Leu Gin 255 Phe Leu Ser Ser Pro 335 Gin Arg Asn Ser Ser Asp Asp Gly Ser Pro Val Gly Asn Ala 2.60 Asn His Leu Thr Asn 240 Phe Pro Thr Gly Ala 320 Giu Tyr Gly Pro Phe Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp LibC/5O7084Dlspeci 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 25 595 600 605 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 S* 645 650 655 Ala Leu Phe Thr Ser Ser Asn Gin Ile Gly Leu Lys Thr Asp Val Thr .660 665 670 I" 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 40 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 Phe Val Asn Ser 920 Met Ile His Ala 905 Gin Ala Tyr Asp Arg Leu 925 Asp Lys Arg Vai 910 Gin Ala Asp His Ser Ile Aila 930 Arg Giu 935 Giu Aia Tyr Leu 945 Ala Pro 950 Leu Leu Ser Val Ile 955 Phe Pro Gly Val Asn Ala 960 Ile Phe Giu Giu Giu Gly Arg 965 As n Ile 970 Gly Thr Ala Phe Ser Leu 975 Tyr Asp Ser Cys Ala Val Ile Lys Asn 985 Asp Phe Asn His Arg 1010 Giu Val 1025 Tyr Lys Trp Asn Val Lys Gly His Val 995 1000 Ser Val Leu Val Val Pro Giu 1015 Arg Val Cys Pro Gly Arg Gly 1030 Giu Gly Tyr Giy Giu Gly Cys 1045 Thr Asp Giu Leu Lys Phe Ser Trp Giu Tyr Ile 1035 Val Thr Asp Vai Giu Glu Asn Gly Leu 990 Gin Asn Asn Val Ser Gin 1005 *Ala Giu 1020 Leu Arg Val Thr Aia 1040 Ile His Glu Ile Giu 1055 10S0 Asn Asn Asn Cys Val Giu 1060 Tyr Pro Asn Asn Thr Val Thr Cys 1075 1080 Giu Tyr Giu Gly Thr Tyr Thr Ser 1065 As n Asp Tyr Thr Ala 1085 Gly Tyr Giu Giu Val 1070 Thr Gin Giu Asp Gly Aia Arg Asn Arg 1090 1095 1100 Tyr Giu Ser Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu 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 Giy Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Vai Thr Lys 1140 1145 1150 Giu Leu Giu Tyr Phe Pro Glu 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 gaacctttct tgatccattc tggagttctt agagctttgg gattcgtcaa tggaaactct tgctagaact cgctatgcca agctgctaac acttacttct Ctctgactac tgagtcttgg tgttgccttg tactagagag atcatcaacg actgatgcta gtttctgctt ggagttccat cctaggggaa caagttactg ttcagagctt agatctgtgt ttgttcgcta cttcatcttc caagagatt c tgcgctaggt cttagataca ttcccatctt atctacactg ctctttctat gaatcgagga ctactgttca tcgctggaca gagatccttg agaacactag accaacaatc tgtacactca tcagaaacca tt ct t cttag aaagatacta ggtacaacac accagttcag acgatactag atccaatcgg cccagctgtt ttctctttgc aactggaatc gattgcttct ggagat ctt c 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/5O7OB4D1 speci gctccatctg atogaggctg ttotctgttc ottgagtcta tctatcaaoo gctggaatca tggagaaacc gttggtaooo aactacgagt agagctocag gattctatca gtttctggac ct t totatgg tacgctgctt oaaggattoo go tgagt too aacaacgctg actctcgag <210> 17 gattcgcttc cagtgatcag tttotagatg gaaotatoag oagttaotot aoattottot oattgaaoto agttgttoga ottaototoa tgtaotottg oto aga ttoo oaggattoao gattgaaott otoaaaotat oatotaotat oagttggaat gaagaoaaao taotaaotgg aooaooaoat gtctaacact aggatotott toagttoaot taotaotcoa tottagaggt ttotgagaot tagatttot gaotoataga aottgtgaag tggaggagao oaaoaaoaot ggt tottaga gtctgotaac ototgottot tttooacttc 20/23 ttoaaoaaoa ottcttgact oagtaoatga totaottota totagagatg gtgaaoggag toot tgt tgt gagottooao aaoattogtt totgotgata totttoaaot atoatoagaa totottoaaa gttaotgttg gagtototta ggatotoaaa gaoaagattg aogotooato tcccagagca aotaotgggt otoatggaaa tgtaoagaao ttoottgggo aoaooattgg oagagaotao tgatototgg gaaotaaoao tgaaototgg otaacgtgaa gataoagagt gaggatotao ottotoaato otgotggaat agttoattcc tttototgot acttactatc tggaoataga oaotaacact tgagtcttto tagattoaao atacactgga tgagagacca aaacactctt oatctcttct aa t to tgt t oggatotgtt tagagttaga taotttogat tttoagatto ototatotot aatcactgct 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1929 <21 1 >643 <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 1 Ser Asn Arg 5 Lys-Asn Giu Asn Giu Ile Ile Asn Ala Leu Ser Ile Pro Ala Ala Arg Ile Pro Phe Val Val Glu Ser Asn His Ser Asp Ser Leu Cys Ala 25 Ile Met Asn Leu Ala Glu Gly 40 Val Asn Asn Ser Thr Asp Asn Ile Asp Ile Ala Gly Ser Ala Ser Gin Thr Gly Arg Ile Ile Gly Leu Gly Val Phe Leu 70 Val Val Pro Phe Gin Ile Ala Ser Tyr Ser Phe Leu Leu Giy Giu Leu Trp 90 Gin Arg Gly Arg Asp Pro Trp Giu Ile Thr Giu Asn 115 Asn Ser Phe Phe 100 Thr Glu His Val Leu Ile Arg Arg Asp Thr Aia 120 Gin Ala Arg Leu Gin Gin Vai 110 Gly Leu Gly Leu Giu Asn Arg Ala Tyr Ser Leu Giu 130 Arg Asp Asp 140 Thr Asp Aia Arg 145 Leu Thr 150 Leu Ser Val Leu Tyr 155 Gin Tyr Ile Ala 160 Glu Leu Asp Phe 165 Leu Asn Ala Met Pro 170 Ala Leu Phe Ala Ile Arg Asn 175 Gin Ala Ala Asn Leu His Gin Giu Val Leu Leu Leu 195 Thr Ser Gin Leu Met Val Tyr 185 Leu Arg Asp Ala Ser 200 Tyr Phe Gly Ser Giu 205 Val 190 Phe Gly Leu Giu Lys Thr Giu Ile Gin 210 Arg Giu Arg 215 Cys Tyr Glu Arg Tyr Ser Asp Ala Arg Trp 225 Asn Tyr 235 Leu Thr Gly Leu Asn 240 Phe Leu Arg Gly Thr Asn Ala Giu Ser Trp Arg Tyr Asn Gin LibC/507084D1 speci

I,

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 Glu 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 Glu Ala Ala Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro Glu 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 Glu 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 Glu Ser Phe 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val 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 25 435 440 445 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 SThr 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 S545 550 555 560 40 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 <210> 18 <211> 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/507084D1speci aactacaagg aac ctCt cc a ctcggcttcc 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 acaccaccot agctcgagat gacctacgac cgacgtcctc gttcgccggc cgacaacgcc ctccgcccag ggagt acct C cgtgacccag gacggggcca caacctccac gcagggccag ccactgcgtg ctggctcaac cctcttcccg cgagatctac ctggggcaac gc ac ctc tt c caccaactcc cccgaccacc caccaacggc cctctacggc cgccgccaac caacttcaac actcgcgacg caacaccatc cggcggcacg cgacggcggc ccgcatccgc ctccgctgcc 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 gctgtgggca ctccgctacg ggcttcaccc accctggagg atcttcccgt aac ccgac cc ccggctccct 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 catcaaccg ggggcgcatc cttcctcctc ccagatcgag cgacctcacc ggagaggccg cgc ct tcgtg cgcgctcctc cgagatctac ccagcaggag ggaggacgtg gatgaccctc cccgaacggc cc cg ccggc c 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 a a.

<212> PRT <213> Artificial Sequence <220> <223> Toxin encoded by synthetic B.t. gene <400> 19 Met Asn Arg Asn Asn Pro Asn Giu Tyr Ile Ile Asp Ala Pro Tyr 1 Cys Gly Cys Pro Ser Asp Asp Asp Val1 As n Tyr Pro Leu Ala Ser Asp Leu Gin Thr Pro Asn Ala Tyr Asp Gly Ala Phe Gin Asn Met Ser Tyr Lys Giu Asp Tyr Thr Pro Gly 55 Gin Leu Ile Asn Pro Ile Leu Ser Ile Asn Leu Arg Asp Val Thr Gly Ile Val Giy Arg Ile Gly Phe Leu Gly Asn Val Pro Phe Ala Gin Leu Val Thr Phe Tyr Thr Phe Leu Glu Ala Phe Gin Leu Trp Pro 105 Glu Asn Asp Asn Ala Val Trp 110 Lys Ile Ser Ala Gin Ile Leu Ile Asp 115 Val1 Gin 125 Gly Ala Gin 130 Tyr Tyr Val Arg Asn Ala 135 Ala Asp Asp Leu Thr 140 Trp Leu His Asp Giu Giu Tyr Ala Leu Giu Glu Leu Giu Arg 145 As n 155 Arg Pro 160 His Gly Ala Arg Ala Leu Val Thr Gin Phe Glu Asn Leu LibC/50708401 speci 23/23 0 :o4: 0400* :00.0.

V-0..

0 0.

Thr Gin Leu Gly 225 Arg Leu Arg Phe Leu 305 Asn Ser Leu Asn Ala 385 30 Asn Arg Ser 35 Ala Glu 465 40 Asn Tyr Asp Gly Arg 545 Ala Thr Thr Phe Thr 6251 Tyr Al a Arg His 210 Leu Thr Giu Phe Pro 290 Thr Gin Giu Asn Ser 370 Asn Thr Ala Leu Asn 450 As n Thr Val1 Arg Thr 530 Arg Pro Ser Leu Asn 610 Thr Val Phe Asp 195 Leu Gin Arg Asp Arg 275 Phe Arg Gly Leu Arg 355 Phe Asn Arg Arg Asn 435 Thr Asn Thr Trp Ile 515 Thr Thr Leu Phe Pro 595 Thr Leu Asp Val 180 Ala Leu Gin Ile Val1 260 Arg Tyr Giu Ile Giu 340 Leu Leu Pro Leu Asn 420 Ile Cys Asn Gin Thr 500 Thr Val1 Asp Thr Val 580 Ser Leu Arg Lys 165 Thr Val Leu Gly Tyr 245 Arg Giu Asn Ile Cys 325 Asn Thr Asp Thr Phe 405 Phe Phe Arg Phe Gly 485 Arg Gin Val1 Gly Gin 565 Val1 Thr Giu Leu Leu 645 Arg Ala Leu Gin 230 Thr Gly Met Val Tyr 310 Arg Ala Ile Tyr Thr 390 Asn Gly Pro Gin Asn 470 Gly Glu Leu Lys Gly 550 Gin Asn Met Val Asn 630 Glu Met Leu Lys 215 Ile Asn Thr Thr Arg 295 Thr Arg Phe Ser Trp 375 Tyr Thr Asn Thr Asp 455 Leu Pro Asp Pro Gly 535 Ala Tyr Leu Ala Thr 615 Ile Ile Pro Leu 200 Asp Asn His Asn Leu 280 Gin Asp Trp Ile Arg 360 Ser Giu Thr Leu Gly 440 Leu Leu Leu Val1 Trp 520 Pro Val Arg Phe Gin 600 His Phe Val1 Ser 185 Thr Ala Leu Cys Thr 265 Met Tyr Pro Gly Arg 345 Asn Gly Thr Asn Tyr 425 Val Thr Ser Ala Asp 505 Val Gly Gly Ile Vai 585 Asn Thr Pro Pro Phe Val Giu Tyr Val1 250 Giu Ala Pro Ile Asn 330 Pro Arg His Ser Gly 410 Al a Met Thr His Thr 490 Phe Lys Phe Thr Arg 570 Asn Gly Ile Ser Ile 650 Gly Tyr Ile Phe 235 Glu Ser Met As n Val1 315 Asn Pro Tyr Thr Tyr 395 Al a Asn Ser Thr Val1 475 Leu Thr Al a Thr Ile 555 Leu Asn Ser Arg Ile 635 Asn Thr Ala Tyr 220 Asn Thr Trp Asp Gly 300 Tyr Pro His Thr Leu 380 Gly Arg Leu Giu Giu 460 Thr Gly Asn Ser Gly 540 Arg Arg Ser Leu Phe 620 Ser Pro Gly Gin 205 Gly Ala Tyr Leu Leu 285 Ala Asn Tyr Leu Ala 365 Gin Gin Ala Tyr Ile 445 Glu Phe Phe Thr Giu 525 Gly Ala Tyr Ala Thr 605 Ser Gly Thr Pro 190 Ala Al a Gin Asn Asn 270 Val1 Asn Pro Asn Phe 350 Pro Ser Ile Ile Gly 430 Thr Leu Leu Val1 Ile 510 Ile Asp Asn Al a Ala 590 Tyr Gin Gin Arg Gly Ala Arg Gin Arg 255 Tyr Ala Pro Pro Thr 335 Giu Thr Gin Thr Asp 415 Val Asn Pro Arg Pro 495 Thr Gly Ile Val Ser 575 Gly Giu Ser Giu Giu 655 Ser As n Trp Giu 240 Gly His Leu Gin Ala 320 Phe Arg Thr His Ser 400 Ser Ser Ala Leu Phe 480 Thr Ala Gly Leu Asn 560 Thr Phe Ser Asp Val1 640 LibC/507084D1 speci

Claims (23)

1. A polynucleotide optimised for expression in a plant and which encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, and wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO.

2. The polynucleotide according to claim 1 which encodes the amino acid sequenceof SEQ ID NO 7.

3. A DNA construct comprising a promoter region capable of expression in a plant cell and a polynucleotide optimised for expression in a plant, wherein said polynucleotide is to under the control of said promoter region, and wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, and wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO.

4. The DNA construct according to claim 3, wherein said polynucleotide encodes a pesticidal toxin comprising the amino acid sequence of SEQ ID NO. 7. A DNA construct comprising a promoter region capable of expression in a plant cell and a polynucleotide optimised for expression in a plant, wherein said polynucleotide is under the control of said promoter region, and wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, substantially as hereinbefore described with reference to the example.

6. A method for transforming a host cell to a phenotype capable of expressing a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, said method comprising introducing into said cell a polynucleotide according to claim 1 or claim 2, or a DNA construct S. according to any one of claims 3 to 25 7. The method according to claim 6, wherein said cell is a plant cell.

8. The method according to claim 7, wherein said plant is cotton.

9. The method according to claim 7, wherein said plant is maize. The method according to any one of claims 6 to 9, wherein said polynucleotide DNA construct is introduced into said cell using Agrobacterium-mediated transfer. 30 11. A method for transforming a host cell to a phenotype capable of expressing a o CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, said method comprising introducing 99 9 into said cell a polynucleotide optimised for expression in a plant, wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin and wherein :.said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, said method being substantially as hereinbefore described with reference to the example.

12. A transformed host cell comprising a polynucleotide optimised for expression in a plant, wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB LibC/507084D1speci pesticidal toxin, and wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO.

13. The transformed host cell according to claim 12, wherein said polynucleotide encodes a pesticidal toxin comprising the amino acid sequence of SEQ ID NO. 7.

14. The transformed host cell according to claim 12, which is transformed by the method of any one of claims 6 to 11. The transformed host cell according to any one of claims 12 to 14, wherein said cell is capable of expressing said Cry Ac or chimeric CrylAc/CrylAB pesticidal toxin.

16. The transformed host cell according to claim 15, wherein said cell is a plant cell.

17. The transformed host cell according to claim 16, wherein said plant is cotton.

18. The transformed host cell according to claim 16, wherein said plant is maize.

19. A transformed plant regenerated from a transformed cell according to any one of claims 16 to 18. Is 20. A transformed plant comprising a plurality of transformed cells according to any one of claims 16 to 18.

21. The transformed plant according to claim 20, wherein said plant is cotton.

22. The transformed plant according to claim 20, wherein said plant is maize.

23. Transgenic seed of a plant according to any one of claims 19 to 22, wherein said seed comprises said polynucleotide encoding said CrylAc or chimeric CrylAc/CrylAB pesticidal toxin.

24. A method for producing a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, said method comprising culturing a transformed cell according to any one of claims 15 to 18 or a transformed plant according to any one of claims 19 to 22 under conditions promoting 25 expression of the pesticidal toxin and optionally isolating the pesticidal toxin.

25. A CrylAc or chimeric CrylAc/CrylAB pesticidal toxin produced by a method according to claim 24.

26. A pesticidal composition comprising a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin obtained by a method according to claim 24. 30 27. A pesticidal composition comprising transformed cell(s) according to any one of claims 15 to 18.

28. A pesticidal composition comprising transformed plant(s) according to any one of claims 19 to 22, or parts thereof comprising a CrylAc or chimeric CrylAc/CrylAB o pesticidal toxin. 35 29. 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 optimised for expression in a plant, and wherein said polynucleotide encodes a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin, and wherein said polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ LibC/507084D1 spec 16 ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. The method according to claim 29, wherein said polynucleotide encodes the amino acid sequence of SEQ ID NO. 7.

31. The method according to claim 29 or claim 30, wherein said transformed host is a host cell according to any one of claims 15 to 18 or a transformed plant according to any one of claims 19 to 22.

32. A method for controlling a pest, wherein said method comprises contacting said pest with a CrylAc or chimeric CrylAc/CrylAB pesticidal toxin produced by a method according to claim 24 or a pesticidal composition according to claim 26 or claim 27. Dated 28 April 2004 MYCOGEN CORPORATION Patent Attorneys for the Applicant/Nominated Person SPRUSON&FERGUSON S S r~ 4 S 4* 6C CC .6 S S S *505 I LibC/507084D1speci