Compositions and Methods for Control of Insect Pests

Abstract

Novel insecticidal proteins that are toxic to lepidopteran pests are disclosed. The polynucleotides encoding the insecticidal proteins can be used to transform prokaryotic and eukaryotic organisms to express the insecticidal proteins. The recombinant organisms or compositions containing the recombinant organisms or the insecticidal proteins alone or in combination with other pest control agents and an appropriate agricultural carrier can be used to control lepidopteran pests in various environments.

Description

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] This application is a divisional of U.S. patent application Ser. No. 16/473,467, filed Jun. 25, 2019, which is a 371 of International Application No. PCT/US2017/064897, filed Dec. 6, 2017, which claims priority to U.S. Provisional Application No. 62/442,155, filed Jan. 4, 2017.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "81065-US-ORG-NAT-1_SeqList_ST25.txt", created on Apr. 22, 2022, and having a size of 224 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] This invention relates to pesticidal proteins and the polynucleotides that encode them, as well as compositions and methods for controlling plant pests.

BACKGROUND

[0004] Plant pests are a major factor in the loss of the world's important agricultural crops. About $8 billion are lost every year in the U.S. alone due to infestations of invertebrate pests including insects. In addition to losses in field crops, insect pests are also an economic problem in commodities derived from crop plants, in vegetable and fruit production, and in home gardens.

[0005] Insect pests are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Biological pest control agents, such as Bacillus thuringiensis strains expressing pesticidal toxins such as Cry proteins, have also been applied to crop plants with satisfactory results, offering an alternative or compliment to chemical pesticides. The genes coding for some of these Cry proteins have been isolated and their expression in heterologous hosts such as transgenic plants have been shown to provide another tool for the control of economically important insect pests.

[0006] Good insect control can thus be reached, but certain chemicals can sometimes also affect non-target beneficial insects and certain biologicals have a very narrow spectrum of activity. In addition, the continued use of certain chemical and biological control methods heightens the chance for insect pests to develop resistance to such control measures. This has been partially alleviated by various resistance management practices, but there remains a need to develop new and effective pest control agents that provide an economic benefit to farmers and that are environmentally acceptable. Particularly needed are control agents that can target different spectrums of economically important insect pests and that efficiently control insect strains that are or could become resistant to existing insect control agents.

SUMMARY

[0007] In view of these needs, it is an object of the present invention to provide new pest control agents by providing new Bacillus thuringiensis (Bt) isolates as well as novel genes and pesticidal proteins that may be used to control a variety of plant and commodity pests.

[0008] The invention provides compositions and methods for conferring pesticidal activity to bacteria, plants, plant parts, plant cells, tissues and seeds. In particular, novel polynucleotides that encode Cry proteins isolated from Bt and sequences substantially identical thereto are provided. The invention is further drawn to chimeric genes comprising the novel polynucleotides whose expression results in Cry proteins with toxicity to economically important insect pests, particularly insect pests that infest plants or commodities derived from plants. The invention is further drawn to the novel Cry proteins resulting from the expression of the polynucleotides, and to compositions and formulations containing the Cry proteins, which are toxic to insects by inhibiting the ability of insect pests to survive, grow and reproduce, or of limiting insect-related damage or loss to crop plants or commodities derived from crop plants. Cry proteins of the invention include native Cry proteins and their variants as well as modified Cry proteins that have one or more amino acid substitutions, additions or deletions. Examples of modified Cry proteins include without limitation those that are mutated to modulate their biological activity. Such modulation may, for example, broaden or narrow their spectrum of activity, or increase or decrease their specificity compared to their native Cry protein counterparts. Cry proteins of the invention may be mutated to introduce an epitope to generate antibodies that differentially recognize the modified protein from the native protein or they may be mutated to modify expression in a transgenic organism, such as a plant or bacteria. The Cry proteins of the invention are highly active against economically important insect pests, for example, insect pests of crop plants such as black cutworm (BCW; Agrotis ipsilon), European corn borer (ECB; Ostrinia nubilalis), fall armyworm (FAW; Spodoptera frugiperda), corn earworm (CEW; Helicoverpa zea), sugarcane borer (SCB; Diatraea saccharalis), velvetbean caterpillar (VBC; Anticarsia gemmatalis), soybean looper (SBL; Chrysodeixis includens), southwest corn borer (SWCB; Diatraea grandiosella), western bean cutworm (WBC; Richia albicosta), tobacco budworm (TBW; Heliothis virescens), Asian corn borer (ACB; Ostrinia furnacalis), cotton bollworm (CBW; Helicoverpa armigera), striped stem borer (SSB; Chilo suppressalis), pink stem borer (PSB; Sesamia calamistis), rice leaffolder (RLF; Cnaphalocrocis medinalis), and the like, or economically important insect pests of stored-products or commodity products derived from crop plants such as whiteshouldered house moth (WHM; Endrosis sarcitrella), brown hose moth (BHM; Hofinannophila pseudospretella), Angoumois grain moth (AGM; Sitotroga cerealella), almond moth (ADM; Cadra cautella), Mediterranean flour moth (MFM; Ephestia kuehniella), Indianmeal moth (IMM; Plodia interpunctella), European grain moth (EGM; Nemapogon granella), and the like.

[0009] The invention also provides synthetic polynucleotides that encode the Cry proteins of the invention that have one or more codons optimized for expression in transgenic organisms such as transgenic bacteria or transgenic plants.

[0010] The invention is further drawn to expression cassettes and recombinant vectors comprising a polynucleotide that encodes a Cry protein of the invention. The invention also provides transformed bacteria, plants, plant parts, plant cells, tissues, and seeds comprising a chimeric gene, or an expression cassette or a recombinant vector which are useful in expressing a Cry protein of the invention in the transformed bacteria, plants, plant cells, tissues and seeds.

[0011] The invention is also drawn to isolated Bacillus thuringiensis (Bt) strains that produce the Cry proteins of the invention. Such Bt strains may be a naturally occurring isolate or a recombinant Bt strain which produces one or more of the Cry proteins of the invention.

[0012] The invention is also drawn to methods of using polynucleotides of the invention, for example in DNA constructs or chimeric genes or expression cassettes or recombinant vectors for transformation and expression in organisms, including plants and microorganisms, such as bacteria. The nucleotide or amino acid sequences may be native or synthetic sequences that have been designed for expression in an organism such as a plant or bacteria or in making hybrid Cry toxins with enhanced pesticidal activity. The invention is further drawn to methods of making the Cry proteins and to methods of using the polynucleotide sequences and Cry proteins, for example in microorganisms to control insects or in transgenic plants to confer protection from insect damage.

[0013] Another aspect of the invention includes insecticidal compositions and formulations comprising the Cry proteins or the Bacillus thuringiensis strains of the invention, and methods of using the compositions or formulations to control insect pest populations, for example by applying the compositions or formulations to insect-infested areas, or to prophylactically treat insect-susceptible areas or plants to confer protection against the insect pests. Optionally, the compositions or formulations of the invention may, in addition to the Cry protein or Bt strain of the invention, comprise other pesticidal agents such as chemical or biological pesticides in order to augment or enhance the insect-controlling capability of the compositions or formulations of the invention.

[0014] The compositions and methods of the invention are useful for controlling insect pests that attack plants, particularly crop plants or commodity products derived from crop plants. The compositions of the invention are also useful for generating altered or improved Cry proteins that have pesticidal activity, or for detecting the presence of a Cry protein or polynucleotides in commercial products or transgenic organisms.

[0015] These and other features, aspects, and advantages of the invention will become better understood with reference to the following detailed description and claims.

BRIEF DESCRIPTION OF THE SEVERAL DRAWINGS

[0016] FIG. 1A-E shows an alignment of a BT2Cry1J protein and various embodiments of modified BT2Cry1J proteins.

[0017] FIG. 2A-B shows an alignment of a Cry1Ig1 protein, a BT25Cry1I protein and a variant BT25Cry1I protein.

[0018] FIG. 3A-B shows an alignment of a Cry1Ja1 protein, a BT2Cry1J protein and a variant BT2Cry1J protein.

[0019] FIG. 4A-B shows an alignment of a Cry1k1 protein, a BT53Cry1J protein and a variant B T53 Cry1J protein.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0020] SEQ ID NO:1 is a BT2Cry1J amino acid sequence.

[0021] SEQ ID NO:2 is a BT25Cry1I amino acid sequence.

[0022] SEQ ID NO:3 is a BT53Cry1J amino acid sequence.

[0023] SEQ ID NO:4 is a variant BT2Cry1J amino acid sequence.

[0024] SEQ ID NO:5 is a variant BT25Cry1I amino acid sequence.

[0025] SEQ ID NO:6 is a variant BT53Cry1J amino acid sequence.

[0026] SEQ ID NO:7 is a .alpha.-helix3 modified BT2Cry1J amino acid sequence.

[0027] SEQ ID NO:8 is a .alpha.-helix4 modified BT2Cry1J amino acid sequence.

[0028] SEQ ID NO:9 is a .alpha.-helix5/6 modified BT2Cry1J amino acid sequence.

[0029] SEQ ID NO:10 is a .alpha.-helix3/4 modified BT2Cry1J amino acid sequence.

[0030] SEQ ID NO:11 is a .alpha.-helix4/5/6 modified BT2Cry1J amino acid sequence.

[0031] SEQ ID NO:12 is a .alpha.-helix 3/5/6 modified BT2Cry1J amino acid sequence.

[0032] SEQ ID NO:13 is a .alpha.-helix 3/4/5/6 modified BT2Cry1J amino acid sequence.

[0033] SEQ ID NO:14 is a nucleotide sequence encoding SEQ ID NO:1.

[0034] SEQ ID NO:15 is a nucleotide sequence encoding SEQ ID NO:2.

[0035] SEQ ID NO:16 is a nucleotide sequence encoding SEQ ID NO:3.

[0036] SEQ ID NO:17 is a codon optimized sequence encoding SEQ ID NO:1.

[0037] SEQ ID NO:18 is a codon optimized sequence encoding SEQ ID NO:2.

[0038] SEQ ID NO:19 is a codon optimized sequence encoding SEQ ID NO:3.

[0039] SEQ ID NO:20 is a nucleotide sequence encoding SEQ ID NO: 4.

[0040] SEQ ID NO:21 is a nucleotide sequence encoding SEQ ID NO:5.

[0041] SEQ ID NO:22 is a nucleotide sequence encoding SEQ ID NO:6.

[0042] SEQ ID NO: 23 is a nucleotide sequence encoding SEQ ID NO:7.

[0043] SEQ ID NO: 24 is a nucleotide sequence encoding SEQ ID NO:8.

[0044] SEQ ID NO: 25 is a nucleotide sequence encoding SEQ ID NO:9.

[0045] SEQ ID NO: 26 is a nucleotide sequence encoding SEQ ID NO:10.

[0046] SEQ ID NO: 27 is a nucleotide sequence encoding SEQ ID NO:11.

[0047] SEQ ID NO: 28 is a nucleotide sequence encoding SEQ ID NO:12.

[0048] SEQ ID NO: 29 is a nucleotide sequence encoding SEQ ID NO:13.

[0049] SEQ ID NO:30 is a nucleotide sequence of a shuttle vector.

[0050] SEQ ID NO:31 is a nucleotide sequence of a binary transformation vector.

DETAILED DESCRIPTION

[0051] This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Definitions

[0053] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" is a reference to one or more plants and includes equivalents thereof known to those skilled in the art, and so forth. As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list (i.e., includes also "and").

[0054] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). With regard to a temperature the term "about" means.+-.1.degree. C., preferably .+-.0.5.degree. C. Where the term "about" is used in the context of this invention (e.g., in combinations with temperature or molecular weight values) the exact value (i.e., without "about") is preferred.

[0055] An "active" Cry protein or the "activity" of a Cry protein of the invention is meant that the Cry protein functions as an insect control agent, has a toxic effect, or is able to disrupt or deter insect feeding, which may or may not cause death of the insect. When a Cry protein of the invention is delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the Cry protein available to the insect.

[0056] As used herein, the term "amplified" means the construction of multiple copies of a polynucleotide or multiple copies complementary to the polynucleotide using at least one of the polynucleotides as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleotide sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an "amplicon."

[0057] The term "chimeric construct" or "chimeric gene" or "chimeric polynucleotide" or "chimeric nucleic acid" (or similar terms) as used herein refers to a construct or molecule comprising two or more polynucleotides of different origin assembled into a single polynucleotide. The term "chimeric construct", "chimeric gene", "chimeric polynucleotide" or "chimeric nucleic acid" refers to any construct or molecule that contains, without limitation, (1) polynucleotides (e.g., DNA), including regulatory and coding polynucleotides that are not found together in nature (i.e., at least one of the polynucleotides in the construct is heterologous with respect to at least one of its other polynucleotides), or (2) polynucleotides encoding parts of proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Further, a chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid may comprise regulatory polynucleotides and coding polynucleotides that are derived from different sources, or comprise regulatory polynucleotides and coding polynucleotides derived from the same source, but arranged in a manner different from that found in nature. In some embodiments of the invention, the chimeric construct, chimeric gene, chimeric polynucleotide or chimeric nucleic acid comprises an expression cassette comprising a polynucleotide of the invention under the control of regulatory polynucleotides, particularly under the control of regulatory polynucleotides functional in plants or bacteria.

[0058] A "coding sequence" is a nucleotide sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.

[0059] As used herein, a "codon optimized" sequence means a nucleotide sequence of a recombinant, transgenic, or synthetic polynucleotide wherein the codons are chosen to reflect the particular codon bias that a host cell or organism may have. This is typically done in such a way so as to preserve the amino acid sequence of the polypeptide encoded by the nucleotide sequence that is codon optimized. In certain embodiments, the DNA sequence of the recombinant DNA construct includes sequence that has been codon optimized for the cell (e.g., an animal, plant, or fungal cell) in which the construct is to be expressed. For example, a construct to be expressed in a plant cell can have all or parts of its sequence (e.g., the first gene suppression element or the gene expression element) codon optimized for expression in a plant. See, for example, U.S. Pat. No. 6,121,014, incorporated herein by reference.

[0060] To "control" insects means to inhibit, through a toxic effect, the ability of insect pests to survive, grow, feed, or reproduce, or to limit insect-related damage or loss in crop plants or to protect the yield potential of a crop when grown in the presence of insect pests. To "control" insects may or may not mean killing the insects, although it preferably means killing the insects.

[0061] The terms "comprises" or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or groups thereof.

[0062] As used herein, the transitional phrase "consisting essentially of (and grammatical variants) means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim" and those that do not materially alter the basic and novel characteristic(s)" of the claimed invention. Thus, the term "consisting essentially of" when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

[0063] In the context of the invention, "corresponding to" or "corresponds to" means that when the amino acid sequences of variant or modified Cry proteins are aligned with each other, the amino acids that "correspond to" certain enumerated positions in the variant or modified protein are those that align with these positions in a reference protein but that are not necessarily in these exact numerical positions relative to the particular reference amino acid sequence of the invention. For example, if SEQ ID NO:1 is the reference sequence and is aligned with SEQ ID NO:3, Pro419 of SEQ ID NO:3 "corresponds to" Pro421 of SEQ ID NO:1 or Tyr417 of SEQ ID NO:3 "corresponds to" Asn419 of SEQ ID NO:1.

[0064] As used herein, the term "Cry protein" means an insecticidal protein of a Bacillus thuringiensis crystal delta-endotoxin type. The term "Cry protein" can refer to the protoxin form or any insecticidally active fragment or toxin thereof. Cry proteins from Bacillus thuringiensis have potent insecticidal activity against predominantly lepidopteran, dipteran, and coleopteran pest insects. These proteins also have shown activity against pests in the Orders Hymenoptera, Homoptera, Phthiraptera, Mallophaga, and Acari pest orders, as well as other invertebrate orders such as Nemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson, J. 1993. The Bacillus Thuringiensis family tree. In Advanced Engineered Pesticides. Marcel Dekker, Inc., New York, N.Y.). These proteins were originally classified based primarily on their insecticidal activity. The major classes were Lepidoptera-specific (CryI), Lepidoptera- and Diptera-specific (CryII), Coleoptera-specific (CryIII), Diptera-specific (CryIV), and nematode-specific (CryV) and (CryVI). The proteins were further classified into subfamilies; more highly related proteins within each family were assigned divisional letters such as CryIA, CryIB, CryIC, etc. Even more closely related proteins within each division were given names such as CryIC(a), CryIC(b), etc. The terms "Cry toxin" and "delta-endotoxin" have been used interchangeably with the term "Cry protein." Current nomenclature for Cry proteins and genes is based upon amino acid sequence homology rather than insect target specificity (Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In this more accepted classification, each Cry protein is assigned a unique name incorporating a primary rank (an Arabic number) if it has <45% sequence identity to known named Cry proteins, for example Cry1 or Cry2 and the like; a secondary rank (an uppercase letter) if it has .gtoreq.45% and <75% sequence identity to known named Cry proteins, for example Cry1I or Cry1J and the like; a tertiary rank (a lowercase letter) if it has from 75% to 95% sequence identity to known named Cry proteins, for example Cry1Ja or Cry1Jc and the like; and a quaternary rank (another Arabic number) if it has >95% sequence identity to known named Cry proteins, for example Cry1Ja1 or Cry1Ja2 and the like. In the current classification, Roman numerals have been exchanged for Arabic numerals in the primary rank. For example, "CryIA(a)" under the older nomenclature is now "Cry1Aa" under the current nomenclature. According to Ibrahim et al. (2010, Bioeng. Bugs, 1:31-50), the Cry toxins can still be separated into six major classes according to their insect host specificities and include: Group 1--lepidopteran e.g., Cry1, Cry9 and Cry15); group 2--lepidopteran and dipteran (e.g., Cry2); group 3--coleopteran (Cry3, Cry7 and Cry8); group 4--dipteran (Cry4, Cry10, Cry 11, Cry16, Cry17, Cry19 and Cry20); group 5--lepidopteran and coleopteran (Cry1I); and group 6--nematodes (Cry6). The Cry1I, Cry2, Cry3, Cry10 and Cry11 toxins (73-82 kDa) are unique because they appear to be natural truncations of the larger Cry1 and Cry4 proteins (130-140 kDa).

[0065] Cry proteins are globular protein molecules which accumulate as protoxins in crystalline form during the sporulation stage of Bt. After ingestion by a pest, the crystals are typically solubilized to release protoxins, which can range in size, for example, from 130-140 kDa for many of the lepidopteran-active Cry proteins, such as Cry1 and Cry9, and 60-80 kDa for the coleopteran-active Cry3 proteins and the lepidopteran/dipteran-active Cry2 proteins. After the crystals are solubilized by a susceptible insect the released protoxins are processed by proteases in the insect gut, for example trypsin and chymotrypsin, to produce a protease-resistant core Cry protein toxin. This proteolytic processing involves the removal of amino acids from different regions of the various Cry protoxins. For example, Cry protoxins that are 130-140 kDa are typically activated through the proteolytic removal of an N-terminal peptide of 25-30 amino acids and approximately half of the remaining protein from the C-terminus resulting in an approximately 60-70 kDa mature Cry toxin. The protoxins that are 60-80 kDa, e.g. Cry2 and Cry3, are also processed but not to the same extent as the larger protoxins. The smaller protoxins typically have equal or more amino acids removed from the N-terminus than the larger protoxins but less amino acids removed from the C-terminus. For example, proteolytic activation of Cry2 family members typically involves the removal of approximately 40-50 N-terminal amino acids. Many of the Cry proteins are quite toxic to specific target insects, but many have narrow spectrums of activity.

[0066] Cry proteins generally have five conserved sequence domains, and three conserved structural domains (see, for example, de Maagd et al. (2001) Trends Genetics 17:193-199). The first conserved structural domain, called Domain I, typically consists of seven alpha helices and is involved in membrane insertion and pore formation. Domain II typically consists of three beta-sheets arranged in a Greek key configuration, and domain III typically consists of two antiparallel beta-sheets in `jelly-roll` formation (de Maagd et al., 2001, supra). Domains II and III are involved in receptor recognition and binding, and are therefore considered determinants of toxin specificity.

[0067] The term "Cry1I" refers to any member of a group of Cry proteins having at least 75% sequence identity to the holotype Cry1I protein (NCBI Accession No. CAA44633) and the term "Cry1Ig" refers to any member of a family of Cry1I proteins having at least 95% sequence identity to the holotype Cry1Ig protein (NCBI Accession No. KC156701) according to Crickmore et al. supra., incorporated herein by reference.

[0068] The term "Cry1J" refers to any member of a group of Cry proteins having at least 75% sequence identity to the holotype Cry1J protein (NCBI Accession No. AA22341) and the term "Cry1Ja" refers to any member of a family of Cry proteins having at least 95% sequence identity to the above identified holotype Cry1Ja protein, according to Crickmore et al. supra., incorporated herein by reference. The term "Cry1Jc" refers to any member of a family of Cry proteins having at least 95% sequence identity to the holotype Cry1k1 protein (NCBI Accession No. AAC31092).

[0069] To "deliver" a composition or toxic protein means that the composition or toxic protein comes in contact with an insect, which facilitates the oral ingestion of the composition or toxic Cry protein, resulting in a toxic effect and control of the insect. The composition or toxic Cry protein can be delivered in many recognized ways, including but not limited to, transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized protein delivery system.

[0070] The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide group.

[0071] "Effective insect-controlling amount" means that concentration of a Cry protein that inhibits, through a toxic effect, the ability of insects to survive, grow, feed or reproduce, or limits insect-related damage or loss in crop plants or protects the yield potential of a crop when grown in the presence of insect pests. "Effective insect-controlling amount" may or may not mean an amount that kills the insects, although it preferably means an amount that kills insects.

[0072] "Expression cassette" as used herein means a polynucleotide capable of directing expression of at least one polynucleotide of interest, such as a polynucleotide that encodes a Cry protein of the invention, in an appropriate host cell, comprising a promoter operably linked to the polynucleotide of interest which is operably linked to a termination signal. An "expression cassette" also typically comprises additional polynucleotides required for proper translation of the polynucleotide of interest. The expression cassette may also comprise other polynucleotides not necessary in the direct expression of a polynucleotide of interest but which are present due to convenient restriction sites for removal of the cassette from an expression vector. The expression cassette comprising the polynucleotide(s) of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e. the polynucleotide of interest in the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation process or a breeding process. The expression of the polynucleotide(s) of interest in the expression cassette is generally under the control of a promoter. In the case of a multicellular organism, such as a plant, the promoter can also be specific or preferential to a particular tissue, or organ, or stage of development. An expression cassette, or fragment thereof, can also be referred to as "inserted polynucleotide" or "insertion polynucleotide" when transformed into a plant.

[0073] A "gene" is defined herein as a hereditary unit comprising one or more polynucleotides that occupies a specific location on a chromosome or plasmid and that contains the genetic instruction for a particular characteristic or trait in an organism.

[0074] A "gut protease" is a protease naturally found in the digestive tract of an insect. The gut protease is typically involved in the digestion of ingested proteins. Examples of insect gut proteases include trypsin, which typically cleaves peptides on the C-terminal side of lysine (K) or arginine (R) residues, and chymotrypsin, which typically cleaves peptides on the C-terminal side of phenylalanine (F), tryptophan (W) or tyrosine (Y).

[0075] The term "heterologous" when used in reference to a gene or a polynucleotide or a polypeptide refers to a gene or a polynucleotide or a polypeptide that is or contains a part thereof not in its natural environment (i.e., has been altered by the hand of man). For example, a heterologous gene may include a polynucleotide from one species introduced into another species. A heterologous gene may also include a polynucleotide native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.). Heterologous genes further may comprise plant gene polynucleotides that comprise cDNA forms of a plant gene; the cDNAs may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). In one aspect of the invention, heterologous genes are distinguished from endogenous plant genes in that the heterologous gene polynucleotide are typically joined to polynucleotides comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene polynucleotide in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed). Further, a "heterologous" polynucleotide refers to a polynucleotide not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.

[0076] "Homologous recombination" is the exchange ("crossing over") of DNA fragments between two DNA molecules or chromatids of paired chromosomes in a region of identical polynucleotides. A "recombination event" is herein understood to mean a meiotic crossing-over.

[0077] A nucleotide sequence is "isocoding" with a reference nucleotide sequence when the nucleotide sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleotide sequence. For example, SEQ ID NO:17 is isocoding with SEQ ID NO: 14 because they both encode the amino acid sequence represented by SEQ ID NO:1.

[0078] The term "isolated" polynucleotide or protein is a polynucleotide or protein that no longer exists in its natural environment. An isolated polynucleotide or protein of the invention may exist in a purified form or may exist in a recombinant host such as in a transgenic bacteria or a transgenic plant. Therefore, it is intended that a claim directed to an "isolated polynucleotide" encompasses that polynucleotide when the polynucleotide is comprised within a plant.

[0079] "Operably linked" refers to the association of polynucleotides on a single polynucleotide fragment so that the function of one affects the function of the other. For example, a promoter is operably linked with a coding polynucleotide or functional RNA when it is capable of affecting the expression of that coding polynucleotide or functional RNA (i.e., that the coding polynucleotide or functional RNA is under the transcriptional control of the promoter). Coding polynucleotide in sense or antisense orientation can be operably linked to regulatory polynucleotides.

[0080] As used herein "pesticidal," insecticidal," and the like, refer to the ability of a Cry protein of the invention to control a pest organism or an amount of a Cry protein that can control a pest organism as defined herein. Thus, a pesticidal Cry protein can kill or inhibit the ability of a pest organism (e.g., insect pest) to survive, grow, feed, or reproduce.

[0081] A "plant" is any plant at any stage of development, particularly a seed plant.

[0082] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0083] "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

[0084] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0085] A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.

[0086] "Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0087] A "polynucleotide" refers to a polymer composed of many nucleotide monomers covalently bonded in a chain. Such "polynucleotides" includes DNA, RNA, modified oligo nucleotides (e.g., oligonucleotides comprising bases that are not typical to biological RNA or DNA, such as 2'-O-methylated oligonucleotides), and the like. In some embodiments, a polynucleotide can be single-stranded, double-stranded, multi-stranded, or combinations thereof. Unless otherwise indicated, a particular polynucleotide of the present invention optionally comprises or encodes complementary polynucleotides, in addition to any polynucleotide explicitly indicated.

[0088] "Polynucleotide of interest" refers to any polynucleotide which, when transferred to an organism, e.g., a plant, confers upon the organism a desired characteristic such as insect resistance, disease resistance, herbicide tolerance, antibiotic resistance, improved nutritional value, improved performance in an industrial process, production of commercially valuable enzymes or metabolites or altered reproductive capability.

[0089] The term "promoter" refers to a polynucleotide, usually upstream (5') of its coding polynucleotide, which controls the expression of the coding polynucleotide by providing the recognition for RNA polymerase and other factors required for proper transcription.

[0090] A "protoplast" is an isolated plant cell without a cell wall or with only parts of the cell wall.

[0091] As used herein, the term "recombinant" refers to a form of polynucleotide (e.g., DNA or RNA) or protein or an organism that would not normally be found in nature and as such was created by human intervention. As used herein, a "recombinant polynucleotide" is a polynucleotide comprising a combination of polynucleotides that would not naturally occur together and is the result of human intervention, e.g., a polynucleotide that is comprised of a combination of at least two polynucleotides heterologous to each other, or a polynucleotide that is artificially synthesized and comprises a polynucleotide that deviates from the polynucleotide that would normally exist in nature, or a polynucleotide that comprises a transgene artificially incorporated into a host cell's genomic DNA and the associated flanking DNA of the host cell's genome. An example of a recombinant polynucleotide is a DNA molecule resulting from the insertion of a transgene into a plant's genomic DNA, which may ultimately result in the expression of a recombinant RNA or protein molecule in that organism. A "recombinant protein" is a protein encoded by a recombinant polynucleotide that has been cloned in a system that supports expression of the polynucleotide and translation of mRNA. For example, a Cry protein having a native amino acid sequence or a mutated amino acid sequence and expressed in a plant is a recombinant Cry protein. As used herein, a "recombinant plant" is a plant that would not normally exist in nature, is the result of human intervention, and contains a transgene or heterologous polynucleotide incorporated into its genome. As a result of such genomic alteration, the recombinant plant is distinctly different from the related wild-type plant.

[0092] "Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

[0093] The term "identity" or "identical" or "substantially identical," in the context of two nucleotide or amino acid sequences, refers to two or more sequences or subsequences that have at least 60%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues or bases in length, more preferably over a region of at least about 100 residues or bases, and most preferably the sequences are substantially identical over at least about 150 residues or bases. In an especially preferred embodiment, the sequences are substantially identical over the entire length of the coding regions. Furthermore, substantially identical nucleotide or amino acid sequences perform substantially the same function.

[0094] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0095] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0096] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, Md. 20894 USA). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89: 10915 (1989)).

[0097] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0098] Another indication that two nucleotide sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleotide and a target nucleotide and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleotide sequence.

[0099] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of polynucleotide hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleotides is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but not to other sequences.

[0100] The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T.sub.m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary polynucleotides which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42.degree. C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15M NaCl at 72.degree. C. for about 15 minutes. An example of stringent wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6.times.SSC at 40.degree. C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30.degree. C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2.times. (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Polynucleotides that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a polynucleotide is created using the maximum codon degeneracy permitted by the genetic code.

[0101] The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree. C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC, 0.1% SDS at 50.degree. C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C., preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

[0102] A further indication that two proteins are substantially identical is that an antibody raised against a protein encoded by a first polynucleotide is immunologically cross reactive with, or specifically binds to, a protein encoded by a second polynucleotide. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

[0103] "Synthetic" refers to a nucleotide sequence comprising bases or structural features that are not present in the natural sequence. For example, an artificial sequence encoding a Cry protein of the invention that resembles more closely the G+C content and the normal codon distribution of dicot or monocot plant genes is said to be synthetic.

[0104] As used herein, "toxic" is synonymous with "insecticidal" and is meant that a Cry protein of the invention has a negative effect on an insect pest by killing the insect pest, or by disrupting or deterring feeding of the insect pest, or causing growth inhibition to the insect pest, both of which may or may not cause death of the insect. When a Cry protein of the invention is delivered to an insect or an insect comes into oral contact with the Cry protein, the toxic effect is typically death of the insect, or the insect's growth is slowed, or the insect stops feeding upon the source that makes the toxic Cry protein available to the insect.

[0105] "Transformation" is a process for introducing a heterologous polynucleotide into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of an organism of interest.

[0106] "Transformed/transgenic/recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous polynucleotide has been introduced. The polynucleotide can be stably integrated into the genome of the host or the polynucleotide can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous polynucleotide.

[0107] Nucleotides are indicated herein by their bases by the following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0108] This invention provides compositions and methods for controlling harmful pests of crop plants and commodities derived from crop plants. Particularly, the invention relates to Cry proteins that may be isolated from bacteria, such as Bacillus thuringiensis, that are toxic to insect pests and to polynucleotides that comprise nucleotide sequences that encode the Cry proteins, and to the making and using of the polynucleotides and Cry proteins to control insect pests.

[0109] Polynucleotides that are fragments of Cry protein protoxin-encoding polynucleotides are also encompassed by the invention. By "fragment" is intended a portion of the nucleotide sequence encoding a Cry protein. A fragment of a nucleotide sequence may encode a biologically active portion of a Cry protein, the so called "toxin fragment," or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. Polynucleotides that are fragments of a Cry protein encoding nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 contiguous nucleotides, or up to the number of nucleotides present in a full-length Cry protein encoding nucleotide sequence disclosed herein (for example, 3504 nucleotides for SEQ ID NO:14) depending upon the intended use. By "contiguous" nucleotides is intended nucleotide residues that are immediately adjacent to one another. Some fragments of the nucleotide sequences of the invention will encode toxin fragments that retain the biological activity of the Cry protein and, hence, retain insecticidal activity. By "retains insecticidal activity" is intended that the fragment will have at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 80% of the insecticidal activity of the Cry protein. Methods for measuring insecticidal activity are well known in the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No. 5,743,477, all of which are herein incorporated by reference in their entirety.

[0110] A toxin fragment of a Cry protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, and 450 contiguous amino acids, or up to the total number of amino acids present in a full-length Cry protein of the invention (for example, 1167 amino acids for SEQ ID NO:1). Thus, in some embodiments, Cry proteins which have been activated by means of proteolytic processing, for example, by proteases prepared from the gut of an insect, may be characterized and the N-terminal or C-terminal amino acids of the activated toxin fragment identified. In this aspect of the invention, the skilled person can determine that, for example, a toxin fragment of SEQ ID NO:1 may comprise amino acids from about position 28, 30, 36, 41, 43, 44, 45, 47 or 48 to about position 610, 615, 616, 622, 623, 624 or 625 of SEQ ID NO:1, or a toxin fragment of SEQ ID NO:2 may comprise amino acids from about position 21, 23, 28, 35, 38, 46 or 61 to about position 607, 611, 618, 625 or 628 of SEQ ID NO:2, or a toxin fragment of SEQ ID NO:3 may comprise amino acids from about position 23, 24, 28, 30, 32, 33, 35, 36, 37 or 40 to about position 611, 616, 617, 625, 626, 647, 653 or 654 of SEQ ID NO:3. Cry protein variants produced by introduction or elimination of protease processing sites at appropriate positions in the coding sequence to allow, or eliminate, proteolytic cleavage of a larger variant protein by insect, plant or microorganism proteases are also within the scope of the invention. The end result of such manipulation is understood to be the generation of toxin fragment molecules having the same or different activity as the intact protoxin Cry protein.

[0111] According to some embodiments, the invention provides a polynucleotide or optionally an isolated polynucleotide comprising a nucleotide sequence encoding a Cry protein in its protoxin form or a toxin fragment thereof that is toxic to a lepidopteran pest, wherein the nucleotide sequence (a) has at least 80% to at least 99% sequence identity with any of SEQ ID NOs:14-16 or a toxin-encoding fragment of any of SEQ ID NOs:14-16; or (b) encodes a protein comprising an amino acid sequence that has at least 80% to at least 99% sequence identity with any of SEQ ID NOs:1-3 or a toxin fragment of any of SEQ ID NOs:1-3; or (c) is a synthetic sequence of (a) or (b) that has codons optimized for expression in a transgenic organism.

[0112] In other embodiments, the lepidopteran pest is selected from the group consisting of European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella), western bean cutworm (Richia albicosta), tobacco budworm (Heliothis virescens), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armigera), striped stem borer (Chilo suppressalis), pink stem borer (Sesamia calamistis) and rice leaffolder (Cnaphalocrocis medinalis).

[0113] In still other embodiments, the nucleotide sequence or the synthetic nucleotide sequence comprises any of SEQ ID NOs:14-29 or a toxin-encoding fragment of any of SEQ ID NOs:14-29. In other embodiments, the synthetic nucleotide sequence comprises any of SEQ ID NOs:17-22 or a toxin-encoding fragment of any of SEQ ID NOs:17-22.

[0114] In some embodiments, a polynucleotide of the invention comprises, consists essentially of or consists of a nucleotide sequence encoding a Cry protein comprising an amino acid sequence that has at least 80% to at least 99% sequence identity with any of SEQ ID NOs:1-3 or a toxin fragment of any of SEQ ID NOs:1-3. In some other embodiments, the amino acid sequence comprises, consists essentially of or consists of any of SEQ ID NOs:1-13 or a toxin fragment of any of SEQ ID NOs:1-13.

[0115] In some embodiments, the polynucleotide of the invention encodes a Cry protein that is a Cry1I or a Cry1J protein. In other embodiments the Cry1I protein is a Cry1Ig protein. In still other embodiments, the Cry1Ig protein comprises SEQ ID NO:2, SEQ ID NO:5, or a toxin fragment of SEQ ID NO:2 or SEQ ID NO:5. In other embodiments, the Cry1Ig protein is toxic to a lepidopteran pest selected from the group consisting of European corn borer (Ostrinia nubilalis), sugarcane borer (Diatraea saccharalis), soybean looper (Chrysodeixis includes) and southwest corn borer (Diatraea grandiosella). In still other embodiments, the synthetic sequence comprises, consists essentially of or consists of SEQ ID NO:18, or a toxin-encoding fragment thereof.

[0116] In some embodiments, the polynucleotide of the invention encodes a Cry1J protein that is a Cry1Ja or a Cry1Jc protein. In other embodiments, the Cry1Ja protein comprises, consists essentially of or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NOs:7-13 and a toxin fragment of SEQ ID NO:1, SEQ ID NO:4 or any of SEQ ID NOs:7-13. In other embodiments, the Cry1Ja protein is toxic to a lepidopteran pest selected from the group consisting of European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella) and tobacco budworm (Heliothis virescens). In still other embodiments, the synthetic sequence comprises SEQ ID NO:17 or a toxin-encoding fragment thereof.

[0117] In other embodiments, the Cry1Jc protein comprises, consists essentially of or consists of SEQ ID NO:3, SEQ ID NO:6, or a toxin fragment of SEQ ID NO:3 or SEQ ID NO:6. In still other embodiments, the Cry1Jc protein is toxic to a lepidopteran pest selected from the group consisting of European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella) and tobacco budworm (Heliothis virescens). In other embodiments, the synthetic sequence comprises, consists essentially of or consists of SEQ ID NO:19 or a toxin-encoding fragment thereof.

[0118] In some embodiments of the invention, a chimeric gene is provided that comprises a heterologous promoter operably linked to a polynucleotide comprising, consisting essentially of or consisting of a nucleotide sequence that encodes a Cry protein toxic to a lepidopteran pest, wherein the nucleotide sequence (a) has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) to at least 99% (99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) sequence identity with any one of SEQ ID NOs:14-16, or a toxin-encoding fragment of any of SEQ ID NOs:14-16; or (b) encodes a protein comprising an amino acid sequence that has at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) to at least 99% (99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) sequence identity with any one of SEQ ID NOs:1-3, or a toxin fragment of any of SEQ ID NOs:1-3; or (c) is a synthetic sequence of (a) or (b) that has codons optimized for expression in a transgenic organism.

[0119] In other embodiments, the heterologous promoter in the chimeric gene of the invention is operable in multiple bacterial species. In other embodiments, the bacterial species is Bacillus thuringiensis or Escherichia coli. In still other embodiments, the heterologous promoter is a Cry1Ac promoter or variant thereof. In further embodiments, the Cry1Ac promoter comprises, consists essentially of or consists of nucleotides 12-197 of SEQ ID NO:30 or a fragment thereof. In still other embodiments, the heterologous promoter is a Cry1Ac promoter and the polynucleotide comprises, consists essentially of or consists of any of SEQ ID NOs:1-3 or a toxin fragment of any of SEQ ID NOs:1-3.

[0120] In other embodiments, the heterologous promoter in the chimeric gene of the invention is a plant-expressible promoter. For example, without limitation, the plant-expressible promoter can be selected from the group of promoters consisting of ubiquitin, cestrum yellow virus, corn TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5' UTR, corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, potato patatin, lectin, CaMV 35S and S-E9 small subunit RuBP carboxylase promoter.

[0121] In additional embodiments, the protein encoded by the chimeric gene is toxic to one or more lepidopteran pests selected from the group consisting of European corn borer (ECB; Ostrinia nubilalis), black cutworm (BCW; Agrotis ipsilon), fall armyworm (FAW; Spodoptera frugiperda), corn earworm (CEW; Helicoverpa zea), sugarcane borer (SCB; Diatraea saccharalis), velvetbean caterpillar (VBC; Anticarsia gemmatalis), soybean looper (SBL; Chrysodeixis includens), southwest corn borer (SWCB; Diatraea grandiosella), western bean cutworm (WBC; Richia albicosta), tobacco budworm (TBW; Heliothis virescens), Asian corn borer (ACB; Ostrinia furnacalis), cotton bollworm (CBW; Helicoverpa armigera), striped stem borer (SSB; Chilo suppressalis), pink stem borer (PSB; Sesamia calamistis) and rice leaffolder (RLF; Cnaphalocrocis medinalis).

[0122] In further embodiments, the chimeric gene comprises a polynucleotide that comprises, consists essentially of or consists of a nucleotide sequence that has at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:14, or a toxin-encoding fragment thereof, or has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:15, or a toxin-encoding fragment thereof, or has at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:16, or a toxin-encoding fragment thereof.

[0123] In other embodiments, the polynucleotide comprises, consists essentially of or consists of any of SEQ ID NOs:14-29, or a toxin-encoding fragment of any of SEQ ID NOs:14-29.

[0124] In still other embodiments, the polynucleotide comprises, consists essentially of or consists of a nucleotide sequence that encodes a protein comprising, consisting essentially of or consisting of an amino acid sequence that has at least 80% to at least 99% sequence identity with any one of SEQ ID NOs:1-3, or a toxin fragment of any of SEQ ID NOs:1-3.

[0125] In still other embodiments, the amino acid sequence has at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:1, or a toxin fragment thereof.

[0126] In further embodiments, the amino acid sequence has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:2, or a toxin fragment thereof.

[0127] In still further embodiments, the amino acid sequence has at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:3, or a toxin fragment thereof.

[0128] In other embodiments, the amino acid sequence comprises, consists essentially of or consists of any of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs:1-13.

[0129] In some embodiments, the chimeric gene of the invention comprises a polynucleotide comprising a synthetic nucleotide sequence that has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity over the entire length of any of SEQ ID NOs:14-22, wherein the synthetic sequence has codons optimized for expression is a transgenic organism. In other embodiments, the chimeric gene of the invention comprises a polynucleotide comprising a synthetic sequence of a nucleotide sequence that encodes a protein comprising an amino acid sequence that has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity over the entire length of any of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs:1-13, wherein the synthetic sequence has codons optimized for expression is a transgenic organism. In other embodiments, the protein comprises, consists essentially of or consists of any of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs:1-13. In further embodiments, the transgenic organism is a transgenic bacteria or a transgenic plant. In still other embodiments, the transgenic bacteria is Escherichia coli or Bacillus thuringiensis. In other embodiments, the transgenic plant is Zea mays.

[0130] In some embodiments, the chimeric gene of the invention comprises a polynucleotide that encodes a Cry 1I or a Cry 1J protein. In other embodiments, the Cry 1I protein is a Cry1Ig protein. In other embodiments, the Cry1Ig protein comprises SEQ ID NO:2, SEQ ID NO:5, or a toxin fragment of SEQ ID NO:2 or SEQ ID NO:5. In still other embodiments, the Cry1Ig is toxic to European corn borer (Ostrinia nubilalis), sugarcane borer (Diatraea saccharalis), soybean looper (Chrysodeixis includens) and southwest corn borer (Diatraea grandiosella). In other embodiments, the Cry1Ig protein is encoded by a synthetic polynucleotide comprising, consisting essentially of or consisting of SEQ ID NO:18 or a toxin-encoding fragment thereof.

[0131] In some embodiments, the chimeric gene encodes a Cry1J protein that is a Cry1Ja or a Cry1Jc protein. In other embodiments, the Cry1Ja protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NOs:7-13 and a toxin fragment of SEQ ID NO:1, SEQ ID NO:4 or any of SEQ ID NOs:7-13. In other embodiments, the Cry1Ja protein is toxic to European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella) and tobacco budworm (Heliothis virescens). In other embodiments, the Cry1Ja protein is encoded by a synthetic polynucleotide comprising, consisting essentially of or consisting of SEQ ID NO:17 or a toxin-encoding fragment thereof.

[0132] In some embodiments, the chimeric gene encodes a Cry1J protein that is a Cry1k protein. In other embodiments, the Cry1Jc protein comprises SEQ ID NO:3, SEQ ID NO:6, or a toxin fragment of SEQ ID NO:3 or SEQ ID NO:6. In still other embodiments, the Cry1Jc protein is toxic to European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella) and tobacco budworm (Heliothis virescens). In other embodiments, the Cry1Jc protein is encoded by a synthetic polynucleotide comprising, consisting essentially of or consisting of SEQ ID NO:19 or a toxin-encoding fragment thereof.

[0133] In some embodiments, the invention provides a synthetic polynucleotide comprising, consisting essentially of or consisting of a nucleotide sequence that encodes a protein that is toxic to a lepidopteran pest, wherein the nucleotide sequence has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with any of SEQ ID NOs:14-22, or a toxin-encoding fragment of any of SEQ ID NOs: 14-22.

[0134] In other embodiments, the invention provides a synthetic polynucleotide comprising, consisting essentially of or consisting of a nucleotide sequence that encodes a protein that is toxic to a lepidopteran pest, wherein the nucleotide sequence encodes an amino acid sequence that has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity over the entire length of any one of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs:1-13. In other embodiments, the synthetic polynucleotide encodes a protein comprising, consisting essentially of or consisting of any of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs:1-13.

[0135] Cry proteins of the invention may be isolated from certain Bacillus thuringiensis (Bt) strains such as SC0532, SC0705 and SC0666 described herein. It will be recognized that Cry proteins of the invention may also be isolated from other Bt strains and that such Bt strains can be isolated by standard techniques and tested for the presence of the Cry proteins of the invention or for toxicity to a lepidopteran pest of the invention. Generally Bt strains can be isolated from any environmental sample, including soil, plant, insect, grain elevator dust, and other sample material by methods known in the art. See, for example, Travers et al. (1987) Appl. Environ. Microbiol. 53:1263-1266; Saleh et al. (1969) Can J. Microbiol. 15:1101-1104; DeLucca et al. (1981) Can J. Microbiol. 27:865-870; and Norris, et al. (1981) "The genera Bacillus and Sporolactobacillus," In Starr et al. (eds.), The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria, Vol. II, Springer-Verlog Berlin Heidelberg; all incorporated herein by reference. After isolation, Bt strains can be tested for toxicity to a lepidopteran pest and one or more Cry proteins encompassed by the invention can be identified using, for example, the nucleotide or amino acid sequences disclosed herein, and molecular techniques standard in the art. Therefore, in some embodiments, the invention encompasses a Bacillus thuringiensis (Bt) strain that produces a Cry protein or a recombinant Cry protein comprising, consisting essentially of or consisting of an amino acid sequence having at least 80% to at least 99% sequence identity to any of SEQ ID NOs: 1-13, or a toxin fragment of any of SEQ ID NOs:1-13. In other embodiments, the Bt strain is selected from the group consisting of SC0532, SC0705 and SC0666. In still further embodiments, the Cry protein or recombinant Cry protein comprises, consists essentially of or consists of any of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs:1-13.

[0136] According to some embodiments, the invention provides a Cry protein or an optionally isolated Cry protein or a recombinant Cry protein that is toxic to a lepidopteran pest, wherein the Cry protein, optionally isolated Cry protein or recombinant Cry protein comprises, consists essentially of or consists of (a) an amino acid sequence that has at least 80% sequence identity to at least 99% sequence identity with any one of SEQ ID NOs:1-6, or a toxin fragment of any of SEQ ID NOs:1-6; or (b) an amino acid sequence that is encoded by a nucleotide sequence that has at least 80% sequence identity to at least 99% sequence identity with a nucleotide sequence represented by any one of SEQ ID NOs:14-19, or a toxin-encoding fragment of any of SEQ ID NOs:14-19.

[0137] In other embodiments, the Cry protein or optionally isolated Cry protein or recombinant Cry protein comprises, consists essentially of or consists of an amino acid sequence that has at least 80% to at least 99% sequence identity with any one of SEQ ID NOs:1-6., or a toxin fragment of any of SEQ ID NOs:1-6. In still other embodiments, the amino acid sequence has at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:1, or a toxin fragment thereof.

[0138] In further embodiments, the amino acid sequence has at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:2, or a toxin fragment thereof.

[0139] In still further embodiments, the amino acid sequence has at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:3, or a toxin fragment thereof.

[0140] In some embodiments, the amino acid sequence comprises, consists essentially of or consists of any one of SEQ ID NOs:1-13, or a toxin fragment thereof. In other embodiments, the amino acid sequence is encoded by a nucleotide sequence comprising, consisting essentially of or consisting of any of SEQ ID NOs:14-29, or a toxin-encoding fragment of any of SEQ ID NOs:14-29.

[0141] In other embodiments, the Cry protein or optionally isolated Cry protein or recombinant Cry protein of the invention is toxic to a lepidopteran pest selected from the group consisting of European corn borer (Ostrinia nubilalis), black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella), western bean cutworm (Richia albicosta), tobacco budworm (Heliothis virescens), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armigera), striped stem borer (Chilo suppressalis), pink stem borer (Sesamia calamistis) and rice leaffolder (Cnaphalocrocis medinalis).

[0142] In other embodiments, the Cry protein, the optionally isolated Cry protein or recombinant Cry protein comprises, consists essentially of or consists of an amino acid sequence that has at least 80% to at least 99% sequence identity with any one of SEQ ID NOs:1-13, or a toxin fragment of any of SEQ ID NOs; 1-13.

[0143] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:1, or a toxin fragment thereof.

[0144] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:2, or a toxin fragment thereof.

[0145] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:3, or a toxin fragment thereof.

[0146] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:4, or a toxin fragment thereof.

[0147] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:5, or a toxin fragment thereof.

[0148] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:6, or a toxin fragment thereof.

[0149] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:7, or a toxin fragment thereof.

[0150] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:8, or a toxin fragment thereof.

[0151] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:9, or a toxin fragment thereof.

[0152] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:10, or a toxin fragment thereof.

[0153] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:11, or a toxin fragment thereof.

[0154] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:12, or a toxin fragment thereof.

[0155] In still other embodiments, the amino acid sequence of the Cry protein, optionally isolated Cry protein or recombinant Cry protein has at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9% sequence identity with SEQ ID NO:13, or a toxin fragment thereof.

[0156] In still further embodiments, the Cry protein, optionally isolated Cry protein or recombinant Cry protein comprises, consists essentially of or consists of an amino acid sequence of any of SEQ ID NOs:4-13, or a toxin fragment thereof. In other embodiments, the recombinant Cry protein is encoded by a nucleotide sequence that comprises, consists essentially of or consists of any of SEQ ID NOs:17-29, or a toxin-encoding fragment thereof.

[0157] In some embodiments, the Cry protein, optionally isolated Cry protein or recombinant Cry protein of the invention is a Cry1I or a Cry1J protein. In other embodiments, the Cry1I protein is a Cry1Ig protein. In other embodiments, the Cry1Ig protein comprises SEQ ID NO:2, SEQ ID NO:5, or a toxin fragment of SEQ ID NO:2 or SEQ ID NO:5. In still other embodiments, the Cry1Ig is toxic to European corn borer (Ostrinia nubilalis), sugarcane borer (Diatraea saccharalis), soybean looper (Chrysodeixis includes) and southwest corn borer (Diatraea grandiosella). In other embodiments, the Cry1Ig protein is encoded by a synthetic polynucleotide comprising, consisting essentially of or consisting of SEQ ID NO:18 or a toxin-encoding fragment thereof.

[0158] In some embodiments, the Cry1J protein, optionally isolated Cry1J protein or recombinant Cry1J protein is a Cry1Ja or a Cry1Jc protein. In other embodiments, the Cry1Ja protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NOs:7-13 and a toxin fragment of SEQ ID NO:1, SEQ ID NO:4 or any of SEQ ID NOs:7-13. In other embodiments, the Cry1Ja protein is toxic to European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella) and tobacco budworm (Heliothis virescens). In other embodiments, the Cry1Ja protein is encoded by a synthetic polynucleotide comprising, consisting essentially of or consisting of SEQ ID NO:17 or a toxin-encoding fragment thereof.

[0159] In some embodiments, the Cry1J protein, optionally isolated Cry1J protein or recombinant Cry1J protein that is a Cry1Jc protein. In other embodiments, the Cry1Jc protein comprises SEQ ID NO:3, SEQ ID NO:6, or a toxin fragment of SEQ ID NO:3 or SEQ ID NO:6. In still other embodiments, the Cry1Jc protein is toxic to European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includes), southwest corn borer (Diatraea grandiosella) and tobacco budworm (Heliothis virescens). In other embodiments, the Cry1Jc protein is encoded by a synthetic polynucleotide comprising, consisting essentially of or consisting of SEQ ID NO:19 or a toxin-encoding fragment thereof.

[0160] Antibodies raised in response to immune challenge by a native or mutant BT2Cry1J, BT25Cry1I and BT53Cry1J Cry protein or related Cry proteins are also encompassed by the invention. Such antibodies may be produced using standard immunological techniques for production of polyclonal antisera and, if desired, immortalizing the antibody-producing cells of the immunized host for sources of monoclonal antibody production. Techniques for producing antibodies to any substance of interest are well known, e.g., as in Harlow and Lane (1988. Antibodies a laboratory manual. pp. 726. Cold Spring Harbor Laboratory) and as in Goding (Monoclonal Antibodies: Principles & practice. 1986. Academic Press, Inc., Orlando, Fla.), both of which are incorporated herein by reference. The present invention encompasses insecticidal proteins that cross-react with antibodies, particularly monoclonal antibodies, raised against one or more of the insecticidal Cry proteins of the present invention.

[0161] The antibodies produced in the invention are also useful in immunoassays for determining the amount or presence of a native or mutant BT2Cry1J, BT25Cry1I and BT53Cry1J or related Cry protein in a biological sample. Such assays are also useful in quality-controlled production of compositions containing one or more of the Cry proteins of the invention or related Cry proteins. In addition, the antibodies can be used to assess the efficacy of recombinant production of one or more of the Cry proteins of the invention or a related protein, as well as for screening expression libraries for the presence of a nucleotide sequence encoding one or more of the Cry proteins of the invention or related protein coding sequences. Antibodies are useful also as affinity ligands for purifying or isolating any one or more of the proteins of the invention and related proteins. The Cry proteins of the invention and proteins containing related antigenic epitopes may be obtained by over expressing full or partial lengths of a sequence encoding all or part of a Cry protein of the invention or a related protein in a preferred host cell.

[0162] It is recognized that DNA sequences that encode a native Cry protein of the invention may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by a native Cry protein of the invention. A Cry protein may be altered in various ways to make a mutant Cry protein including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of any of SEQ ID NOs:1-3, or a toxin fragment of any of SEQ ID NOs:1-3, including up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, or more amino acid substitutions, deletions or insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a native Cry protein can be prepared by mutations in a polynucleotide that encodes the protein. This may also be accomplished by one of several forms of mutagenesis or in directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will possess a desired insecticidal activity. In some embodiments of the invention, nucleotide sequences represented by SEQ ID NOs: 14-16 are altered to introduce amino acid substitutions in the encoded protein resulting in a mutant protein having essentially the same insecticidal properties as the native protein. In other embodiments, the resulting mutant protein is encoded by a synthetic mutant polynucleotide comprising a nucleotide sequence represented by any one of SEQ ID NOs:17-29, or a toxin-encoding fragment of any of SEQ ID NOs:17-29. In other embodiments, the mutant proteins comprise, consist essentially of or consist of an amino acid sequence represented by any one of SEQ ID NOs:4-13, or a toxin fragment of any of SEQ ID NOs:4-13.

[0163] In other embodiments, the insecticidal activity of a native Cry protein of the invention can be modulated by inserting, deleting or substituting amino acids in the native Cry protein amino acid sequence resulting in a modified Cry protein of the invention. For example, a Cry protein's insecticidal activity is modulated where the modified Cry protein is toxic to a wider or narrower range of insects compared to the range of insects that is affected by a native Cry protein. It may be desirable to create a modified Cry protein with toxicity to a wider range of insect pests than the native Cry protein where multiple pests feed on a single crop plant of interest, while a modified Cry protein with toxicity to a narrower range of insects may be desirable where, for example, a particular insect pest that a native Cry protein is active against does not feed on the crop plant into which the modified Cry protein will be expressed. This reduction in target pest range may help to mitigate insect resistance development in multiple cropping system environments, for example, where transgenic corn, transgenic soybean and transgenic cotton are grown in close proximity to each other.

[0164] In some other embodiments the insecticidal activity of a native Cry protein can be modulated by substituting at least one amino acid in alpha-helix 3, alpha-helix 4, alpha-helix 5 or alpha-helix 6 of domain I of the native Cry amino acid sequence with a different amino acid than the one at that position in the native Cry protein. In other embodiments, the native Cry protein is a Cry1J protein. In other embodiments, the Cry1J protein is a Cry1Ja or a Cry1k protein. In still other embodiments, the amino acids in alpha-helix 3 of a native Cry1J protein corresponding to amino acid positions 97, 105, 108, 110, 118 and 119 of SEQ ID NO:1 or SEQ ID NO:3 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1J protein resulting in a modified Cry1J protein. In some embodiments, the amino acid substitutions in alpha-helix 3 of a Cry1Ja protein result in a modified Cry1Ja protein that is toxic is a narrower range of insects than a native Cry1Ja protein. In other embodiments the native Cry1Ja protein comprises SEQ ID NO:1 and the modified Cry1Ja has an A97T, an S105N, an L108I, a G110A, a K118S and a T119D substitution in alpha-helix 3. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:7 or a toxin fragment thereof. In still other embodiments, the modified Cry1Ja has activity against soybean looper and tobacco budworm but little or no activity against European corn borer, sugarcane borer, southwest corn borer, black cutworm, fall armyworm, corn earworm or velvetbean caterpillar compared to a native Cry1Ja protein.

[0165] In some embodiments, the amino acids in alpha-helix 4 of a native Cry1Ja protein corresponding to amino acid positions 123, 126, 130, 131, 136, 138, 139, 149 and 150 of SEQ ID NO:1 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1Ja protein resulting in a modified Cry1Ja protein. In other embodiments, the amino acid substitutions in alpha-helix 4 of the Cry1Ja protein result in a modified Cry1Ja protein that is toxic is a narrower range of insects than a native Cry1Ja protein. In other embodiments the native Cry1Ja protein comprises SEQ ID NO:1 or a toxin fragment thereof and the modified Cry1Ja has a T123E, an R126K, a T130I, an E131D, an I136L, an A138G, a Q139L, a V149I and a V150I substitution in alpha-helix 4. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:8 or a toxin fragment thereof. In still other embodiments, the modified Cry1Ja has activity against soybean looper, velvetbean caterpillar and tobacco budworm but little or no activity against European corn borer, sugarcane borer, southwest corn borer, black cutworm, fall armyworm and corn earworm compared to a native Cry1Ja protein.

[0166] In some embodiments, the amino acids in alpha-helix 5/6 of a native Cry1Ja protein corresponding to amino acid positions 158, 161, 176, 186, 196, 197, 198 and 200 of SEQ ID NO:1 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1Ja protein resulting in a modified Cry1Ja protein. In other embodiments, the amino acid substitutions in alpha-helix 5/6 of the Cry1Ja protein result in a modified Cry1Ja protein having essentially the same insecticidal spectrum of activity as a native Cry1Ja. In other embodiments the native Cry1Ja protein comprises SEQ ID NO:1 or a toxin fragment thereof and the modified Cry1Ja has an L158S; a T161V; a V176I; a T186K; a V196I; an N197R; an R198E and a G200H substitution in alpha-helix 5/6. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:9 or a toxin fragment thereof.

[0167] In some embodiments, the amino acids in alpha-helix 3 and alpha-helix 4 of a native Cry1Ja protein corresponding to amino acid positions 97, 105, 108, 110, 118, 119, 123, 126, 130, 131, 136, 138, 139, 149 and 150 of SEQ ID NO:1 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1Ja protein resulting in a modified Cry1Ja protein. In some embodiments, the amino acid substitutions in alpha-helix 3 and alpha-helix 4 of the Cry1Ja protein result in a modified Cry1Ja protein that is toxic is a narrower range of insects than a native Cry1Ja protein. In other embodiments, the native Cry1Ja protein comprises SEQ ID NO:1 or a toxin fragment thereof and the modified Cry1Ja has an A97T, an S105N, an L108I, a G110A, a K118S, a T119D, a T123E, an R126K, a T130I, an E131D, an I136L, an A138G, a Q139L, a V149I and a V150I substitution in alpha-helix 3 and alpha-helix 4. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:10 or a toxin fragment thereof. In still other embodiments, the modified Cry1J has no or reduced activity against sugarcane borer, FAW and CEW and essentially the same activity against European corn borer, southwest corn borer, black cutworm, soybean looper, velvet bean caterpillar and tobacco budworm compared to a native Cry1Ja protein.

[0168] In some embodiments, the amino acids in alpha-helix 4 and alpha-helix 5/6 of a native Cry1Ja protein corresponding to amino acid positions 123, 126, 130, 131, 136, 138, 139, 149, 150, 158, 161, 176, 186, 196, 197, 198 and 200 of SEQ ID NO:1 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1Ja protein resulting in a modified Cry1Ja protein. In other embodiments, the amino acid substitutions in alpha-helix 4 and alpha-helix 5/6 of the Cry1Ja protein result in a modified Cry1Ja protein that is toxic to a narrower range of insects than a native Cry1Ja protein. In other embodiments, the modified Cry1Ja protein is active against insect pests in the Family Noctuidae but not active against insect pests in the Family Crambidae. In other embodiments the native Cry1Ja protein comprises SEQ ID NO:1 or a toxin fragment thereof and the modified Cry1Ja has a T123E, an R126K, a T130I, an E131D, an I136L, an A138G, a Q139L, a V149I, a V150I, an L158S; a T161V; a V176I; a T186K; a V196I; an N197R; an R198E and a G200H substitution in alpha-helix 4 and alpha-helix 5/6. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:11 or a toxin fragment thereof. In still other embodiments, the modified Cry1Ja protein has no activity against the Crambidae Family members European corn borer, sugarcane borer and SWCB. In other embodiments, the modified Cry1Ja protein has activity against the Noctuidae members black cutworm, fall armyworm, corn earworm, soybean looper, velvetbean caterpillar and tobacco budworm. In still other embodiments, the modified Cry1Ja protein has reduced activity against black cutworm, fall armyworm and corn earworm compared to the native Cry1Ja protein. In still other embodiments, the modified Cry1Ja protein has no activity against European corn borer, sugarcane borer and southwest corn borer and has activity against black cutworm, fall armyworm, corn earworm, soybean looper, velvetbean caterpillar and tobacco budworm.

[0169] In some embodiments, the amino acids in alpha-helix 3 and alpha-helix 5/6 of a native Cry1Ja protein corresponding to amino acid positions 97, 105, 108, 110, 118, 119, 158, 161, 176, 186, 196, 197, 198 and 200 of SEQ ID NO:1 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1Ja protein resulting in a modified Cry1Ja protein. In some embodiments, the amino acid substitutions in alpha-helix 3 and alpha-helix 5/6 of the Cry1Ja protein result in a modified Cry1Ja protein with no insecticidal activity compared to the native Cry1Ja protein. In other embodiments, the native Cry1Ja protein comprises SEQ ID NO:1 or a toxin fragment thereof and the modified Cry1Ja has an A97T, an S105N, an L108I, a G110A, a K118S, a T119D, an L158S; a T161V; a V176I; a T186K; a V196I; an N197R; an R198E and a G200H substitution in alpha-helix 3 and alpha-helix 5/6. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:12 or a toxin fragment thereof.

[0170] In some embodiments, the amino acids in alpha-helix 3, alpha-helix 4 and alpha-helix 5/6 of a native Cry1Ja protein corresponding to amino acid positions 97, 105, 108, 110, 118, 119, 123, 126, 130, 131, 136, 138, 139, 149, 150, 158, 161, 176, 186, 196, 197, 198 and 200 of SEQ ID NO:1 are substituted with an amino acid different from the amino acid that is present at these positions in the native Cry1Ja protein resulting in a modified Cry1Ja protein. In some embodiments, the amino acid substitutions in alpha-helix 3, alpha-helix 4 and alpha-helix 5/6 of the Cry1Ja protein result in a modified Cry1Ja protein with the same insecticidal activity as a native Cry1Ja protein. In other embodiments, the native Cry1Ja protein comprises SEQ ID NO:1 or a toxin fragment thereof and the modified Cry1Ja has an A97T, an S105N, an L108I, a G110A, a K118S, a T119D, a T123E, an R126K, a T130I, an E131D, an I136L, an A138G, a Q139L, a V149I, a V150I, an L158S; a T161V; a V176I; a T186K; a V196I; an N197R; an R198E and a G200H substitution in alpha-helix 3, alpha-helix 4 and alpha-helix 5/6. In still other embodiments, the modified Cry1Ja comprises SEQ ID NO:13 or a toxin fragment thereof.

[0171] It is understood that the ability of an insecticidal protein to confer insecticidal activity may be improved by the use of such techniques upon the compositions of this invention. For example, one may express a Cry protein in host cells that exhibit high rates of base mis-incorporation during DNA replication, such as XL-1 Red (Stratagene, La Jolla, Calif.). After propagation in such strains, one can isolate the DNA (for example by preparing plasmid DNA, or by amplifying by PCR and cloning the resulting PCR fragment into a vector), culture the Cry protein mutations in a non-mutagenic strain, and identify mutated genes with insecticidal activity, for example by performing an assay to test for insecticidal activity. Generally, the protein is mixed and used in feeding assays. See, for example Marrone et al. (1985) J. of Economic Entomology 78:290-293. Such assays can include contacting plants with one or more pests and determining the plant's ability to survive or cause the death of the pests. Examples of mutations that result in increased toxicity are found in Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.

[0172] Alternatively, alterations may be made to an amino acid sequence of the invention at the amino or carboxy terminus without substantially affecting activity. This can include insertions, deletions, or alterations introduced by modern molecular methods, such as PCR, including PCR amplifications that alter or extend the protein coding sequence by virtue of inclusion of amino acid encoding sequences in the oligonucleotides utilized in the PCR amplification. Alternatively, the protein sequences added can include entire protein-coding sequences, such as those used commonly in the art to generate protein fusions. Such fusion proteins are often used to (1) increase expression of a protein of interest (2) introduce a binding domain, enzymatic activity, or epitope to facilitate either protein purification, protein detection, or other experimental uses known in the art (3) target secretion or translation of a protein to a subcellular organelle, such as the periplasmic space of Gram-negative bacteria, or the endoplasmic reticulum of eukaryotic cells, the latter of which often results in glycosylation of the protein.

[0173] A Cry protein of the invention can also be mutated to introduce an epitope to generate antibodies that recognize the mutated protein. Therefore, in some embodiments, the invention provides a mutated Cry protein, wherein an amino acid substitution in a native Cry protein produces a mutant Cry protein having an antigenic region that allows the mutant Cry protein to be distinguished from the native Cry protein in a protein detection assay.

[0174] In some embodiments, the invention provides a method of making an antibody that differentially recognizes a mutated Cry protein from the native Cry protein from which the mutated Cry protein is derived, the method comprising the steps of substituting amino acids in an antigenic loop of a native Cry protein and raising antibodies that specifically recognize the mutated antigenic loop in the mutated Cry protein and does not recognize the native Cry protein. In one embodiment, the antigenic loop is identified in non-conserved regions outside of domain I of the native Cry protein. In another embodiment, the antigenic loop is not a loop involved in the Cry protein's insect gut receptor recognition or involved in the protease activation of the Cry protein.

[0175] Variant nucleotide and amino acid sequences of the invention also encompass sequences derived from mutagenic and recombinogenic procedures such as DNA shuffling. With such a procedure, one or more different toxic protein coding regions can be used to create a new toxic protein possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between a pesticidal gene of the invention and other known pesticidal genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased insecticidal activity. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0176] Domain swapping or shuffling is another mechanism for generating altered Cry proteins of the invention. Domains may be swapped between Cry proteins, resulting in hybrid or chimeric toxic proteins with improved pesticidal activity or target spectrum. Methods for generating recombinant proteins and testing them for pesticidal activity are well known in the art (see, for example, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol. 65:2918-2925). In some embodiments, the invention provides hybrid Cry proteins comprising at a C-terminus, amino acids from a first Cry protein of the invention and at an N-terminus, amino acids from a second Cry protein different from the first Cry protein of the invention. In other embodiments, the invention provides hybrid Cry proteins comprising at an N-terminus, amino acids from a Cry protein of the invention and at a C-terminus, amino acids from a second Cry protein different from the first Cry protein of the invention.

[0177] When a heterologous polynucleotide sequence encoding a Cry protein encompassed by the invention is introduced into a plant the introduced polynucleotide is stably integrated into the genome of the now transgenic plant. Thus, according to the invention, the encoded Cry protein can be mutated in situ by targeted DNA editing using various genome editing techniques such as zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENS), meganucleases and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (U.S. Pat. No. 8,697,359; Ran et al., incorporated by reference). The CRISPR system can be used to introduce specific nucleotide modifications at the target sequence. Originally discovered in bacteria, where several different CRISPR cascades function as innate immune systems and natural defense mechanisms, the engineered CRISPR-Cas9 system can be programmed to target specific stretches of genetic code and to make cuts at precise locations. Over the past few years, those capabilities have been harnessed and used as genome editing tools, enabling researchers to permanently modify genes in plant cells.

[0178] Thus, the invention encompasses a method for generating a mutant polynucleotide encoding a modified Cry protein wherein said method comprises modifying a plant genome comprising a polynucleotide encoding a Cry protein using CRISPR. The method involves targeting of Cas9 to the specific genomic locus, in this case a Cry protein-encoding polynucleotide, via a 20 nt guide sequence of the single-guide RNA. An online CRISPR Design Tool can identify suitable target sites (world wide web at tools.genome-engineering.org. Ran et al. Genome engineering using the CRISPR-Cas9 system nature protocols, VOL. 8 NO. 11, 2281-2308, 2013). Target plants for the mutagenesis/genome editing methods according to the invention are any monocot or dicot plants into which a Cry protein-encoding polynucleotide of the invention has been introduced.

[0179] In an exemplary embodiment, the activity of a Cry1J protein can be modulated by mutating a cry1J gene, for example a gene encoding BT2Cry1Ja (SEQ ID NO:1) or BT53Cry1J (SEQ ID NO:3), comprised in a transgenic maize genome by engineering recombinant DNA restriction enzymes by fusing a nuclease, for example FokI, with a structure that binds to a site in the cry1J gene to make a double strand cut within the cry1J gene and replace with an engineered polynucleotide that comprises the mutations of interest. FokI is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain, which can be made to bind to specific DNA sequences in the genome and a non-specific DNA cleavage domain at the C-terminus. Plants expressing the mutated Cry1J protein with modulated activity can be selected using insect bioassays as disclosed herein.

[0180] In some embodiments, the invention provides a recombinant vector comprising a polynucleotide, an expression cassette or a chimeric gene of the invention. In other embodiments, the vector is further defined as a plasmid, cosmid, phagemid, artificial chromosome, phage or viral vector. Certain vectors comprising on or more expression cassettes for use in transformation of plants and other organisms are known in the art.

[0181] Thus, some embodiments of the invention are directed to expression cassettes designed to express the Cry protein-encoding polynucleotides of the invention. As used herein, "expression cassette" means a polynucleotide having at least a control sequence operatively linked to a nucleotide sequence of interest. In this manner, for example, plant promoters operably linked to the nucleotide sequences to be expressed are provided in expression cassettes for expression in a plant, plant part or plant cell.

[0182] An expression cassette comprising a polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one other of its other components. An expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleotide sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.

[0183] In addition to the promoters operatively linked to the nucleotide sequences of the invention, an expression cassette of this invention also can include other regulatory sequences. As used herein, "regulatory sequences" means nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences, termination signals, and polyadenylation signal sequences.

[0184] In some embodiments, an expression cassette of the invention also can include polynucleotides that encode other desired traits in addition to the Cry proteins of the invention. Such expression cassettes comprising the stacked traits may be used to create plants, plant parts or plant cells having a desired phenotype with the stacked traits (i.e., molecular stacking). Such stacked combinations in plants can also be created by other methods including, but not limited to, cross breeding plants by any conventional methodology. If stacked by genetically transforming the plants, the nucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The additional nucleotide sequences can be introduced simultaneously in a co-transformation protocol with a nucleotide sequence, polynucleotide, polynucleotide construct, or composition of this invention, provided by any combination of expression cassettes. For example, if two nucleotide sequences will be introduced, they can be incorporated in separate cassettes (trans) or can be incorporated on the same cassette (cis). Expression of polynucleotides can be driven by the same promoter or by different promoters. It is further recognized that polynucleotides can be stacked at a desired genomic location using a site-specific recombination system. See, e.g., Int'l Patent Application Publication Nos. WO 99/25821; WO 99/25854; WO 99/25840; WO 99/25855 and WO 99/25853.

[0185] The expression cassette also can include an additional coding sequence for one or more polypeptides or double stranded RNA molecules (dsRNA) of interest for agronomic traits that primarily are of benefit to a seed company, grower or grain processor. A polypeptide of interest can be any polypeptide encoded by a nucleotide sequence of interest. Non-limiting examples of polypeptides of interest that are suitable for production in plants include those resulting in agronomically important traits such as herbicide resistance (also sometimes referred to as "herbicide tolerance"), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, or fungal resistance. See, e.g., U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. The polypeptide also can be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., a selectable marker, seed coat color, etc.). Various polypeptides of interest, as well as methods for introducing these polypeptides into a plant, are described, for example, in U.S. Pat. Nos. 4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374; 5,013,659; 5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798; 5,561,236; 5,569,823; 5,767,366; 5,879,903, 5,928,937; 6,084,155; 6,329,504 and 6,337,431; as well as US Patent Publication No. 2001/0016956. See also, on the World Wide Web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.

[0186] Polynucleotides conferring resistance/tolerance to an herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea can also be suitable in some embodiments of the invention. Exemplary polynucleotides in this category code for mutant ALS and AHAS enzymes as described, e.g., in U.S. Pat. Nos. 5,767,366 and 5,928,937. U.S. Pat. Nos. 4,761,373 and 5,013,659 are directed to plants resistant to various imidazalinone or sulfonamide herbicides. U.S. Pat. No. 4,975,374 relates to plant cells and plants containing a polynucleotide encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that are known to inhibit GS, e.g., phosphinothricin and methionine sulfoximine. U.S. Pat. No. 5,162,602 discloses plants resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The resistance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).

[0187] Polypeptides encoded by nucleotides sequences conferring resistance to glyphosate are also suitable for the invention. See, e.g., U.S. Pat. Nos. 4,940,835 and 4,769,061. U.S. Pat. No. 5,554,798 discloses transgenic glyphosate resistant maize plants, which resistance is conferred by an altered 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene.

[0188] Polynucleotides coding for resistance to phosphono compounds such as glufosinate ammonium or phosphinothricin, and pyridinoxy or phenoxy propionic acids and cyclohexones are also suitable. See, European Patent Application No. 0 242 246. See also, U.S. Pat. Nos. 5,879,903, 5,276,268 and 5,561,236.

[0189] Other suitable polynucleotides include those coding for resistance to herbicides that inhibit photosynthesis, such as a triazine and a benzonitrile (nitrilase) See, U.S. Pat. No. 4,810,648. Additional suitable polynucleotides coding for herbicide resistance include those coding for resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil. Also suitable are polynucleotides conferring resistance to a protox enzyme, or that provide enhanced resistance to plant diseases; enhanced tolerance of adverse environmental conditions (abiotic stresses) including but not limited to drought, excessive cold, excessive heat, or excessive soil salinity or extreme acidity or alkalinity; and alterations in plant architecture or development, including changes in developmental timing. See, e.g., U.S. Patent Publication No. 2001/0016956 and U.S. Pat. No. 6,084,155.

[0190] Additional suitable polynucleotides include those coding for pesticidal (e.g., insecticidal) polypeptides. These polypeptides may be produced in amounts sufficient to control, for example, insect pests (i.e., insect controlling amounts). It is recognized that the amount of production of a pesticidal polypeptide in a plant necessary to control insects or other pests may vary depending upon the cultivar, type of pest, environmental factors and the like. Polynucleotides useful for additional insect or pest resistance include, for example, those that encode toxins identified in Bacillus organisms. Polynucleotides comprising nucleotide sequences encoding Bacillus thuringiensis (Bt) Cry proteins from several subspecies have been cloned and recombinant clones have been found to be toxic to lepidopteran, dipteran and coleopteran insect larvae. Examples of such Bt insecticidal proteins include the Cry proteins such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1Ea, Cry1Fa, Cry3A, Cry9A, Cry9B, Cry9C, and the like, as well as vegetative insecticidal proteins such as vip1, vip2, vip3, and the like. A full list of Bt-derived proteins can be found on the worldwide web at Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also, Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813).

[0191] Polypeptides that are suitable for production in plants further include those that improve or otherwise facilitate the conversion of harvested plants or plant parts into a commercially useful product, including, for example, increased or altered carbohydrate content or distribution, improved fermentation properties, increased oil content, increased protein content, improved digestibility, and increased nutraceutical content, e.g., increased phytosterol content, increased tocopherol content, increased stanol content or increased vitamin content. Polypeptides of interest also include, for example, those resulting in or contributing to a reduced content of an unwanted component in a harvested crop, e.g., phytic acid, or sugar degrading enzymes. By "resulting in" or "contributing to" is intended that the polypeptide of interest can directly or indirectly contribute to the existence of a trait of interest (e.g., increasing cellulose degradation by the use of a heterologous cellulase enzyme).

[0192] In some embodiments, the polypeptide contributes to improved digestibility for food or feed. Xylanases are hemicellulolytic enzymes that improve the breakdown of plant cell walls, which leads to better utilization of the plant nutrients by an animal. This leads to improved growth rate and feed conversion. Also, the viscosity of the feeds containing xylan can be reduced. Heterologous production of xylanases in plant cells also can facilitate lignocellulosic conversion to fermentable sugars in industrial processing.

[0193] Numerous xylanases from fungal and bacterial microorganisms have been identified and characterized (see, e.g., U.S. Pat. No. 5,437,992; Coughlin et al. (1993) "Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases" Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial Fermentation Research 8:125-135; U.S. Patent Publication No. 2005/0208178; and PCT Publication No. WO 03/16654). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in T. reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio/Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).

[0194] In other embodiments, a polypeptide useful for the invention can be a polysaccharide degrading enzyme. Plants of this invention producing such an enzyme may be useful for generating, for example, fermentation feedstocks for bioprocessing. In some embodiments, enzymes useful for a fermentation process include alpha amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolyzing enzyme and other glucoamylases.

[0195] Polysaccharide-degrading enzymes include: starch degrading enzymes such as .alpha.-amylases (EC 3.2.1.1), glucuronidases (E.C. 3.2.1.131); exo-1,4-.alpha.-D glucanases such as amyloglucosidases and glucoamylase (EC 3.2.1.3), .beta.-amylases (EC 3.2.1.2), .alpha.-glucosidases (EC 3.2.1.20), and other exo-amylases; starch debranching enzymes, such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), and the like; b) cellulases such as exo-1,4-3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-.beta.-D-glucanase (EC 3.2.1.39), .beta.-glucosidase (EC 3.2.1.21); c) L-arabinases, such as endo-1,5-.alpha.-L-arabinase (EC 3.2.1.99), .alpha.-arabinosidases (EC 3.2.1.55) and the like; d) galactanases such as endo-1,4-.beta.-D-galactanase (EC 3.2.1.89), endo-1,3-.beta.-D-galactanase (EC 3.2.1.90), .alpha.-galactosidase (EC 3.2.1.22), .beta.-galactosidase (EC 3.2.1.23) and the like; e) mannanases, such as endo-1,4-.beta.-D-mannanase (EC 3.2.1.78), .beta.-mannosidase (EC 3.2.1.25), .alpha.-mannosidase (EC 3.2.1.24) and the like; f) xylanases, such as endo-1,4-.beta.-xylanase (EC 3.2.1.8), .beta.-D-xylosidase (EC 3.2.1.37), 1,3-.beta.-D-xylanase, and the like; and g) other enzymes such as .alpha.-L-fucosidase (EC 3.2.1.51), .alpha.-L-rhamnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulanase (EC 3.2.1.7), and the like. In one embodiment, the .alpha.-amylase is the synthetic .alpha.-amylase, Amy797E, described is U.S. Pat. No. 8,093,453, herein incorporated by reference in its entirety.

[0196] Further enzymes which may be used with the invention include proteases, such as fungal and bacterial proteases. Fungal proteases include, but are not limited to, those obtained from Aspergillus, Trichoderma, Mucor and Rhizopus, such as A. niger, A. awamori, A. oryzae and M. miehei. In some embodiments, the polypeptides of this invention can be cellobiohydrolase (CBH) enzymes (EC 3.2.1.91). In one embodiment, the cellobiohydrolase enzyme can be CBH1 or CBH2.

[0197] Other enzymes useful with the invention include, but are not limited to, hemicellulases, such as mannases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (e.g., E.C. 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha 1,6 glucosidases (e.g., E.C. 3.2.1.20); esterases such as ferulic acid esterase (EC 3.1.1.73) and acetyl xylan esterases (EC 3.1.1.72); and cutinases (e.g. E.C. 3.1.1.74).

[0198] Double stranded RNA molecules useful with the invention include, but are not limited to those that suppress target insect genes. As used herein the words "gene suppression", when taken together, are intended to refer to any of the well-known methods for reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA. Gene suppression is also intended to mean the reduction of protein expression from a gene or a coding sequence including posttranscriptional gene suppression and transcriptional suppression. Posttranscriptional gene suppression is mediated by the homology between of all or a part of a mRNA transcribed from a gene or coding sequence targeted for suppression and the corresponding double stranded RNA used for suppression, and refers to the substantial and measurable reduction of the amount of available mRNA available in the cell for binding by ribosomes. The transcribed RNA can be in the sense orientation to effect what is called co-suppression, in the anti-sense orientation to effect what is called anti-sense suppression, or in both orientations producing a dsRNA to effect what is called RNA interference (RNAi). Transcriptional suppression is mediated by the presence in the cell of a dsRNA, a gene suppression agent, exhibiting substantial sequence identity to a promoter DNA sequence or the complement thereof to effect what is referred to as promoter trans suppression. Gene suppression may be effective against a native plant gene associated with a trait, e.g., to provide plants with reduced levels of a protein encoded by the native gene or with enhanced or reduced levels of an affected metabolite. Gene suppression can also be effective against target genes in plant pests that may ingest or contact plant material containing gene suppression agents, specifically designed to inhibit or suppress the expression of one or more homologous or complementary sequences in the cells of the pest. Such genes targeted for suppression can encode an essential protein, the predicted function of which is selected from the group consisting of muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, digestive enzyme synthesis, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, hemolymph synthesis, hemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, and apoptosis.

[0199] In some embodiments, the invention provides a transgenic non-human host cell comprising a polynucleotide, a chimeric gene, an expression cassette or a recombinant vector of the invention. The transgenic non-human host cell can include, but is not limited to, a plant cell, a yeast cell, a bacterial cell or an insect cell. Accordingly, in some embodiments, the invention provides a bacterial cell selected from the genera Bacillus, Brevibacillus, Clostridium, Xenorhabdus, Photorhabdus, Pasteuria, Escherichia, Pseudomonas, Envinia, Serratia, Klebsiella, Salmonella, Pasteurella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, or Alcaligenes. Thus, for example, as biological insect control agents, the Cry proteins of the invention can be produced by expression of a chimeric gene encoding the Cry proteins of the invention in a bacterial cell. For example, in some embodiments, a Bacillus thuringiensis cell comprising a chimeric gene of the invention is provided.

[0200] In further embodiments, the invention provides a transgenic plant cell that is a dicot plant cell or a monocot plant cell. In additional embodiments, the dicot plant cell is selected from the group consisting of a soybean cell, sunflower cell, tomato cell, cole crop cell, cotton cell, sugar beet cell and tobacco cell. In further embodiments, the monocot cell is selected from the group consisting of a barley cell, maize cell, oat cell, rice cell, sorghum cell, sugar cane cell and wheat cell. In some embodiments, the invention provides a plurality of dicot cells or monocot cells expressing a Cry protein of the invention that is encoded by a chimeric gene of the invention. In other embodiments the plurality of cells are juxtaposed to form an apoplast and are grown in natural sunlight.

[0201] In other embodiments of the invention, an insecticidal Cry protein of the invention is expressed in a higher organism, for example, a plant. In this case, transgenic plants expressing effective amounts of the insecticidal protein protect themselves from plant pests such as insect pests. When an insect starts feeding on such a transgenic plant, it ingests the expressed insecticidal Cry protein. This can deter the insect from further biting into the plant tissue or may even harm or kill the insect. A polynucleotide of the invention is inserted into an expression cassette, which is then stably integrated in the genome of the plant. In other embodiments, the polynucleotide is included in a non-pathogenic self-replicating virus. Plants transformed in accordance with the invention may be monocots or dicots and include, but are not limited to, corn (maize), soybean, rice, wheat, barley, rye, oats, sorghum, millet, sunflower, safflower, sugar beet, cotton, sugarcane, oilseed rape, alfalfa, tobacco, peanuts, vegetables, including, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, carrot, eggplant, cucumber, radish, spinach, potato, tomato, asparagus, onion, garlic, melons, pepper, celery, squash, pumpkin, zucchini, fruits, including, apple, pear, quince, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, and specialty plants, such as Arabidopsis, and woody plants such as coniferous and deciduous trees. Preferably, plants of the of the invention are crop plants such as maize, sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco, barley, oilseed rape, and the like.

[0202] Once a desired polynucleotide has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques.

[0203] A polynucleotide of the invention is expressed in transgenic plants, thus causing the biosynthesis of the corresponding Cry protein, either in protoxin or mature toxin form, in the transgenic plants. In this way, transgenic plants with enhanced yield protection in the presence of insect pressure are generated. For their expression in transgenic plants, the nucleotide sequences of the invention may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that living organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention can be changed to conform with plant preferences, while maintaining the amino acids encoded thereby. Furthermore, high expression in plants, for example corn plants, is best achieved from coding sequences that have at least about 35% GC content, or at least about 45%, or at least about 50%, or at least about 60%. Microbial nucleotide sequences that have low GC contents may express poorly in plants due to the existence of ATTTA motifs that may destabilize messages, and AATAAA motifs that may cause inappropriate polyadenylation. Although certain gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleotide sequences are screened for the existence of illegitimate splice sites that may cause message truncation. All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described for example in U.S. Pat. Nos. 5,625,136; 5,500,365 and 6,013,523.

[0204] In some embodiments, the invention provides synthetic coding sequences or polynucleotide made according to the procedure disclosed in U.S. Pat. No. 5,625,136, herein incorporated by reference. In this procedure, maize preferred codons, i.e., the single codon that most frequently encodes that amino acid in maize, are used. The maize preferred codon for a particular amino acid can be derived, for example, from known gene sequences from maize. For example, maize codon usage for 28 genes from maize plants is found in Murray et al., Nucleic Acids Research 17:477-498 (1989), the disclosure of which is incorporated herein by reference. Specifically exemplified synthetic sequences of the present invention made with maize optimized codons are represented by any one of SEQ ID NOs: 17-22. It is recognized that codons optimized for expression in one plant species will also function in other plant species but possibly not at the same level as the plant species for which the codons were optimized. In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of a nucleotide sequence may be optimized or synthetic. That is, a polynucleotide may comprise a nucleotide sequence that is part native sequence and part codon optimized sequence.

[0205] For efficient initiation of translation, sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi has suggested an appropriate consensus for plants (NAR 15:6643-6653 (1987)) and Clonetech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the nucleotide sequences of this invention. The sequences are incorporated into constructions comprising the nucleotide sequences, up to and including the ATG (while leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility of modifying the second amino acid of the transgene).

[0206] The novel Cry protein coding sequences of the invention, either as their native sequence or as synthetic sequences as described above, can be operably fused to a variety of promoters for expression in plants including constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters to prepare recombinant DNA molecules, i.e., chimeric genes. The choice of promoter will vary depending on the temporal and spatial requirements for expression, and also depending on the target species. Thus, expression of the nucleotide sequences of this invention in leaves, in stalks or stems, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, or seedlings is preferred. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired cell.

[0207] Suitable constitutive promoters include, for example, CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. W004081173A2); maize Ubi 1 (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant J November; 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); Rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); Maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al., Plant J. 10(1); 107-121, 1996), constitutive root tip CT2 promoter (PCT application No. IL/2005/000627) and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0208] Tissue-specific or tissue-preferential promoters useful for the expression of the novel cry protein coding sequences of the invention in plants, particularly maize, are those that direct expression in root, pith, leaf or pollen. Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], seed-preferred promoters [e.g., from seed specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al., Plant Mol Biol, 143). 323-32 1990), napA (Stalberg, et al., Planta 199: 515-519, 1996), Wheat SPA (Albani et al, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-876, 1992)], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMB03:1409-15, 1984), Barley ltr1 promoter, barley B1, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), Barley DOF (Mena et al., The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice-globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgum gamma-kafirin (Plant Mol. Biol 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSH1 (Sato et al., Proc. Nati. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245; 1989), apetala-3, plant reproductive tissues [e.g., OsMADS promoters (U.S. Patent Application publication No. 2007/0006344)].

[0209] The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemically regulated. This enables the Cry proteins of the invention to be synthesized only when the crop plants are treated with the inducing chemicals. Examples of such technology for chemical induction of gene expression is detailed in the published application EP 0 332 104 and U.S. Pat. No. 5,614,395. In one embodiment, the chemically regulated promoter is the tobacco PR-la promoter.

[0210] Another category of promoters useful in the invention is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. Ideally, such a promoter should only be active locally at the sites of insect invasion, and in this way the insecticidal proteins only accumulate in cells that need to synthesize the insecticidal proteins to kill the invading insect pest. Examples of promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), and Warner et al. Plant J. 3:191-201 (1993).

[0211] Non-limiting examples of promoters that cause tissue specific expression patterns that are useful in the invention include green tissue specific, root specific, stem specific, or flower specific. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. One such promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12:579-589 (1989)). Another promoter for root specific expression is that described by de Framond (FEBS 290:103-106 (1991) or U.S. Pat. No. 5,466,785). Another promoter useful in the invention is the stem specific promoter described in U.S. Pat. No. 5,625,136, which naturally drives expression of a maize trpA gene.

[0212] In addition to the selection of a suitable promoter, constructs for expression of an insecticidal toxin in plants require an appropriate transcription terminator to be operably linked downstream of the heterologous nucleotide sequence. Several such terminators are available and known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any available terminator known to function in plants can be used in the context of this invention.

[0213] Numerous other sequences can be incorporated into expression cassettes described in this invention. These include sequences that have been shown to enhance expression such as intron sequences (e.g. from Adh1 and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).

[0214] It may be preferable to target expression of the nucleotide sequences of the present invention to different cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Any mechanism for targeting gene products, e.g., in plants, can be used to practice this invention, and such mechanisms are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detail. Sequences have been characterized which cause the targeting of gene products to other cell compartments Amino terminal sequences can be responsible for targeting a protein of interest to any cell compartment, such as, a vacuole, mitochondrion, peroxisome, protein bodies, endoplasmic reticulum, chloroplast, starch granule, amyloplast, apoplast or cell wall of a plant (e.g. Unger et. al. Plant Molec. Biol. 13: 411-418 (1989); Rogers et. al. (1985) Proc. Natl. Acad. Sci. USA 82: 6512-651; U.S. Pat. No. 7,102,057; WO 2005/096704, all of which are hereby incorporated by reference. Optionally, the signal sequence may be an N-terminal signal sequence from waxy, an N-terminal signal sequence from gamma-zein, a starch binding domain, a C-terminal starch binding domain, a chloroplast targeting sequence, which imports the mature protein to the chloroplast (Comai et. al. (1988) J. Biol. Chem. 263: 15104-15109; van den Broeck, et. al. (1985) Nature 313: 358-363; U.S. Pat. No. 5,639,949) or a secretion signal sequence from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et. al. (1990) Plant Molec. Biol. 14: 357-368). In one embodiment, the signal sequence selected includes the known cleavage site, and the fusion constructed takes into account any amino acids after the cleavage site(s), which are required for cleavage. In some cases this requirement may be fulfilled by the addition of a small number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. These construction techniques are well known in the art and are equally applicable to any cellular compartment.

[0215] It will be recognized that the above-described mechanisms for cellular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific cell-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.

Plant Transformation

[0216] Procedures for transforming plants are well known and routine in the art and are described throughout the literature. Non-limiting examples of methods for transformation of plants include transformation via bacterial-mediated polynucleotide delivery (e.g., via Agrobacterium), viral-mediated polynucleotide delivery, silicon carbide or whisker-mediated polynucleotide delivery, liposome mediated polynucleotide delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated polynucleotide uptake, as well as any other electrical, chemical, physical (mechanical) or biological mechanism that results in the introduction of polynucleotide into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0217] For Agrobacterium-mediated transformation, binary vectors or vectors carrying at least one T-DNA border sequence are suitable, whereas for direct gene transfer (e.g., particle bombardment and the like) any vector is suitable and linear DNA containing only the construction of interest can be used. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al., Biotechnology 4:1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker that may be a positive selection (Phosphomannose Isomerase), provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (glyphosate or glufosinate). However, the choice of selectable marker is not critical to the invention.

[0218] Agrobacterium-mediated transformation is a commonly used method for transforming plants because of its high efficiency of transformation and because of its broad utility with many different species. Agrobacterium-mediated transformation typically involves transfer of the binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain that may depend on the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomally (Uknes et al. (1993) Plant Cell 5:159-169). The transfer of the recombinant binary vector to Agrobacterium can be accomplished by a triparental mating procedure using Escherichia coli carrying the recombinant binary vector, a helper E. coli strain that carries a plasmid that is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by polynucleotide transformation (Hagen & Willmitzer (1988) Nucleic Acids Res. 16:9877).

[0219] Dicots as well as monocots may be transformed using Agrobacterium. Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The polynucleotides or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hagen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0220] Transformation of a plant by recombinant Agrobacterium usually involves co-cultivation of the Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is regenerated on selection medium carrying an antibiotic or herbicide resistance marker between the binary plasmid T-DNA borders.

[0221] As discussed previously, another method for transforming plants, plant parts and plant cells involves propelling inert or biologically active particles at plant tissues and cells. See, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006 and 5,100,792. Generally, this method involves propelling inert or biologically active particles at the plant cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the polynucleotide of interest. Alternatively, a cell or cells can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., a dried yeast cell, a dried bacterium or a bacteriophage, each containing one or more polynucleotides sought to be introduced) also can be propelled into plant tissue.

[0222] In other embodiments, a polynucleotide of the invention can be directly transformed into the plastid genome. A major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride et al. (1994) Proc. Nati. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin or streptomycin can be utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45). The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P. (1993) EMBO J. 12, 601-606). Substantial increases in transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-cletoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab, Z., and Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. Typically, approximately 15-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readily exceed 10% of the total soluble plant protein. In one embodiment, a polynucleotide of the invention can be inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Thus, plants homoplastic for plastid genomes containing a nucleotide sequence of the invention can be obtained, which are capable of high expression of the polynucleotide.

[0223] Methods of selecting for transformed, transgenic plants, plant cells or plant tissue culture are routine in the art and can be employed in the methods of the invention provided herein. For example, a recombinant vector of the invention also can include an expression cassette comprising a nucleotide sequence for a selectable marker, which can be used to select a transformed plant, plant part or plant cell. As used herein, "selectable marker" means a nucleotide sequence that when expressed imparts a distinct phenotype to the plant, plant part or plant cell expressing the marker and thus allows such transformed plants, plant parts or plant cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic, herbicide, or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., the R-locus trait). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0224] Examples of selectable markers include, but are not limited to, a nucleotide sequence encoding neo or nptII, which confers resistance to kanamycin, G418, and the like (Potrykus et al. (1985) Mol. Gen. Genet. 199:183-188); a nucleotide sequence encoding bar, which confers resistance to phosphinothricin; a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915-922); a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; or a nucleotide sequence encoding hph that confers resistance to hygromycin. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of this invention.

[0225] Additional selectable markers include, but are not limited to, a nucleotide sequence encoding .beta.-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; an R-locus nucleotide sequence that encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., "Molecular cloning of the maize R-nj allele by transposon-tagging with Ac" 263-282 In: Chromosome Structure and Function: Impact of New Concepts, 18th Stadler Genetics Symposium (Gustafson & Appels eds., Plenum Press 1988)); a nucleotide sequence encoding .beta.-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737-3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding .beta.-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding aequorin which may be employed in calcium-sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm. 126:1259-1268); or a nucleotide sequence encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14:403-406). One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette of this invention.

[0226] Further, as is well known in the art, intact transgenic plants can be regenerated from transformed plant cells, plant tissue culture or cultured protoplasts using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue culture or cultured protoplasts is described, for example, in Evans et al. (Handbook of Plant Cell Cultures, Vol. 1, MacMilan Publishing Co. New York (1983)); and Vasil I. R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I (1984), and Vol. II (1986)).

[0227] Additionally, the genetic properties engineered into the transgenic seeds and plants, plant parts, or plant cells of the invention described above can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling.

[0228] A polynucleotide therefore can be introduced into the plant, plant part or plant cell in any number of ways that are well known in the art, as described above. Therefore, no particular method for introducing one or more polynucleotides into a plant is relied upon, rather any method that allows the one or more polynucleotides to be stably integrated into the genome of the plant can be used. Where more than one polynucleotide is to be introduced, the respective polynucleotides can be assembled as a single polynucleotide, or as separate polynucleotides, and can be located on the same or different expression cassettes or vectors. Accordingly, the polynucleotides can be introduced into the cell of interest in a single transformation event, in separate transformation events, or, for example, in plants, as part of a breeding protocol.

[0229] Additional embodiments of the invention encompasses harvested products produced from the transgenic plants or parts thereof comprising a Cry protein-encoding polynucleotide of the invention, as well as a processed product produced from the harvested products. A harvested product can be a whole plant or any plant part, as described herein. Thus, in some embodiments, non-limiting examples of a harvested product include a seed, a fruit, a flower or part thereof (e.g., an anther, a stigma, and the like), a leaf, a stem, and the like. In other embodiments, a processed product includes, but is not limited to, flour, meal, oil, starch, cereal, and the like produced from a harvested seed or other plant part of the invention, wherein the seed or other plant part comprises a polynucleotide or nucleotide sequence of the invention. In some embodiments, the invention encompasses harvested products and processed products, such as meal or flour that comprise a Cry protein of the invention, where the Cry protein continues to perform the insecticidal function it had in the transgenic plant from which the harvested product or processed product was derived.

[0230] In other embodiments, the invention provides an extract from a transgenic seed or a transgenic plant of the invention, wherein the extract comprises a polynucleotide or a Cry protein of the invention. Extracts from plants or plant parts can be made according to procedures well known in the art (See, de la Torre et al., Food, Agric. Environ. 2(1):84-89 (2004); Guidet, Nucleic Acids Res. 22(9): 1772-1773 (1994); Lipton et al., Food Agric. Immun. 12:153-164 (2000)).

Insecticidal Compositions

[0231] In some embodiments, the invention provides an insecticidal composition comprising a Cry protein of the invention in an agriculturally acceptable carrier. As used herein an "agriculturally-acceptable carrier" can include natural or synthetic, organic or inorganic material which is combined with the active Cry protein to facilitate its application to or in the plant, or part thereof. Examples of agriculturally acceptable carriers include, without limitation, powders, dusts, pellets, granules, sprays, emulsions, colloids, and solutions. Agriculturally-acceptable carriers further include, but are not limited to, inert components, dispersants, surfactants, adjuvants, tackifiers, stickers, binders, or combinations thereof, that can be used in agricultural formulations. Such compositions can be applied in any manner that brings the pesticidal proteins or other pest control agents in contact with the pests. Accordingly, the compositions can be applied to the surfaces of plants or plant parts, including seeds, leaves, flowers, stems, tubers, roots, and the like. In other embodiments, a plant producing a Cry protein of the invention in planta is an agricultural-carrier of the expressed Cry protein.

[0232] In further embodiments, the insecticidal composition comprises a bacterial cell or a transgenic bacterial cell of the invention, wherein the bacterial cell or transgenic bacterial cell produces a Cry protein of the invention. Such an insecticidal composition can be prepared by desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, sedimentation, or concentration of a culture of Bacillus thuringiensis (Bt). Such Bt cultures can be selected from the group of Bt strains consisting of SC0532, SC0705, SC0666 described below in the Examples or transgenic Bt cultures. In additional embodiments, the composition comprises from about 1% to about 99% by weight of the Cry protein of the invention.

[0233] The Cry proteins of the invention can be used in combination with other pest control agents to increase pest target range or for the prevention or management of insect resistance. Therefore, in some embodiments, the invention provides a composition that controls one or more plant pests, wherein the composition comprises a first Cry protein of the invention and a second pest control agent different from the first Cry protein. In other embodiments, the composition is a formulation for topical application to a plant. In still other embodiments, the composition is a transgenic plant. In further embodiments, the composition is a combination of a formulation topically applied to a transgenic plant. In some embodiments, the formulation comprises the first Cry protein of the invention when the transgenic plant comprises the second pest control agent. In other embodiments, the formulation comprises the second pest control agent when the transgenic plant comprises the first Cry protein of the invention.

[0234] In some embodiments, the second pest control agent can be an agent selected from the group consisting of a chemical pesticide, such as an insecticide, a Bacillus thuringiensis (Bt) insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Brevibacillus laterosporus insecticidal protein, a Bacillus sphaericus insecticidal protein, a protease inhibitors (both serine and cysteine types), lectins, alpha-amylase, peroxidase, cholesterol oxidase and a double stranded RNA (dsRNA) molecule.

[0235] In other embodiments, the second pest control agent is a chemical pesticide selected from the group consisting of pyrethroids, carbamates, neonicotinoids, neuronal sodium channel blockers, insecticidal macrocyclic lactones, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas and juvenile hormone mimics. In other embodiments, the chemical pesticide is selected from the group consisting of abamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin, carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin, beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron, dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole, fenothicarb, fenoxycarb, fenpropathrin, fenproximate, fenvalerate, fipronil, flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid, indoxacarb, isofenphos, lufenuron, malathion, metaldehyde, methamidophos, methidathion, methomyl, methoprene, methoxychlor, monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine, pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060), sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin, trichlorfon and triflumuron, aldicarb, oxamyl, fenamiphos, amitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad. In still other embodiments, the chemical pesticide is selected from the group consisting of cypermethrin, cyhalothrin, cyfluthrin and beta-cyfluthrin, esfenvalerate, fenvalerate, tralomethrin, fenothicarb, methomyl, oxamyl, thiodicarb, clothianidin, imidacloprid, thiacloprid, indoxacarb, spinosad, abamectin, avermectin, emamectin, endosulfan, ethiprole, fipronil, flufenoxuron, triflumuron, diofenolan, pyriproxyfen, pymetrozine and amitraz.

[0236] In additional embodiments, the second pest control agent can be one or more of any number of Bacillus thuringiensis insecticidal proteins including but not limited to a Cry protein, a vegetative insecticidal protein (VIP) and insecticidal chimeras of any of the preceding insecticidal proteins. In other embodiments, the second pest control agent is a Cry protein selected from the group consisting of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Af, Cry1Ag, Cry1Ah, Cry1Ai, Cry1Aj, Cry1Ba, Cry1Bb, Cry1Bc, Cry1Bd, Cry1Be, Cry1Bf, Cry1Bg, Cry1Bh, Cry1Bi, Cry1Ca, Cry1Cb, Cry1Da, Cry1db, Cry1Dc, Cry1Dd, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Gb, Cry1Gc, Cry1Ha, Cry1Hb, Cry1Hc, Cry1Ia, Cry1Ib, Cry1Ic, Cry1Id, Cry1Ie, Cry1If, Cry1Ig, Cry1Ja, Cry1Ib, Cry1k, Cry1Id, Cry1Ka, Cry1La, Cry1Ma, Cry1Na, Cry 1Nb, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Aj, Cry2Ak, Cry2A1, Cry2Ba, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry4Aa, Cry4Ba, Cry4Ca, Cry4Cb, Cry4Cc, Cry5Aa, Cry5Ab, Cry5Ac, Cry5Ad, Cry5Ba, Cry5Ca, Cry5 Da, Cry5Ea, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry7Ac, Cry7Ba, Cry7Bb, Cry7Ca, Cry7Cb, Cry7 Da, Cry7Ea, Cry7Fa, Cry7Fb, Cry7Ga, Cry7Gb, Cry7Gc, Cry7Gd, Cry7Ha, Cry7Ia, Cry7Ja, Cry7Ka, Cry7Kb, Cry7La, Cry8Aa, Cry8Ab, Cry8Ac, Cry8Ad, Cry8Ba, Cry8Bb, Cry8Bc, Cry8Ca, Cry8 Da, Cry8db, Cry8Ea, Cry8Fa, Cry8Ga, Cry8Ha, Cry8Ia, Cry8Ib, Cry8Ja, Cry8Ka, Cry8Kb, Cry8La, Cry8Ma, Cry8Na, Cry8 Pa, Cry8Qa, Cry8Ra, Cry8Sa, Cry8Ta, Cry9Aa, Cry9Ba, Cry9Bb, Cry9Ca, Cry9 Da, Cry9db, Cry9Dc, Cry9Ea, Cry9Eb, Cry9Ec, Cry9Ed, Cry9Ee, Cry9Fa, Cry9Ga, Cry10Aa, Cry11Aa, Cry11Ba, Cry11Bb, Cry12Aa, Cry13Aa, Cry14Aa, Cry14Ab, Cry15Aa, Cry16Aa, Cry17Aa, Cry18Aa, Cry18Ba, Cry18Ca, Cry19Aa, Cry19Ba, Cry19Ca, Cry20Aa, Cry20Ba, Cry21Aa, Cry21Ba, Cry21Ca, Cry21 Da, Cry21Ea, Cry21Fa, Cry21Ga, Cry21Ha, Cry22Aa, Cry22Ab, Cry22Ba, Cry22Bb, Cry23Aa, Cry24Aa, Cry24Ba, Cry24Ca, Cry25Aa, Cry26Aa, Cry27Aa, Cry28Aa, Cry29Aa, Cry29Ba, Cry30Aa, Cry30Ba, Cry30Ca, Cry30 Da, Cry30db, Cry30Ea, Cry30Fa, Cry30Ga, Cry31Aa, Cry31Ab, Cry31Ac, Cry31Ad, Cry32Aa, Cry32Ab, Cry32Ba, Cry32Ca, Cry32Cb, Cry32 Da, Cry32Ea, Cry32Eb, Cry32Fa, Cry32Ga, Cry32Ha, Cry32Hb, Cry32Ia, Cry32Ja, Cry32Ka, Cry32La, Cry32Ma, Cry32 Mb, Cry32Na, Cry32Oa, Cry32 Pa, Cry32Qa, Cry32Ra, Cry32Sa, Cry32Ta, Cry32Ua, Cry33Aa, Cry34Aa, Cry34Ab, Cry34Ac, Cry34Ba, Cry35Aa, Cry35Ab, Cry35Ac, Cry35Ba, Cry36Aa, Cry37Aa, Cry38Aa, Cry39Aa, Cry40Aa, Cry40Ba, Cry40Ca, Cry40 Da, Cry41Aa, Cry41Ab, Cry41Ba, Cry42Aa, Cry43Aa, Cry43Ba, Cry43Ca, Cry43Cb, Cry43Cc, Cry44Aa, Cry45Aa, Cry46Aa Cry46Ab, Cry47Aa, Cry48Aa, Cry48Ab, Cry49Aa, Cry49Ab, Cry50Aa, Cry50Ba, Cry51Aa, Cry52Aa, Cry52Ba, Cry53Aa, Cry53Ab, Cry54Aa, Cry54Ab, Cry54Ba, Cry55Aa, Cry56Aa, Cry57Aa, Cry57Ab, Cry58Aa, Cry59Aa, Cry59Ba, Cry60Aa, Cry60Ba, Cry61Aa, Cry62Aa, Cry63Aa, Cry64Aa, Cry65Aa, Cry66Aa, Cry67Aa, Cry68Aa, Cry69Aa, Cry69Ab, Cry70Aa, Cry70Ba, Cry70Bb, Cry71Aa, Cry72Aa and Cry73Aa.

[0237] In further embodiments, the second pest control agent is a Vip3 vegetative insecticidal protein selected from the group consisting of Vip3Aa1, Vip3Aa2, Vip3Aa3, Vip3Aa4, Vip3Aa5, Vip3Aa6, Vip3Aa7, Vip3Aa8, Vip3Aa9, Vip3Aa10, Vip3Aa11, Vip3Aa12, Vip3Aa13, Vip3Aa14, Vip3Aa15, Vip3Aa16, Vip3Aa17, Vip3Aa18, Vip3Aa19, Vip3Aa20, Vip3Aa21, Vip3Aa22, Vip3Aa2, Vip3Aa24, Vip3Aa25, Vip3Aa26, Vip3Aa27, Vip3Aa28, Vip3Aa29, Vip3Aa30, Vip3Aa31, Vip3Aa32, Vip3Aa33, Vip3Aa34, Vip3Aa35, Vip3Aa36, Vip3Aa37, Vip3Aa38, Vip3Aa39, Vip3Aa40, Vip3Aa41, Vip3Aa42, Vip3Aa43, Vip3Aa44, Vip3Ab1, Vip3Ab2, Vip3Ac1, Vip3Ad1, Vip3Ad2, Vip3Ae1, Vip3Af1, Vip3Af2, Vip3Af3, Vip3Ag1, Vip3Ag2, Vip3Ag3 HM117633, Vip3Ag4, Vip3Ag5, Vip3Ah1, Vip3Ba1, Vip3Ba2, Vip3Bb1, Vip3Bb2 and Vip3Bb3.

[0238] In still further embodiments, the first Cry protein of the invention and the second pest control agent are co-expressed in a transgenic plant. This co-expression of more than one pesticidal principle in the same transgenic plant can be achieved by genetically engineering a plant to contain and express all the genes necessary. Alternatively, a plant, Parent 1, can be genetically engineered for the expression of the Cry protein of the invention. A second plant, Parent 2, can be genetically engineered for the expression of a second pest control agent. By crossing Parent 1 with Parent 2, progeny plants are obtained which express all the genes introduced into Parents 1 and 2.

[0239] In other embodiments, the invention provides a stacked transgenic plant resistant to plant pest infestation comprising a DNA sequence encoding a dsRNA for suppression of an essential gene in a target pest and a DNA sequence encoding a Cry protein of the invention exhibiting biological activity against the target pest. It has been reported that dsRNAs are ineffective against certain lepidopteran pests (Rajagopol et al. 2002. J. Biol. Chem. 277:468-494), likely due to the high pH of the midgut which destabilizes the dsRNA. Therefore, in some embodiments where the target pest is a lepidopteran pest, a Cry protein of the invention acts to transiently reduce the midgut pH which serves to stabilize the co-ingested dsRNA rendering the dsRNA effective in silencing the target genes.

[0240] In addition to providing compositions, the invention provides methods of producing a Cry protein toxic to a lepidopteran pest. Such a method comprises, culturing a transgenic non-human host cell that comprises a polynucleotide or a chimeric gene or a recombinant vector of the invention under conditions in which the host cell produces a protein toxic to the lepidopteran pest. In some embodiments, the transgenic non-human host cell is a plant cell. In some other embodiments, the plant cell is a maize cell. In other embodiments, the conditions under which the plant cell or maize cell are grown include natural sunlight. In other embodiments, the transgenic non-human host cell is a bacterial cell. In still other embodiments, the transgenic non-human host cell is a yeast cell.

[0241] In other embodiments of the method, the lepidopteran pest is selected from the group consisting of European corn borer (Ostrinia nubilalis), black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella), western bean cutworm (Richia albicosta), tobacco budworm (Heliothis virescens), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armigera), striped stem borer (Chilo suppressalis), pink stem borer (Sesamia calamistis), rice leaffolder (Cnaphalocrocis medinalis), and any combination thereof.

[0242] In further embodiments of the method, the chimeric gene comprises any of SEQ ID NOs:14-29, or a toxin-encoding fragment thereof. In still other embodiments, the produced protein comprises an amino acid sequence of any of SEQ ID NOs: 1-13, or a toxin fragment thereof.

[0243] In some embodiments of the method, the chimeric gene comprises a nucleotide sequence that is codon optimized for expression in a plant. In other embodiments, the chimeric gene comprises any of SEQ ID NOs:17-22, or a toxin-encoding fragment thereof. In further embodiments, the produced protein comprises an amino acid sequence of any of SEQ ID NOs:1-6, or a toxin fragment thereof.

[0244] In further embodiments, the invention provides a method of producing a pest-resistant (e.g., an insect-resistant) transgenic plant, comprising: introducing into a plant a polynucleotide, a chimeric gene, a recombinant vector, an expression cassette or a polynucleotide of the invention comprising a nucleotide sequence that encodes a Cry protein of the invention, wherein the encoded Cry protein is expressed in the plant, thereby conferring to the plant resistance to at least a lepidopteran insect pest, and producing a pest-resistant transgenic plant. In some embodiments the polynucleotide, chimeric gene, recombinant vector, expression cassette or polynucleotide comprises a nucleotide sequence that encodes any of SEQ ID NOs: 1-13. In other embodiments, the polynucleotide, chimeric gene, recombinant vector, expression cassette or polynucleotide comprises any of SEQ ID NOs:17-22. In still other embodiments, the pest-resistant transgenic plant is resistant to at least European corn borer (Ostrinia nubilalis) or black cutworm (Agrotis ipsilon) as compared to a control plant lacking the polynucleotide, chimeric gene, recombinant vector, expression cassette or polynucleotide of the invention. In some embodiments, the introducing is achieved by transforming the plant. In other embodiments, the introducing is achieved by crossing a first plant comprising the polynucleotide, chimeric gene, recombinant vector, expression cassette or polynucleotide of the invention with a different second plant resulting in progeny seed and plants having the polynucleotide, chimeric gene, recombinant vector, expression cassette or polynucleotide incorporated into their genome.

[0245] In some embodiments, a transgenic plant of the invention that is resistant to at least European corn borer (Ostrinia nubilalis) or black cutworm (Agrotis ipsilon) is further resistant to at least one additional insect, wherein the additional insect includes, but is not limited to, fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella), western bean cutworm (Richia albicosta), tobacco budworm (Heliothis virescens), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armigera), striped stem borer (Chilo suppressalis), pink stem borer (Sesamia calamistis) or rice leaffolder (Cnaphalocrocis medinalis), and any combination thereof.

[0246] In further embodiments, a method of controlling at least a lepidopteran insect pest such as European corn borer (Ostrinia nubilalis) or black cutworm (Agrotis ipsilon) is provided, the method comprising delivering to the insects an effective amount of a Cry protein of the invention. To be effective, the Cry protein is first orally ingested by the insect. However, the Cry protein can be delivered to the insect in many recognized ways. The ways to deliver a protein orally to an insect include, but are not limited to, providing the protein (1) in a transgenic plant, wherein the insect eats (ingests) one or more parts of the transgenic plant, thereby ingesting the polypeptide that is expressed in the transgenic plant; (2) in a formulated protein composition(s) that can be applied to or incorporated into, for example, insect growth media; (3) in a protein composition(s) that can be applied to the surface, for example, sprayed, onto the surface of a plant part, which is then ingested by the insect as the insect eats one or more of the sprayed plant parts; (4) a bait matrix; or (5) any other art-recognized protein delivery system. Thus, any method of oral delivery to an insect can be used to deliver the toxic Cry proteins of the invention. In some particular embodiments, the Cry protein of the invention is delivered orally to an insect, wherein the insect ingests one or more parts of a transgenic plant.

[0247] In other embodiments, the Cry protein of the invention is delivered orally to an insect, wherein the insect ingests one or more parts of a plant sprayed with a composition comprising the Cry proteins of the invention. Delivering the compositions of the invention to a plant surface can be done using any method known to those of skill in the art for applying compounds, compositions, formulations and the like to plant surfaces. Some non-limiting examples of delivering to or contacting a plant or part thereof include spraying, dusting, sprinkling, scattering, misting, atomizing, broadcasting, soaking, soil injection, soil incorporation, drenching (e.g., root, soil treatment), dipping, pouring, coating, leaf or stem infiltration, side dressing or seed treatment, and the like, and combinations thereof. These and other procedures for contacting a plant or part thereof with compound(s), composition(s) or formulation(s) are well-known to those of skill in the art.

[0248] In some embodiments, the invention encompasses a method of providing a farmer with a means of controlling a lepidopteran pest, the method comprising supplying or selling to the farmer plant material such as a seed, the plant material comprising a polynucleotide, chimeric gene, expression cassette or a recombinant vector capable of expressing a Cry protein of the invention in a plant grown from the seed, as described above.

[0249] Embodiments of this invention can be better understood by reference to the following examples. The foregoing and following description of embodiments of the invention and the various embodiments are not intended to limit the claims, but are rather illustrative thereof. Therefore, it will be understood that the claims are not limited to the specific details of these examples. It will be appreciated by those skilled in the art that other embodiments of the invention may be practiced without departing from the spirit and the scope of the disclosure, the scope of which is defined by the appended claims.

EXAMPLES

Example 1. Identification of Bt Strains Containing Novel Cry Proteins

[0250] Bacillus thuringiensis isolates present in current collections were cultured from spores and maintained on T3+penicillin agar plates. Each isolate was grown aerobically in 24 well deep blocks for about 10 days at 28.degree. C. until sporulation, which was verified by staining with Coomasie blue/acetic acid and visualization with a microscope. After sporulation both the soluble and insoluble fractions were tested for activity against lepidopteran species of interest. Fractions were tested in a surface contamination bioassay, where the fractions were overlaid onto a multispecies artificial diet. Each isolate was screened against at least four lepidopteran species, including Helicoverpa zea (corn earworm), Agrotis ipsilon (black cutworm), Ostrinia nubilalis (European corn borer), and Spodoptera frugiperda (fall armyworm) with a sample size of 12 neonate larvae. The duration of each assay was about 7 days at room temperature; the plates were scored for mortality as well as larval growth inhibition. Observed mortality at an increase of 30% over the negative control was considered active. Based on the initial insect testing, three Bt strains, designated SC0532, SC0666 and SC0705, were selected for further analysis.

Example 2. Genome Assembly and Analysis

[0251] Bt cry genes of the invention were isolated from the strains identified in Example 1 using a whole genome sequencing approach. Briefly, Bacillus DNA was sheared using a Covaris S2 ultrasonic device (Covaris, Inc., Woburn, Mass.) with the program DNA 400 bp set at duty cycle: 10%; intensity: 4; cycles/burst: 200. The DNA was treated with the NEBNext.RTM. Ultra.TM. End Repair/dA-tailing module (New England Biolabs, Inc. Ipswich, Mass.). Biooscience indexes 1-57 adapters (1-27 Brazil, 28-57 USA, UK and Switzerland) were ligated using NEB Quick Ligation.TM. as described by the supplier (New England Biolabs, Inc. Ipswich, Mass.). Ligations were cleaned up using Agencourt AMPure XP beads as described by the supplier (Beckman Coulter, Inc., Indianapolis, Ind.).

[0252] The library was size fractionated as follows: A 50 .mu.l sample was mixed with 45 .mu.l 75% bead mix (25% AMPure beads plus 75% NaCl/PEG solution; TekNova, Inc. Hollister, Calif., USA; cat #P4136). The mix was stirred and placed on a magnetic rack. The resulting supernatant was transferred to a new well and 45.mu.l 50% bead mix (50% AMPure beads plus 50% NaCl/PEG solution; TekNova cat #P4136) was added. This mix was stirred and placed on a magnetic rack. The resulting supernatant was removed and the beads were washed with 80% ethanol. 25 .mu.l of elution buffer (EB) buffer was added and the mix placed on a magnetic rack. The final resulting supernatant was removed and placed in 1.5 ml tube. This method yielded libraries in the 525 DNA base pairs (bp) (insert plus adapter) size range.

[0253] The sized DNA library was amplified using KAPA Biosystem HiFi Hot Start (Kapa Biosystems, Inc., Wilmington, Mass.) using the following cycle conditions: [98.degree. C., 45 s]; 12.times.[98.degree. C., 15 s, 60.degree. C., 30 s, 72.degree. C., 30 s]; [72.degree. C., 1 min]. Each reaction contained: 5 .mu.l DNA library, 1 .mu.l Bioscience universal primer (25 .mu.M), 18 .mu.l sterile water, 1 .mu.l Bioscience indexed primer (25 .mu.M), 25 .mu.l2.times.KAPA HiFi polymerase.

[0254] Libraries were run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.) using High Sensitivity chips to determine the library size range and average insert size. All libraries were processed for paired end (PE) sequencing (100 cycles per read; 12-24 libraries per lane) on a HiSeq 2500 sequencing system using standard manufacturer's sequencing protocols (Illumina, Inc., San Diego, Calif.).

[0255] A Bacillus computational analysis tool developed to identify and characterize Cry-like genes was used for prioritization of leads for further laboratory testing.

[0256] The genome assembly and analysis as well as the genomic library analysis described above led to the identification of three Cry1-like genes in the Bacillus thuringiensis strains with toxicity to at least European corn borer (Ostrinia nubilalis) or corn earworm (Helicoverpa zea). Identifying characteristics of the Cry1-like genes and proteins are shown in Table 1.

TABLE-US-00001 TABLE 1 Cry genes/proteins identified in Bacillus thuringiensis strains. Nearest Cry Molecular Protein/Gene Family Weight Amino Acid Nucleotide Strain Name Member (kD) SEQ ID NO: SEQ ID NO: SC0532 BT2Cry1J Cry1Ja 132.6 1 14 SC0705 BT25Cry1I Cry1Ig 79.8 2 15 SC0666 BT53Cry1J Cry1Jc 133.3 3 16

Example 3. Homology of BT2Cry1J, BT25Cry1I and BT53Cry1J to Known Bt Cry Proteins

[0257] Comparison of the amino acid sequences of the proteins in Table 1 to the non-redundant (nr) database maintained by the National Center for Biotechnology Information (NCBI) (world wide web at ncbi.nlm.nih.gov) using the BLAST algorithm revealed that the proteins have the highest identity to Cry proteins, particularly those in the Cry1 family. More specifically, BT2Cry1J has about 98% identity to Cry1Ja proteins, example sequences of which may be found at NCBI under accession numbers AAA22341 (Cry1Ja1), HM070030 (Cry1Ja2) and JQ228425 (Cry1Ja3). BT25Cry1I has about 95% identity to Cry1Ig proteins, an example sequence of which may be found at NCBI under accession number KC156701 (cry1Ig1). BT53Cry1J has about 99% identity to Cry1Jc proteins, examples of which may be found at NCBI under accession numbers AAC31092 (Cry1Jc1) and AAQ52372 (Cry1Jc2).

Example 4. Bt Protein Expression in Recombinant Host Cells

[0258] The Cry proteins described in Examples 2 and 3 were expressed in recombinant bacterial host cells via a shuttle vector designated pCIB5634', designed for expression in both E. coli and Bacillus thuringiensis. Vector pCIB5634' comprises a variant modified Cry1Ac promoter (bp 12-97 of SEQ ID NO:30) that improves expression of the cloned Bt Cry gene and a erythromycin resistance marker over the native Cry1Ac promoter. For example, a BT25Cry1I coding sequence was cloned into the pCIB5634' vector using BamHI and SacI restriction sites resulting in an Cry expression shuttle vector comprising the sequence of SEQ ID NO:30.

[0259] Bacillus Expression. Expression cassettes comprising the Cry protein coding sequence of interest were transformed into a crystal-minus Bacillus thuringiensis (Bt) strain having no observable background insecticidal activity via electroporation and transgenic Bt strains were selected on erythromycin containing agar plates. Selected transgenic Bt strains were grown to the sporulation phase in T3 media at 28.degree. C. for 4-5 days. Cell pellets were harvested and washed iteratively before solubilization of the expressed protein in high pH carbonate buffer (50 mM) containing 2 mM DTT.

[0260] E. coli Expression. Cry proteins were expressed in E. coli strains using pET28a or pET29a vectors (Merck KGaA, Darmstadt, Germany). Constructs were transformed by electroporation and transgenic E. coli clones were selected on kanamycin-containing agar plates. Selected transgenic E. coli strains were grown and Cry protein expression induced using IPTG induction at 28.degree. C. Cells were resuspended in high pH carbonate buffer (50 mM) containing 2 mM DTT and then broken using a Microfluidics LV-1 homogenizer.

[0261] Expression Analysis. Resulting cell lysates from either transgenic Bt or E. coli strains were then clarified via centrifugation and samples were analyzed for purity via SDS-PAGE and electropherogram using a BioRad Experion system (Biorad, Hercules, Calif.). Total protein concentrations were determined via Bradford or Thermo 660 assay. Purified Cry proteins were then tested in bioassays described below.

Example 5. Activity of Cry Proteins in Bioassays

[0262] The Cry proteins produced in Example 4 were tested against one or more of the following insect pest species using an art-recognized artificial diet bioassay method: fall armyworm (FAW; Spodoptera frugiperda), corn earworm (CEW; Helicoverpa zea), European corn borer (ECB; Ostrinia nubilalis), black cutworm (BCW; Agrotis sugarcane borer (SCB; Diatraea saccharlis), velvet bean caterpillar (VBC; Anticarsia gemmatalis), soybean looper (SBL; Pseudoplusia includens), southwest corn borer (SWCB; Diatraea grandiosella), western bean cutworm (WBCW; Striacosta albicosta), tobacco budworm (TBW; Heliothis virescens), Asian corn borer (ACB; Ostrinia furnacalis), cotton bollworm (CBW; Helicoverpa armigera), striped stem borer (SSB; Chilo suppressalis), pink stem borer (PSB; Sesamia inferens) or rice leaf folder (RLF; Cnaphalocrocis medinails).

[0263] An equal amount of protein in solution was applied to the surface of an artificial insect diet (Bioserv, Inc., Frenchtown, N.J.) in 24 well plates. After the diet surface dried, larvae of the insect species being tested were added to each well. The plates were sealed and maintained at ambient laboratory conditions with regard to temperature, lighting and relative humidity. A positive-control group consisted of larvae exposed to a very active and broad-spectrum wild-type Bacillus strain. Negative control groups consisted of larvae exposed to insect diet treated with only the buffer solution or empty vector and larvae on untreated insect diet; i.e. diet alone. Mortality was assessed after about 120 hours and scored relative to the controls.

[0264] Results are shown in Table 2, where a "-" means no mortality compared to the control group, a "+1-" means 0-10% mortality compared to the control group (this category also includes 0% mortality with strong larval growth inhibition), a "+" means 10-25% activity compared to the control group, a "++" means 26-75% mortality compared to the control group, and a "+++" means 76-100% mortality compared to the control group.

TABLE-US-00002 TABLE 2 Results of bioassays with Cry Proteins. BT Insect Species Proteins FAW CEW ECB BCW SCB VBC SBL SWCB TBW BT2Cry1J +/- + +++ +++ +++ +++ +++ ++ +++ BT25Cry1I - - + - ++ - +/- +/- - BT53Cry1J - +++ ++ +++ +++ +++ +++ +/- +++

Example 6. Mutagenesis of the BT2Cry1J protein

[0265] The BT2Cry1J protein has 98% identity to the known Cry1Ja proteins, Cry1Ja1 (NCBI Accession No. AAA22341), Cry1Ja2 (NCBI Accession No. HM070030) and Cry1Ja3 (NCBI Accession No. JQ228425). Based on standard Bt Cry protein nomenclature (Crickmore et al. 1998. Microbiol. Molecular Biol. Rev. 62:807-813), BT2Cry1J most likely would be designated a Cry1Ja protein. Given the very high identity between BT2Cry1Ja and Cry1Ja1, having only 24 amino acid differences over 1167 total amino acids, one may expect that the two proteins would have the same spectrum of activity and specificity. Surprisingly, BT2Cry1Ja appears to have a broader spectrum of activity or higher specific activity than the known Cry1Ja proteins. For example, results of some research suggest that Cry1Ja1 has minimal activity against fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea) and European corn borer (Ostrinia nubilalis) and no activity against black cutworm (Agrotis ipsilon) (U.S. Pat. Nos. 5,322,687 and 6,593,293), whereas BT2Cry1Ja has high activity against fall armyworm, corn earworm, European corn borer and black cutworm. Other reports suggest that Cry1Ja1 has some activity against cotton bollworm (Helicoverpa armigera), but no activity against diamondback moth or beet armyworm (Spodoptera exigua) (Choi et al. 2007. J. Microbiol. Biotechnol. 17:1498-1503). Still other reports suggest that Cry1Ja2 is active against diamondback moth and that Cry1Ja3 is active against Asian corn borer (Ostrinia furnacalis) (Hai-Shou et al. 2015. Genetics 11:1145-1451.

[0266] Twenty-three of the twenty-four amino acid differences between BT2Cry1J of the invention and Cry1Ja1 are in the region of domain I spanning alpha-helices 3 to 6. The last amino acid difference is in a region in domain II known as Loop alpha-8, which is a region that is believed to be important in insect gut receptor binding. To determine which of the domain I amino acids may be important in modulating the activity of Cry1J proteins, mutations were made in the BT2Cry1J amino acid sequence (SEQ ID NO:1) in three blocks, which essentially correspond to regions spanning alpha-helix 3, alpha-helix 4 and alpha-helices 5&6, respectively. The three mutation blocks were designated: BLK-1 comprising the following amino acid substitutions, A97T, S105N, L108I, G110A, K118S and T119D, which spans alpha-helix 3 and one additional amino acid in the loop between alpha-helices 3 and 4; BLK-2 comprising the following amino acid substitutions, T123E, R126K, T130I, E131D, I136L, A138G, Q139L, V149I and V150I, which spans alpha-helix 4 and two amino acids in the loop between alpha-helices 4 and 5; and BLK-3 comprising the following amino acid substitutions, L158S, T161V, V176I, T186K, V196I, N197R, R198E and G200H, which spans alpha-helix 5 and a portion of alpha-helix 6. BT2Cry1J proteins comprising combinations of the three mutation blocks were also made resulting in a total of seven modified BT2Cry1J proteins that were tested against target insects

[0267] Constructs comprising each of the mutation blocks and combinations of the mutation blocks were made by synthesizing approximately 663 bp NcoI-BglII polynucleotide fragments of SEQ ID NO:14 encoding the substituted amino acids in the desired region of the BT2Cry1J amino acid sequence. Each polynucleotide fragment was cloned into a vector comprising the full-length native BT2Cry1J coding sequence cut with NcoI-BglII enzymes. Each fragment then replaced the 5' end of the full-length gene resulting in a modified full-length coding sequence (SEQ ID NOs:23-29) encoding a modified BT2Cry1J protein (SEQ ID NOs:7-13). Each vector was cloned into a crystal-minus Bacillus thuringiensis strain as described above.

[0268] Modified BT2Cry1J proteins were expressed as described above and tested against three insect pest species in the Family Crambidae, European corn borer (Ostrinia nubilalis), sugarcane borer (Diatraea saccharalis) and southwest corn borer (Diatraea grandiosella), and six insect pest species in the Family Noctuidae, black cutworm (Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), soybean looper (Chrysodeixis includens), velvetbean caterpillar (Anticarsia gemmatalis) and tobacco budworm (Heliothis virescens). The presence of each modified BT2Cry1J protein was confirmed by Coomassie-stained SDS-PAGE using the Bt cells carrying the empty vector as a negative control. The insecticidal activity, as corrected percent mortality, of the seven modified BT2Cry1J proteins compared to a native BT2Cry1J protein (SEQ ID NO:1) and an empty-vector control are shown in Table 3.

TABLE-US-00003 TABLE 3 Insecticidal activity of modified BT2Cry1J proteins. Modified Protein Corrected Percent Mortality or Controls ECB SCB SWCB BCW FAW CEW SBL VBC TBW BT0002Cry1Ja 83 100 75 75 100 75 100 100 100 BT21J-BLK-1 0 0 0 0 0 8 42 0 92 BT21J-BLK-2 8 0 0 8 0 8 100 67 42 BT21J-BLK-3 75 92 75 92 83 100 100 100 100 BT21J-BLK-1/2 83 75 75 67 25 42 100 100 92 BT21J-BLK-2/3 8 8 0 42 83 50 100 100 92 BT21J-BLK-1/3 0 0 0 0 0 8 0 0 0 BT21J-BLK-1/2/3 83 100 75 92 92 92 100 100 100 Empty Vector 0 0 0 0 0 0 0 0 0

Example 7. Vectoring of Genes for Plant Expression

[0269] Prior to expression in plants, a synthetic polynucleotide comprising a nucleotide sequence having codons optimized for expression in the plant and encoding a Cry protein of the invention, such as a BT2Cry1J (SEQ ID NO:1 or SEQ ID NO) or a variant sequence (SEQ ID NO:4), BT25Cry1I (SEQ ID NO:2) or a variant sequence (SEQ ID NO:5) or BT53Cry1J (SEQ ID NO:3) or a variant sequence (SEQ ID NO:6), is synthesized by methods known in the art. For this example, a first expression cassette was made comprising a maize ubiquitin promoter (Ubi1) operably linked to a BT53Cry1J synthetic coding sequence (SEQ ID NO:19) which is operably linked to a maize ubiquitin (Ubi361) terminator and a second expression cassette was made comprising a maize ubiquitin 1 (Ubi1) promoter operably linked to a phosphomannose isomerase (PMI) coding sequence which is operably linked to a maize Ubi 1 terminator. Expression of PMI allows for positive selection of transgenic plants on mannose. Both expression cassettes were cloned into a binary vector (SEQ ID NO:31) for use in Agrobacterium-mediated maize transformation.

Example 8. Expression and Activity of Cry Proteins in Maize Plants

[0270] Transformation of immature maize embryos was performed essentially as described in Negrotto et al., 2000, Plant Cell Reports 19: 798 803. Briefly, Agrobacterium strain LBA4404 (pSB1) comprising a binary vector described in Example 7 was grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl (5 g/L), 15 g/1 agar, pH 6.8) solid medium for 2-4 days at 28.degree. C. Approximately 0.8.times.10.sup.9 Agrobacterium cells were suspended in LS-inf media supplemented with 100 .mu.M As. Bacteria were pre-induced in this medium for approximately 30-60 minutes.

[0271] Immature embryos from an inbred maize line were excised from about 8-12 day old ears into liquid LS-inf+100 .mu.M As. Embryos were rinsed once with fresh infection medium. Agrobacterium solution was then added and embryos were vortexed for about 30 seconds and allowed to settle with the bacteria for 5 minutes. The embryos were then transferred scutellum side up to LSAs medium and cultured in the dark for two to three days. Subsequently, between approximately 20 and 25 embryos per petri plate were transferred to LSDc medium supplemented with cefotaxime (250 mg/1) and silver nitrate (1.6 mg/1) and cultured in the dark at approximately 28.degree. C. for 10 days.

[0272] Immature embryos, producing embryogenic callus were transferred to LSD1M0.5S medium. The cultures were selected on this medium for approximately 6 weeks with a subculture step at about 3 weeks. Surviving calli were transferred to Reg1 medium supplemented with mannose. Following culturing in the light (16 hour light/8 hour dark regiment), green tissues were then transferred to Reg2 medium without growth regulators and incubated for about 1-2 weeks. Plantlets were transferred to Magenta GA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grown in the light. After about 2-3 weeks, plants were tested for the presence of the PMI genes and the Bt53cry1J gene by PCR. Positive plants from the PCR assay were transferred to a greenhouse for further evaluation.

[0273] Transgenic plants from multiple independent events were evaluated for copy number (determined by Taqman analysis), protein expression level (determined by ELISA), and efficacy against insect pest species of interest in leaf excision bioassays. Specifically, plant tissue was excised from 25 single copy events (V3-V4 stage) and infested with neonate larvae of a target pest, then incubated at room temperature for about 5 days. Leaf disks from transgenic plants expressing the variant BT53Cry1J protein (SEQ ID NO:6) were tested against corn earworm (Helicoverpa zea; CEW), black cutworm (Agrotis ipsilon; BCW), and sugarcane borer (Diatraea saccharalis; SCB).

[0274] The expression levels of the BT53Cry1J protein in the 25 transgenic events ranged from about 3 ng/mg TSP to about 11 ng/mg TSP. Results of the plant bioassay confirmed that stably transformed maize plants expressing a BT53Cry1J protein are toxic to one or more lepidopteran insect pests with 15/25 plants having activity against CEW, 7/25 plants having activity against BCW and 11/25 plants having activity against SCB.

Example 9. Mutation of a Cry Protein-Encoding Gene Comprised in a Transgenic Plant

[0275] The following example illustrates the use of genome editing to incorporate mutations into a gene encoding a Cry1J protein of the invention, including but not limited to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:6, comprised in a transgenic maize plant.

[0276] Targeted genome modification, also known as genome editing, is useful for introducing mutations in specific DNA sequences. These genome editing technologies, which include zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENS), meganucleases and clustered regularly interspaced short palindromic repeats (CRISPR) have been successfully applied to over 50 different organisms including crop plants. See, e.g., Belhaj, K., et al., Plant Methods 9, 39 (2013); Jiang, W., et al., Nucleic Acids Res, 41, e188 (2013)). The CRISPR/Cas system for genome editing is based on transient expression of Cas9 nuclease and an engineered single guide RNA (sgRNA) that specifies the targeted polynucleotide sequence.

[0277] Cas9 is a large monomeric DNA nuclease guided to a DNA target sequence with the aid of a complex of two 20-nucleotide (nt) non-coding RNAs: CRIPSR RNA (crRNA) and trans-activating crRNA (tracrRNA), which are functionally available as single synthetic RNA chimera. The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target cry1J DNA.

[0278] When the Cas9 and the sgRNA are transiently expressed in living maize cells, double strand breaks (DSBs) in the specific targeted cry1J DNA is created in the transgenic maize. Mutation at the break site is introduced through the non-homologous end joining and homology-directed DNA repair pathways.

[0279] Specific mutations, for example the BLK1 mutations described above, are introduced into the gene encoding BT2Cry1J or a variant thereof, such as SEQ ID NO:1 or SEQ ID NO:4, through the use of recombinant plasmids expressing the Cas9 nuclease and the sgRNA target that is codon optimized for the cry1J sequence in the transgenic maize. Implementation of the method is by an agroinfiltration method with Agrobacterium tumufaciens carrying the binary plasmid harboring the specified target sequence of interest of cry1J. After the sgRNA binds to the cry1J coding sequence, the Cas9 nuclease makes specific cuts into the coding sequence and introduces the BLK1 mutations during DNA repair. Thus, the now mutated cry1J gene will encode a modified Cry1J protein, such as SEQ ID NO:7, where a mutation at position 97 replaces Ala (A) with Thr (T); a mutation at position 105 replaces Ser (S) with Asn (N); a mutation at position 108 replaces Leu (L) with Ile (I); a mutation at position 110 replaces Gly (G) with Ala (A); a mutation at position 118 replaces the Lys (K) with Ser (S) and a mutation at position 119 replaces Thr (T) with Asp (D). Plant cells comprising mutated cry 1J polynucleotides are screened by PCR and sequencing. Callus that harbor mutations in the cry1J gene are induced to regenerate plants for phenotype evaluation for modulated insecticidal activity of the expressed modified Cry1J protein.

Sequence CWU 1

1

3111167PRTBacillus thuringiensis 1Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 11652710PRTBacillus thuringiensis 2Met Lys Ser Lys Asn Gln Asn Met His Gln Ser Leu Ser Asn Asn Ala1 5 10 15Thr Val Asp Lys Asn Phe Thr Gly Ser Leu Glu Asn Asn Thr Asn Thr 20 25 30Glu Leu Gln Asn Phe Asn His Glu Gly Ile Glu Pro Phe Val Ser Val 35 40 45Ser Thr Ile Gln Thr Gly Ile Gly Ile Ala Gly Lys Ile Leu Gly Asn 50 55 60Leu Gly Val Pro Phe Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile65 70 75 80Leu Gly Glu Leu Trp Pro Lys Gly Lys Ser Gln Trp Glu Ile Phe Met 85 90 95Glu His Val Glu Glu Leu Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg 100 105 110Asn Lys Ala Leu Ala Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val 115 120 125Tyr His Glu Ser Leu Glu Ser Trp Ile Lys Asn Arg Asn Asn Thr Arg 130 135 140Thr Arg Ser Val Val Lys Ser Gln Tyr Ile Thr Leu Glu Leu Met Phe145 150 155 160Val Gln Ser Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu 165 170 175Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Asp Ser Glu Ile 195 200 205Ser Thr Phe Tyr Asn Arg Gln Val Glu Arg Thr Ser Asp Tyr Ser Asp 210 215 220His Cys Thr Lys Trp Phe Asp Thr Gly Leu Asn Arg Leu Lys Gly Ser225 230 235 240Asn Ala Glu Ile Trp Val Lys Tyr Asn Gln Phe Arg Arg Asp Met Thr 245 250 255Leu Met Val Leu Asp Leu Val Ala Leu Phe Gln Ser Tyr Asp Thr His 260 265 270Met Tyr Pro Ile Lys Thr Thr Ala Gln Leu Thr Arg Glu Val Tyr Thr 275 280 285Asn Ala Leu Gly Thr Val His Pro His Pro Ser Phe Thr Ser Thr Thr 290 295 300Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Val305 310 315 320Ile Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gln Val Thr Ile Tyr 325 330 335Ser Leu Leu Ser Arg Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly 340 345 350Gly His Lys Leu Glu Phe Arg Thr Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Pro Phe 370 375 380Thr Ser Arg Asp Ile Tyr Arg Thr Glu Ser Leu Ala Gly Leu Asn Leu385 390 395 400Phe Leu Thr Gln Pro Val Asn Gly Val Pro Arg Val Asp Phe His Trp 405 410 415Lys Phe Val Thr His Pro Ile Ala Ser Asp Asn Phe Tyr Tyr Pro Gly 420 425 430Tyr Ala Gly Ile Gly Thr Gln Leu Gln Asp Ser Glu Asn Glu Leu Pro 435 440 445Pro Glu Ala Thr Gly Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu 450 455 460Ser His Ile Gly Leu Ile Ser Ala Ser His Val Lys Ala Leu Val Tyr465 470 475 480Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile His Ser Asp 485 490 495Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly 500 505 510Thr Thr Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg 515 520 525Arg Thr Ser Gly Gly Pro Phe Ala Phe Ser Asn Val Asn Leu Asp Trp 530 535 540Asn Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr545 550 555 560Asn Leu Arg Met Tyr Val Thr Ile Ala Gly Glu Arg Ile Phe Ala Gly 565 570 575Gln Phe Asn Lys Thr Met Asn Thr Gly Asp Pro Leu Thr Phe Gln Ser 580 585 590Phe Ser Tyr Ala Thr Ile Asp Thr Ala Phe Thr Phe Pro Thr Lys Ala 595 600 605Ser Ser Leu Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val 610 615 620Tyr Val Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala625 630 635 640Val Thr Asp Leu Glu Arg Ala Gln Lys Ala Val His Glu Leu Phe Thr 645 650 655Ser Thr Asn Pro Gly Gly Leu Lys Thr Asp Val Lys Asp Tyr His Ile 660 665 670Asp Gln Val Ser Asn Leu Val Glu Ser Leu Ser Asp Lys Phe Tyr Leu 675 680 685Asp Glu Lys Arg Glu Leu Phe Glu Ile Val Lys Tyr Ala Lys Gln Leu 690 695 700His Ile Glu Arg Asn Met705 71031168PRTBacillus thuringiensis 3Met Glu Ile Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Phe Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Leu Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Asp Trp Glu Leu Phe Leu Glu Gln Ile Glu65 70 75 80Gln Leu Ile Asp Arg Arg Ile Glu Arg Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Lys His Cys Val Asp 195 200 205Thr Tyr Lys Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Val Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Arg Thr Ile Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Asn Asn Ile Ile Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Thr Asn Thr Glu Gly His Gln Arg Ser Phe Pro 325 330 335Leu Ala Gly Thr Ile Gly Asn Ser Ala Pro Pro Val Thr Val Arg Asn 340 345 350Asn Gly Glu Gly Ile Tyr Arg Ile Leu Ser Glu Pro Phe Tyr Ser Ala 355 360 365Pro Phe Leu Gly Thr Ser Val Leu Gly Ser Arg Gly Glu Glu Phe Ala 370 375 380Phe Ala Ser Asn Thr Thr Thr Ser Leu Pro Ser Thr Ile Tyr Arg Asn385 390 395 400Arg Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp Tyr Ser 405 410 415Val Pro Pro His Arg Gly Tyr Ser His Leu Leu Ser His Val Thr Met 420 425 430Arg Asn Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala Thr Pro 435 440 445Arg Asn Thr Ile Asp Pro Asp Ser Ile Thr Gln Ile Pro Ala Val Lys 450 455 460Gly Ala Tyr Ile Phe Asn Ser Pro Val Ile Thr Gly Pro Gly His Thr465 470 475 480Gly Gly Asp Ile Ile Arg Phe Asn Pro Asn Thr Gln Asn Asn Ile Arg 485 490 495Ile Pro Phe Gln Ser Asn Ala Val Gln Arg Tyr Arg Ile Arg Met Arg 500 505 510Tyr Ala Ala Glu Ala Asp Cys Ile Leu Glu Ser Gly Val Asn Ile Val 515 520 525Thr Gly Ala Gly Val Thr Phe Arg Pro Ile Pro Ile Lys Ala Thr Met 530 535 540Thr Pro Gly Ser Pro Leu Thr Tyr Tyr Ser Phe Gln Tyr Ala Asp Leu545 550 555 560Asn Ile Asn Leu Thr Ala Pro Ile Arg Pro Asn Asn Phe Val Ser Ile 565 570 575Arg Arg Ser Asn Gln Pro Gly Asn Leu Tyr Ile Asp Arg Ile Glu Phe

580 585 590Ile Pro Ile Asp Pro Ile Arg Glu Ala Glu His Asp Leu Glu Arg Ala 595 600 605Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu 610 615 620Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val625 630 635 640Ala Cys Leu Ser Asp Lys Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser 645 650 655Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu 660 665 670Gln Asp Gln Asn Phe Thr Gly Ile Asn Arg Gln Val Asp Arg Gly Trp 675 680 685Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys 690 695 700Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr705 710 715 720Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg 725 730 735Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr 740 745 750Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Leu Asn Val Pro Gly Thr 755 760 765Gly Ser Leu Trp Pro Leu Ala Ala Glu Ser Ser Ile Gly Arg Cys Gly 770 775 780Glu Pro Asn Arg Cys Ala Pro His Ile Glu Trp Asn Pro Glu Leu Asp785 790 795 800Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe 805 810 815Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 820 825 830Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly Tyr Ala Arg Leu 835 840 845Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu 850 855 860Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Asp Lys865 870 875 880Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val 885 890 895Asp Ala Leu Phe Val Asp Ser Gln Tyr Asn Arg Leu Gln Thr Asp Thr 900 905 910Asn Ile Ala Met Ile His Val Ala Asp Lys Arg Val His Arg Ile Arg 915 920 925Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala 930 935 940Ile Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr945 950 955 960Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn His Gly Leu Ser 965 970 975Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His 980 985 990Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu 995 1000 1005Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1010 1015 1020Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1025 1030 1035Glu Asp His Thr Asp Glu Leu Lys Phe Arg Asn Cys Glu Glu Glu 1040 1045 1050Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Pro Ala 1055 1060 1065Asn Gln Glu Glu Tyr Arg Ala Ala Glu Thr Ser Arg Asn Arg Gly 1070 1075 1080Tyr Gly Glu Ser Tyr Glu Ser Asn Ser Ser Ile Pro Ala Glu Tyr 1085 1090 1095Ala Pro Ile Tyr Glu Lys Ala Tyr Thr Asp Gly Arg Lys Glu Asn 1100 1105 1110Ser Cys Glu Ser Asn Arg Gly Tyr Gly Asn Tyr Thr Pro Leu Pro 1115 1120 1125Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1130 1135 1140Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val 1145 1150 1155Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1160 116541167PRTArtificial SequenceVariant BT0002 proteinMISC_FEATUREAmino acid substitutions I1146L and L1163I 4Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Leu Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Ile Leu Met Glu Glu 1160 11655710PRTArtificial SequenceVariant BT0025 proteinMISC_FEATUREAmino acid substitutions I672L and I697L 5Met Lys Ser Lys Asn Gln Asn Met His Gln Ser Leu Ser Asn Asn Ala1 5 10 15Thr Val Asp Lys Asn Phe Thr Gly Ser Leu Glu Asn Asn Thr Asn Thr 20 25 30Glu Leu Gln Asn Phe Asn His Glu Gly Ile Glu Pro Phe Val Ser Val 35 40 45Ser Thr Ile Gln Thr Gly Ile Gly Ile Ala Gly Lys Ile Leu Gly Asn 50 55 60Leu Gly Val Pro Phe Ala Gly Gln Val Ala Ser Leu Tyr Ser Phe Ile65 70 75 80Leu Gly Glu Leu Trp Pro Lys Gly Lys Ser Gln Trp Glu Ile Phe Met 85 90 95Glu His Val Glu Glu Leu Ile Asn Gln Lys Ile Ser Thr Tyr Ala Arg 100 105 110Asn Lys Ala Leu Ala Asp Leu Lys Gly Leu Gly Asp Ala Leu Ala Val 115 120 125Tyr His Glu Ser Leu Glu Ser Trp Ile Lys Asn Arg Asn Asn Thr Arg 130 135 140Thr Arg Ser Val Val Lys Ser Gln Tyr Ile Thr Leu Glu Leu Met Phe145 150 155 160Val Gln Ser Leu Pro Ser Phe Ala Val Ser Gly Glu Glu Val Pro Leu 165 170 175Leu Pro Ile Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190Asp Ala Ser Ile Phe Gly Lys Glu Trp Gly Leu Ser Asp Ser Glu Ile 195 200 205Ser Thr Phe Tyr Asn Arg Gln Val Glu Arg Thr Ser Asp Tyr Ser Asp 210 215 220His Cys Thr Lys Trp Phe Asp Thr Gly Leu Asn Arg Leu Lys Gly Ser225 230 235 240Asn Ala Glu Ile Trp Val Lys Tyr Asn Gln Phe Arg Arg Asp Met Thr 245 250 255Leu Met Val Leu Asp Leu Val Ala Leu Phe Gln Ser Tyr Asp Thr His 260 265 270Met Tyr Pro Ile Lys Thr Thr Ala Gln Leu Thr Arg Glu Val Tyr Thr 275 280 285Asn Ala Leu Gly Thr Val His Pro His Pro Ser Phe Thr Ser Thr Thr 290 295 300Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Val305 310 315 320Ile Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gln Val Thr Ile Tyr 325 330 335Ser Leu Leu Ser Arg Trp Ser Asn Thr Gln Tyr Met Asn Met Trp Gly 340 345 350Gly His Lys Leu Glu Phe Arg Thr Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Pro Phe 370 375 380Thr Ser Arg Asp Ile Tyr Arg Thr Glu Ser Leu Ala Gly Leu Asn Leu385 390 395 400Phe Leu Thr Gln Pro Val Asn Gly Val Pro Arg Val Asp Phe His Trp 405 410 415Lys Phe Val Thr His Pro Ile Ala Ser Asp Asn Phe Tyr Tyr Pro Gly 420 425 430Tyr Ala Gly Ile Gly Thr Gln Leu Gln Asp Ser Glu Asn Glu Leu Pro 435 440 445Pro Glu Ala Thr Gly Gln Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu 450 455 460Ser His Ile Gly Leu Ile Ser Ala Ser His Val Lys Ala Leu Val Tyr465 470 475 480Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile His Ser Asp 485 490 495Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly 500 505 510Thr Thr Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg 515 520 525Arg Thr Ser Gly Gly Pro Phe Ala Phe Ser Asn Val Asn Leu Asp Trp 530 535 540Asn Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr545 550 555 560Asn Leu Arg Met Tyr Val Thr Ile Ala Gly Glu Arg Ile Phe Ala Gly 565 570 575Gln Phe Asn Lys Thr Met Asn Thr Gly Asp Pro Leu Thr Phe Gln Ser 580 585 590Phe Ser Tyr Ala Thr Ile Asp Thr Ala Phe Thr Phe Pro Thr Lys Ala 595 600 605Ser Ser Leu Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val 610 615 620Tyr Val Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala625 630 635 640Val Thr Asp Leu Glu Arg Ala Gln Lys Ala Val His Glu Leu Phe Thr 645 650 655Ser Thr Asn Pro Gly Gly Leu Lys Thr Asp Val Lys Asp Tyr His Leu 660 665 670Asp Gln Val Ser Asn Leu Val Glu Ser Leu Ser Asp Lys Phe Tyr Leu 675 680 685Asp Glu Lys Arg Glu Leu Phe Glu Leu

Val Lys Tyr Ala Lys Gln Leu 690 695 700His Ile Glu Arg Asn Met705 71061168PRTArtificial SequenceVariant BT0053 proteinMISC_FEATUREAmino acid substitutions E2A and I1157L 6Met Ala Ile Asn Asn Gln Asn Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Phe Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Leu Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Asp Trp Glu Leu Phe Leu Glu Gln Ile Glu65 70 75 80Gln Leu Ile Asp Arg Arg Ile Glu Arg Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Lys His Cys Val Asp 195 200 205Thr Tyr Lys Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Val Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Arg Thr Ile Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Asn Asn Ile Ile Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Thr Asn Thr Glu Gly His Gln Arg Ser Phe Pro 325 330 335Leu Ala Gly Thr Ile Gly Asn Ser Ala Pro Pro Val Thr Val Arg Asn 340 345 350Asn Gly Glu Gly Ile Tyr Arg Ile Leu Ser Glu Pro Phe Tyr Ser Ala 355 360 365Pro Phe Leu Gly Thr Ser Val Leu Gly Ser Arg Gly Glu Glu Phe Ala 370 375 380Phe Ala Ser Asn Thr Thr Thr Ser Leu Pro Ser Thr Ile Tyr Arg Asn385 390 395 400Arg Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp Tyr Ser 405 410 415Val Pro Pro His Arg Gly Tyr Ser His Leu Leu Ser His Val Thr Met 420 425 430Arg Asn Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala Thr Pro 435 440 445Arg Asn Thr Ile Asp Pro Asp Ser Ile Thr Gln Ile Pro Ala Val Lys 450 455 460Gly Ala Tyr Ile Phe Asn Ser Pro Val Ile Thr Gly Pro Gly His Thr465 470 475 480Gly Gly Asp Ile Ile Arg Phe Asn Pro Asn Thr Gln Asn Asn Ile Arg 485 490 495Ile Pro Phe Gln Ser Asn Ala Val Gln Arg Tyr Arg Ile Arg Met Arg 500 505 510Tyr Ala Ala Glu Ala Asp Cys Ile Leu Glu Ser Gly Val Asn Ile Val 515 520 525Thr Gly Ala Gly Val Thr Phe Arg Pro Ile Pro Ile Lys Ala Thr Met 530 535 540Thr Pro Gly Ser Pro Leu Thr Tyr Tyr Ser Phe Gln Tyr Ala Asp Leu545 550 555 560Asn Ile Asn Leu Thr Ala Pro Ile Arg Pro Asn Asn Phe Val Ser Ile 565 570 575Arg Arg Ser Asn Gln Pro Gly Asn Leu Tyr Ile Asp Arg Ile Glu Phe 580 585 590Ile Pro Ile Asp Pro Ile Arg Glu Ala Glu His Asp Leu Glu Arg Ala 595 600 605Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu 610 615 620Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val625 630 635 640Ala Cys Leu Ser Asp Lys Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser 645 650 655Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu 660 665 670Gln Asp Gln Asn Phe Thr Gly Ile Asn Arg Gln Val Asp Arg Gly Trp 675 680 685Arg Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys 690 695 700Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr705 710 715 720Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Pro Tyr Thr Arg 725 730 735Tyr Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr 740 745 750Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Leu Asn Val Pro Gly Thr 755 760 765Gly Ser Leu Trp Pro Leu Ala Ala Glu Ser Ser Ile Gly Arg Cys Gly 770 775 780Glu Pro Asn Arg Cys Ala Pro His Ile Glu Trp Asn Pro Glu Leu Asp785 790 795 800Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe 805 810 815Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly 820 825 830Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly Tyr Ala Arg Leu 835 840 845Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu 850 855 860Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Asp Lys865 870 875 880Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val 885 890 895Asp Ala Leu Phe Val Asp Ser Gln Tyr Asn Arg Leu Gln Thr Asp Thr 900 905 910Asn Ile Ala Met Ile His Val Ala Asp Lys Arg Val His Arg Ile Arg 915 920 925Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala 930 935 940Ile Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr945 950 955 960Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn His Gly Leu Ser 965 970 975Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His 980 985 990Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu 995 1000 1005Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala 1010 1015 1020Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile 1025 1030 1035Glu Asp His Thr Asp Glu Leu Lys Phe Arg Asn Cys Glu Glu Glu 1040 1045 1050Glu Val Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Pro Ala 1055 1060 1065Asn Gln Glu Glu Tyr Arg Ala Ala Glu Thr Ser Arg Asn Arg Gly 1070 1075 1080Tyr Gly Glu Ser Tyr Glu Ser Asn Ser Ser Ile Pro Ala Glu Tyr 1085 1090 1095Ala Pro Ile Tyr Glu Lys Ala Tyr Thr Asp Gly Arg Lys Glu Asn 1100 1105 1110Ser Cys Glu Ser Asn Arg Gly Tyr Gly Asn Tyr Thr Pro Leu Pro 1115 1120 1125Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp 1130 1135 1140Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Leu Val 1145 1150 1155Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1160 116571167PRTArtificial SequenceModified alpha-helix 3 BT0002MISC_FEATURE(97)..(119)BT0002 block 1 mutations A97T; S105N; L108I; G110A; K118S & T119D 7Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala 100 105 110Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 116581167PRTArtificial SequenceModified alpha-helix 4 BT0002MISC_FEATURE(123)..(150)BT0002 block 2 mutations T123E; R126K; T130I; E131D; I136L; A138G; Q139L; V149I & V150I 8Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile

Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Glu Ala Ala Lys Ser Arg 115 120 125Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Ile Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 116591167PRTArtificial SequenceModified alpha-helix 5/6 BT0002MISC_FEATURE(158)..(200)BT0002 block 3 mutations L158S; T161V; V176I; T186K; V196I; N197R; R198E & G200H 9Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr145 150 155 160Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Lys Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165101167PRTArtificial SequenceModfied alpha-helix 3/4 BT0002MISC_FEATURE(97)..(150)BT0002 blocks 1 & 2 mutations A97T; S105N; L108I; G110A; K118S; T119D; T123E; R126K; T130I; E131D; I136L; A138G; Q139L; V149I & V150I 10Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala 100 105 110Phe Lys Glu Trp Glu Ser Cys Pro Asp Asn Glu Ala Ala Lys Ser Arg 115 120 125Val Ile Asp Arg Phe Arg Ile Leu Asp Gly

Ile Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pro Leu Leu Leu Val Tyr145 150 155 160Thr Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Val 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Thr Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Val Asn Arg Ile Gly Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165111167PRTArtificial SequenceModified alpha-helix 4/5/6 BT0002MISC_FEATURE(123)..(200)BT0002 blocks 2 & 3 mutations; T123E; R126K; T130I; E131D; I136L; A138G; Q139L; V149I; V150I; L158S; T161V; V176I; T186K; V196I; N197R; R198E & G200H 11Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Ala Glu Leu Glu Gly Leu Gly Arg Ser Tyr Gln Leu Tyr Gly Glu Ala 100 105 110Phe Lys Glu Trp Glu Lys Thr Pro Asp Asn Glu Ala Ala Lys Ser Arg 115 120 125Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Ile Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pro Leu Leu Ser Val Tyr145 150 155 160Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Glu Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165121167PRTArtificial SequenceModified alpha-helix 3/5/6 BT0002MISC_FEATURE(97)..(200)BT0002 blocks 1 & 3 mutations A97T; S105N; L108I; G110A; K118S; T119D; L158S; T161V; V176I; T186K; V196I; N197R; R198E & G200H 12Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala 100 105 110Phe Lys Glu Trp Glu Ser Cys Pro Asp Asn Thr Ala Ala Arg Ser Arg 115 120 125Val Thr Glu Arg Phe Arg Ile Ile Asp Ala Gln Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Val Ser Gly Phe Glu Val Pro Leu Leu Ser Val Tyr145 150 155 160Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Glu Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys

Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165131167PRTArtificial SequenceModified alpha-helix 3/4/5/6 BT0002MISC_FEATUREBT0002 blocks 1,2 &3 mutations 97T; S105N; L108I; G110A; K118S; T119D; T123E; R126K; T130I; E131D; I136L;A138G; Q139L; V149I; V150I; L158S; T161V; V176I; T186K; V196I; N197R; R198E & G200HMISC_FEATUREBT0002 blocks 1,2 &3 mutations 97T; S105N; L108I; G110A; K118S; T119D; T123E; R126K; T130I; E131D; I136L; A138G; Q139L; V149I; V150I; L158S; T161V; V176I; T186K; V196I; N197R; R198E & G200H 13Met Glu Ile Asn Asn Gln Lys Gln Cys Ile Pro Tyr Asn Cys Leu Ser1 5 10 15Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Leu Pro Asp Ile 20 25 30Asp Pro Leu Glu Val Ser Leu Ser Leu Leu Gln Phe Leu Leu Asn Asn 35 40 45Phe Val Pro Gly Gly Gly Phe Ile Ser Gly Leu Val Asp Lys Ile Trp 50 55 60Gly Ala Leu Arg Pro Ser Glu Trp Asp Leu Phe Leu Ala Gln Ile Glu65 70 75 80Arg Leu Ile Asp Gln Arg Ile Glu Ala Thr Val Arg Ala Lys Ala Ile 85 90 95Thr Glu Leu Glu Gly Leu Gly Arg Asn Tyr Gln Ile Tyr Ala Glu Ala 100 105 110Phe Lys Glu Trp Glu Ser Asp Pro Asp Asn Glu Ala Ala Lys Ser Arg 115 120 125Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Ile Ile Glu Ala Asn Ile 130 135 140Pro Ser Phe Arg Ile Ile Gly Phe Glu Val Pro Leu Leu Ser Val Tyr145 150 155 160Val Gln Ala Ala Asn Leu His Leu Ala Leu Leu Arg Asp Ser Val Ile 165 170 175Phe Gly Glu Arg Trp Gly Leu Thr Thr Glu Asn Val Asn Asp Ile Tyr 180 185 190Asn Arg Gln Ile Arg Glu Ile His Glu Tyr Ser Asn His Cys Val Asp 195 200 205Thr Tyr Asn Thr Glu Leu Glu Arg Leu Gly Phe Arg Ser Ile Ala Gln 210 215 220Trp Arg Ile Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu225 230 235 240Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Leu Tyr Pro Ile 245 250 255Gln Thr Phe Ser Gln Leu Thr Arg Glu Ile Val Thr Ser Pro Val Ser 260 265 270Glu Phe Tyr Tyr Gly Val Ile Asn Ser Gly Asn Ile Asn Gly Thr Leu 275 280 285Thr Glu Gln Gln Ile Arg Arg Pro His Leu Met Asp Phe Phe Asn Ser 290 295 300Met Ile Met Tyr Thr Ser Asp Asn Arg Arg Glu His Tyr Trp Ser Gly305 310 315 320Leu Glu Met Thr Ala Tyr Phe Thr Gly Phe Ala Gly Ala Gln Val Ser 325 330 335Phe Pro Leu Val Gly Thr Arg Gly Glu Ser Ala Pro Pro Leu Thr Val 340 345 350Arg Ser Val Asn Asp Gly Ile Tyr Arg Ile Leu Ser Ala Pro Phe Tyr 355 360 365Ser Ala Pro Phe Leu Gly Thr Ile Val Leu Gly Ser Arg Gly Glu Lys 370 375 380Phe Asp Phe Ala Leu Asn Asn Ile Ser Pro Pro Pro Ser Thr Ile Tyr385 390 395 400Arg His Pro Gly Thr Val Asp Ser Leu Val Ser Ile Pro Pro Gln Asp 405 410 415Asn Ser Val Pro Pro His Arg Gly Ser Ser His Arg Leu Ser His Val 420 425 430Thr Met Arg Ala Ser Ser Pro Ile Phe His Trp Thr His Arg Ser Ala 435 440 445Thr Thr Thr Asn Thr Ile Asn Pro Asn Ala Ile Ile Gln Ile Pro Leu 450 455 460Val Lys Ala Phe Asn Leu His Ser Gly Ala Thr Val Val Arg Gly Pro465 470 475 480Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe 485 490 495Ala Asp Met Arg Val Asn Ile Thr Gly Pro Leu Ser Gln Arg Tyr Arg 500 505 510Val Arg Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg 515 520 525Ile Asn Gly Thr Ser Val Asn Gln Gly Asn Phe Gln Arg Thr Met Asn 530 535 540Arg Gly Asp Asn Leu Glu Ser Gly Asn Phe Arg Thr Ala Gly Phe Ser545 550 555 560Thr Pro Phe Ser Phe Ser Asn Ala Gln Ser Thr Phe Thr Leu Gly Thr 565 570 575Gln Ala Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Ile Glu Phe Val 580 585 590Pro Ala Glu Val Thr Phe Glu Ala Glu Ser Asp Leu Glu Arg Ala Gln 595 600 605Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys 610 615 620Thr Asp Val Thr Asp Tyr Gln Ile Asp Gln Val Ser Asn Leu Val Glu625 630 635 640Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu 645 650 655Lys Val Lys His Ala Lys Arg Leu Ser Asp Lys Arg Asn Leu Leu Gln 660 665 670Asp Pro Asn Phe Thr Ser Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg 675 680 685Gly Ser Thr Asp Ile Thr Ile Gln Gly Gly Asn Asp Val Phe Lys Glu 690 695 700Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr705 710 715 720Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr 725 730 735Glu Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Val Tyr Leu 740 745 750Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly 755 760 765Ser Leu Trp Pro Leu Ser Val Glu Ser Pro Ile Gly Arg Cys Gly Glu 770 775 780Pro Asn Arg Cys Val Pro His Ile Glu Trp Asn Pro Asp Leu Asp Cys785 790 795 800Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser 805 810 815Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val 820 825 830Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly 835 840 845Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala 850 855 860Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Gln Leu865 870 875 880Gln Phe Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp 885 890 895Ala Leu Phe Val Asp Ser His Tyr Asn Arg Leu Gln Ala Asp Thr Asn 900 905 910Ile Thr Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu 915 920 925Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Asp Ile 930 935 940Phe Glu Glu Leu Glu Gly Leu Ile Phe Thr Ala Phe Ser Leu Tyr Asp945 950 955 960Ala Arg Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys 965 970 975Trp Asn Val Lys Gly His Val Asp Ile Gln Gln Asn Asp His Arg Ser 980 985 990Val Leu Val Val Pro Glu Trp Glu Ser Glu Val Ser Gln Glu Val Arg 995 1000 1005Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys 1010 1015 1020Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asp 1025 1030 1035Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Ile Glu Glu Glu Val 1040 1045 1050Tyr Pro Thr Asp Thr Gly Asn Asp Tyr Thr Ala His Gln Gly Thr 1055 1060 1065Thr Gly Cys Ala Asp Ala Cys Asn Ser Arg Asn Val Gly Tyr Glu 1070 1075 1080Asp Gly Tyr Glu Ile Asn Thr Thr Ala Ser Val Asn Tyr Lys Pro 1085 1090 1095Thr Tyr Glu Glu Glu Met Tyr Thr Asp Val Arg Arg Asp Asn His 1100 1105 1110Cys Glu Tyr Asp Arg Gly Tyr Gly Asn His Thr Pro Leu Pro Ala 1115 1120 1125Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr 1130 1135 1140Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp 1145 1150 1155Ser Val Glu Leu Leu Leu Met Glu Glu 1160 1165143504DNABacillus thuringiensis 14atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcgc tgaattagaa 300ggtttaggga gaagttatca actatatgga gaggcattta aagagtggga aaaaactcca 360gataacacag cggctcggtc tagagtaact gagagatttc gtataattga tgctcaaatt 420gaagcaaata tcccttcgtt tcgggtttcc ggatttgaag tgccacttct attggtttat 480acccaagcag ctaatttgca tctcgctcta ttaagagatt ctgttgtttt tggagagaga 540tggggattga cgactacaaa tgtcaatgat atctataata gacaagttaa tagaattggt 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac

gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504152133DNABacillus thuringiensis 15atgaaatcta agaatcaaaa tatgcatcaa agcttgtcta acaatgcgac agttgataaa 60aactttacag gttcactaga aaataacaca aatacggaat tacaaaactt taatcatgaa 120ggtatagagc cgtttgttag tgtatcaaca attcaaacgg gtattggtat tgctggtaaa 180atccttggta acctaggcgt tccctttgct gggcaagtag ctagcctcta tagttttatc 240ctaggtgagc tttggcccaa agggaaaagc caatgggaaa tttttatgga acatgtagaa 300gagcttatta atcaaaagat atcgacttat gcaagaaaca aagcacttgc agatttaaaa 360ggattaggag atgctttggc tgtctaccat gaatcgctgg aaagttggat taaaaatcgc 420aataacacaa gaactagaag tgttgtcaag agccaataca ttaccttgga acttatgttc 480gtacaatcat taccttcttt tgcagtgtct ggagaggaag taccactatt accaatatat 540gctcaagctg caaatttaca cttgttgcta ttaagagatg cgtctatttt tggaaaagaa 600tggggattat cagactcaga aatttcgaca ttctataatc gtcaagtgga aagaacatca 660gattattccg atcattgcac gaaatggttt gatacgggct tgaatagatt aaagggctca 720aatgctgaaa tctgggtaaa gtataatcaa ttccgtagag acatgacttt aatggtacta 780gatttagtgg cactattcca aagctatgat acacatatgt acccaattaa aactacagcc 840caacttacta gagaagtata tacaaacgca ttggggacag tacatccgca cccaagtttt 900acaagtacga cttggtataa taataatgca ccttcgtttt ctgccataga ggctgccgtt 960atccgaagcc cgcacctact cgattttcta gaacaagtta caatttacag cttattaagc 1020cgatggagta acactcagta tatgaatatg tggggaggac ataaactaga attccgaaca 1080ataggaggaa cgttaaatac ctcaacacaa ggatctacta atacttctat taatcctgta 1140acattaccgt tcacgtctcg agacatctat aggactgaat cattggcagg gctgaatcta 1200tttttaactc aacctgttaa tggagtacct agggttgatt ttcattggaa attcgtcaca 1260catccgatcg catctgataa tttctattat ccagggtatg ctggaattgg gacgcaatta 1320caggattcag aaaatgaatt accacctgaa gcaacaggac agccaaatta tgaatcttat 1380agtcatagat tatctcatat aggactcatt tcagcatcac atgtgaaagc attggtatat 1440tcttggacgc atcgtagtgc agatcgtacg aatacaattc attcagatag tataacacaa 1500ataccactgg taaaagcaca tacccttcag tcaggtacta ctgttgtaaa agggccaggg 1560tttacaggtg gagatatcct ccgacgaact agtggaggac catttgcttt tagtaatgtt 1620aatttagact ggaacttgtc acaaagatat cgtgctagaa tacgctatgc ttctactact 1680aatctaagaa tgtacgtaac gattgcaggg gaacgaattt ttgctggtca atttaataaa 1740acaatgaata ctggtgatcc attaacattc caatctttta gttacgcaac tattgataca 1800gcatttacat tcccaacgaa agcgagcagc ttgactgtag gtgctgatac ttttagctca 1860ggtaatgaag tttatgtaga tagatttgaa ttgatcccag ttactgcaac acttgaggca 1920gtaactgatt tagaaagagc gcagaaggcg gttcatgaac tgtttacatc tacgaatccg 1980ggaggattaa aaacggatgt aaaggattat catattgacc aggtatcaaa tttagtagag 2040tctctatcag ataaattcta tcttgatgaa aagagagaat tattcgagat agttaaatac 2100gcgaagcaac tccatattga gcgtaacatg tag 2133163507DNABacillus thuringiensis 16atggagataa ataatcagaa ccaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtatttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccaggggggg ggtttatttc aggattactt 180gataaaatat ggggggcttt gagaccatct gattgggaat tatttcttga acagattgaa 240cagttgattg atcgaagaat agaaagaaca gtaagagcaa aagcaatcgc tgaattagaa 300ggtttaggga gaagttatca actatatgga gaggcattta aagagtggga aaaaactcca 360gataacacag cggctcggtc tagagtaact gagagatttc gtataattga tgctcaaatt 420gaagcaaata tcccttcgtt tcgggtttcc ggatttgaag tgccacttct attggtttat 480acccaagcag ctaatttgca tctcgctcta ttaagagatt ctgttgtttt tggagagaga 540tggggattga cgactacaaa tgtcaatgat atctataata gacaagttaa tagaattggt 600gaatatagca agcattgtgt agatacgtat aaaacagaat tagaacgtct aggatttaga 660tctatagcgc aatggagaat atataatcag tttagaaggg aattgacact aacggtatta 720gatattgtcg ctgttttccc gaactatgat agtagactgt atccgattcg aacaatttct 780caattgacaa gagaaattta tacatcccca gtaagcgaat tttattatgg tgtcattaat 840agtaataata taattggtac ccttactgaa cagcaaataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggctactaa tactgaggga catcaaaggt cattcccttt agctgggact 1020atagggaatt cagctccacc agtaactgtt agaaataatg gtgagggaat ttatagaata 1080ttatcggaac cattttattc agcacctttt ctaggcacaa gtgtgctagg aagtcgtggg 1140gaagaatttg cttttgcatc taatactact acaagtctgc catctacaat atatagaaat 1200cgtggaacag tagattcatt agtcagcata ccgccacagg attatagcgt accaccgcac 1260agggggtata gtcatttatt aagtcacgtt acgatgcgca atagttctcc tatattccac 1320tggacacatc gtagtgcaac ccctagaaat acaattgatc cagatagtat cactcaaatt 1380ccagcagtta agggagcgta tatttttaat agtccagtca ttactgggcc aggacataca 1440ggtggggata taataaggtt taaccctaat actcagaaca acataagaat tccatttcaa 1500tcaaatgcgg tacagcgtta tcgaattaga atgcgttatg cggcagaagc tgattgtatt 1560ttagaaagtg gagtaaacat tgttactggg gcaggggtca cctttaggcc aattcctatt 1620aaagctacaa tgactcctgg aagtccttta acatattaca gcttccagta tgcagattta 1680aatataaatc ttactgcgcc gataagacct aataattttg tatctattag acgttcaaac 1740caaccaggaa acctttatat agatagaatt gaattcattc caattgaccc aatccgtgag 1800gcagaacatg atttagaaag agcgcaaaag gcggtgaatg cgctgtttac ttcttccaat 1860caaatcgggt taaaaacaga tgtgacggat tatcatattg atcaagtgtc caatttagtt 1920gcgtgtttat cggataaatt ctgcctggat gaaaagcgag aattgtccga gaaagttaaa 1980catgcgaagc gactcagtga tgagagaaat ttactccaag atcaaaactt tacaggcatc 2040aataggcaag tagaccgtgg gtggagagga agtacggata ttaccatcca aggagggaat 2100gatgtattca aagagaatta cgtcacacta ccaggtacct ttgatgagtg ttacccaacg 2160tatttgtatc aaaaaataga tgagtcaaaa ttaaaacctt atactcgcta tgaattaaga 2220gggtatattg aagatagtca agacttagaa gtctatttga tccgttacaa tgcaaaacac 2280gaaacgttaa atgtgccagg tacgggttcc ttatggccac ttgcagccga aagttcaatc 2340gggaggtgcg gcgaaccgaa tcgatgcgcg ccacatattg aatggaatcc tgaactagat 2400tgttcgtgta gggatggaga aaaatgtgca catcattctc atcatttctc cttggatatt 2460gatgttggat gtacagactt aaatgaggat ttaggtgtat gggtgatatt taagattaag 2520acgcaagatg gctatgcaag actaggaaat ttagagtttc tcgaagagaa accattgtta 2580ggagaagcgc tagctcgtgt gaagagagcg gagaaaaaat ggagagacaa acgcgacaaa 2640ttggaatggg aaacaaatat tgtttataaa gaggcaaaag aatctgtaga tgctttattc 2700gtagattctc aatataatag attacaaacg gatacgaaca ttgcgatgat tcatgtggca 2760gataaacgcg ttcatcgaat ccgagaagcg tatttgccag agttatctgt gattccgggt 2820gtcaatgcgg ctattttcga agaattagaa ggtcttattt tcactgcatt ctccctatat 2880gatgcgagaa atgtcattaa aaacggagat ttcaatcatg gtttatcatg ctggaacgtg 2940aaagggcatg tagatgtaga agaacaaaat aaccaccgtt cggtccttgt tgttccggaa 3000tgggaagcag aagtgtcaca agaagtccgc gtatgtccag gacgtggcta tatcctgcgt 3060gttacagcgt acaaagaggg ctacggagaa ggatgcgtaa cgatccatga aattgaagat 3120catacagacg aactgaaatt tagaaactgt gaagaagagg aagtgtatcc gaataacacg 3180gtaacgtgta atgattatcc agcaaatcaa gaagaataca gggctgcgga aacttcccgt 3240aatcgtggat atggcgaatc ttatgaaagt aattcttcca taccagctga gtatgcgcca 3300atttatgaga aagcatatac agatggaaga aaagagaatt cttgtgaatc taacagagga 3360tatggaaatt acacaccgtt accagcaggt tatgtgacaa aagaattaga gtacttccca 3420gaaaccgata aggtatggat agagattgga gaaacggaag gaacattcat cgtagacagt 3480gtggaattac tcctcatgga ggaatag 3507173501DNAArtificial SequenceCodon optimized BT0002 17atggagatta acaaccaaaa gcaatgcatc ccatataact gtttgtcgaa cccggaggag 60gttctgttgg acggtgaaag aatattgccc gacatagatc cgcttgaggt ttctttgagc 120ttgttgcagt ttctgttgaa caacttcgtt cccggcggag gattcatcag cgggcttgtc 180gataagatct ggggagcgct gagaccatcg gaatgggact tgttcctggc ccagatcgaa 240cgccttattg accaaaggat agaagccacg gttcgggcca aggctattgc agaacttgaa 300ggactcggcc ggtcctatca actgtatgga gaggcattca aagaatggga aaaaacgcct 360gacaacactg cagccaggtc gagagtgact gagcgcttta ggataatcga cgcacaaatc 420gaggcaaaca tcccatcgtt ccgcgtctct ggttttgagg tccccctctt gctcgtctat 480acacaagctg caaatcttca tttggcattg ttgagggact ccgttgtctt cggggagcgc 540tggggtctta caacaacgaa cgtgaatgat atctacaaca ggcaggtgaa caggattgga 600gaatactcta atcattgtgt tgacacatac aacacggagc tggagcgcct cggatttcgg 660agcattgcgc agtggcggat ttacaatcag ttcaggagag agctcacgct gacagtgctg 720gacatcgtcg ctctctttcc taactatgac tcgcgcctct atcccatcca gaccttttcg 780cagttgaccc gcgaaatagt cacttcaccc gtgtctgaat tttactacgg cgtcataaat 840tcagggaaca taaatggaac actgactgag cagcaaatca ggaggcccca tctgatggac 900ttcttcaatt ccatgattat gtacacatct gacaaccgcc gcgagcatta ttggtcaggc 960ctggagatga cggcgtactt cacaggtttt gcgggcgcac aggtgtcttt tcctttggtg 1020gggacgaggg gggagtcagc tcctcctctg acagtccgct cagtgaatga cggcatatat 1080aggattttga gcgccccttt ctattcggct ccgtttcttg gtactatagt gcttggctca 1140cggggtgaaa agtttgattt tgcgctgaac aacattagcc cgcctccttc tacaatctac 1200cggcatccgg gaaccgtcga ttctctcgtg tctattccgc cgcaagataa ctcggtgcca 1260cctcatcgcg gctcgtccca ccggttgtca cacgttacaa tgagggcttc atccccgatc 1320ttccactgga ctcatagatc tgccactacg actaatacca tcaatccgaa cgccataata 1380cagatccccc ttgttaaagc ttttaatctc cactccggtg ccaccgttgt tagagggccg 1440ggtttcaccg ggggagatat acttagaaga actaataccg gcacattcgc tgatatgagg 1500gtgaatatta ccgggcccct gtcgcagaga tacagggttc ggatcagata cgcctcaaca 1560actgatttgc aattttttac acgcatcaat gggacctcgg tgaatcaagg aaatttccag 1620cggacaatga atagagggga taacttggaa tccgggaact ttcggacggc aggtttctca 1680actcccttta gcttttccaa cgctcaatct accttcaccc tcgggactca ggcattctct 1740aatcaggaag tctatataga caggattgaa tttgtgccag cggaagttac gttcgaggct 1800gagagcgatc tcgaaagagc gcaaaaagca gttaatgctc tgttcacctc cacgaatcaa 1860ctgggcctga agactgatgt tactgattac cagatcgatc aggtgagcaa tctggtcgaa 1920tgtctctctg atgaattttg tctcgacgag aaaagggaat tgtcagaaaa ggtcaaacat 1980gccaaaaggc tgtctgataa gcgcaatttg ctccaagatc ccaattttac aagcattaat 2040cgccagctcg accgcggctg gcggggttct accgacatta ctatacaggg cgggaacgat 2100gtcttcaaag aaaactacgt gaccttgccg ggcaccttcg atgaatgtta ccctacttac 2160ttgtatcaaa agatagacga gtccaaattg aaggcgtaca ctcgctatga attgaggggg 2220tatatcgaag atagccagga tcttgaagtt tatctcatta gatataatgc taagcatgaa 2280acagtgaacg ttcccggtac aggttctctt tggccactta gcgtcgaatc gccgataggt 2340aggtgtggcg agccgaacag atgcgttccc cacatcgagt ggaatcctga cttggattgt 2400tcctgcaggg acggagagaa atgcgcgcac catagccatc acttttcctt ggacatagat 2460gttggttgta cagacttgaa cgaggatctt ggtgtttggg tgatttttaa aataaagaca 2520caggatggac acgcaagact tggcaatctc gaatttctgg aggagaagcc cctgttggga 2580gaagctctcg ctcgggttaa gcgggcggaa aagaaatgga gagacaagcg ggagcagctc 2640cagttcgaga cgaatatagt ttataaggag gccaaggaga gcgtggacgc cctctttgtt 2700gattcccatt acaacagact gcaggccgat actaacatca ctatgattca cgccgcagat 2760aagagggtcc accggatcag agaagcatac ctgccagaac tctctgtgat accaggggtt 2820aatgctgata tcttcgaaga gctcgaaggg ctgatcttca cagcattttc actctacgac 2880gcccgcaaca taataaagaa tggagacttt aataacggtc tgagctgctg gaacgttaaa 2940ggtcacgttg acatccaaca gaatgaccat cgctctgtgc tcgttgtgcc agaatgggag 3000agcgaggtgt cccaagaagt ccgcgtctgc ccagggcgcg gttacattct cagagttaca 3060gcctataagg aaggctacgg cgaggggtgt gttacgatac atgaaatcga ggataatacc 3120gacgaactca agttctcgaa ctgcatcgag gaagaagttt acccgacgga cactggaaac 3180gattacaccg ctcatcaagg tacaaccggc tgcgctgacg catgtaactc tcgcaacgtg 3240gggtatgagg acggctacga aattaatacc accgcttcgg tgaactacaa acccacgtac 3300gaggaggaga tgtatacgga tgttagacgg gacaatcact gtgagtacga tagaggttac 3360ggcaaccaca cacctcttcc cgcaggatac gttactaaag aattggagta cttcccagaa 3420actgatactg tgtggattga gatcggcgaa acggaaggca cattcatcgt ggattccgtg 3480gaattgctgc tcatggagga a 3501182130DNAArtificial SequenceCodon optimized BT0025 18atgaaatcga aaaatcagaa catgcaccaa agcctgtcga acaatgcaac cgtggataag 60aacttcactg gatcgttgga aaataatacc aatacggagc tgcaaaactt taaccatgaa 120ggcatcgagc cttttgtgtc agtgagcacg atccagacag gtatcggtat cgccggtaaa 180attctcggaa atctgggagt gcctttcgcg ggacaagtcg ctagcctgta ttcattcata 240ctgggtgagc tgtggcccaa aggtaaatcg cagtgggaaa tcttcatgga gcacgttgaa 300gagcttataa atcaaaagat atccacctac gctcgcaaca aagctctggc cgatttgaaa 360gggcttggag acgcgctcgc agtgtatcat gaatctctgg agtcatggat aaagaatcgg 420aataacacga gaacgcggtc tgttgtcaag agccagtaca tcactcttga actcatgttc 480gtccaatctc tcccgtcctt cgcagtgtcc ggcgaagagg tccctttgct gccgatctat 540gcacaggcag cgaacctcca cctgcttttg cttcgggatg cgtccatctt tggaaaagag 600tggggtctta gcgattctga gatatccact ttctataacc ggcaggtgga aaggacctcg 660gattactccg accactgtac aaaatggttt gacacggggt tgaataggct taaggggtcg 720aacgccgaaa tctgggttaa gtacaatcaa ttccggagag atatgacact tatggttctc 780gatctcgtcg cgcttttcca gtcctacgac actcacatgt accctataaa gaccacagcg 840cagttgacac gggaggtgta tactaatgct ctcgggacgg ttcaccccca tccctctttc 900actagcacca cttggtataa taataatgcg ccctcatttt ctgcgataga ggccgcagtc 960atacggagcc cacatctctt ggattttctg gagcaagtca cgatatattc gctcctctct 1020cgctggtcaa acacacaata tatgaatatg tggggcggtc ataagctgga gtttagaact 1080attggaggta cgctcaatac gtccacccag ggatcgacaa acacctccat aaaccccgtt 1140acgctcccat tcacgtctag agacatatat cggactgaga gcctcgccgg cctcaacctc 1200tttctgacgc aacccgtgaa cggggtccca cgcgttgatt tccactggaa attcgtgaca 1260catccgattg cgtcagacaa tttctattac cctgggtacg cgggaatcgg cacgcaactg 1320caggattcgg agaacgaact tcctccggag gcgacagggc agcccaatta tgaaagctac 1380agccatcggc tgagccacat cggtctcatc tccgcctctc atgttaaggc gttggtgtat 1440tcatggacgc acagatccgc agaccggacc aacacgatcc actcggactc gataacccag 1500attccgctcg ttaaggccca cactctccag agcggaacta ctgtcgtcaa agggccaggg 1560ttcaccggag gggatatact gaggagaact agcggtggac cgttcgcttt cagcaacgtc 1620aatctcgact ggaatctctc acagagatac cgcgctcgca taagatatgc ctccaccacg 1680aatctccgga tgtacgtcac cattgcggga gagcgcatct ttgctggtca gtttaacaag 1740acaatgaaca ctggcgatcc actgaccttt caatccttct catatgccac aattgacacg 1800gcgttcacct ttccgaccaa ggcttccagc cttacggttg gtgcggacac cttttctagc 1860ggcaatgagg tttacgtgga ccgcttcgag ttgattcccg ttaccgctac tctcgaagca 1920gtgactgatt tggagcgcgc gcaaaaggcg gtgcatgaac ttttcacgag caccaatcct 1980ggcggcctta aaacggatgt caaagattat catatagacc aggtgtctaa cctggttgag 2040tcactctcgg ataagtttta tttggatgaa aagcgggaac tttttgagat cgtgaagtac 2100gctaaacagc tccatatcga acggaatatg 2130193504DNAArtificial SequenceCodon optimized BT0053 19atggagatta acaaccagaa ccaatgcata ccttataact gtttgagcaa cccagaagaa 60gtgttccttg atggggagcg gatactcccc gacatagacc cgctcgaagt cagcttgagc 120ttgttgcaat tccttctgaa taacttcgtg cctggcgggg gcttcatatc tggtcttctg 180gataagatct ggggggccct tagaccgagc gattgggaac tcttcctgga gcagatcgag 240cagctcatag acagaaggat tgagagaact gtgagggcta aagcaattgc agagctggaa 300ggtttgggca ggagctacca gttgtacggc gaggcgttca aggaatggga aaaaacaccg 360gataacacag ctgcccggtc cagagttacg gagaggttcc ggataattga cgctcagatt 420gaagctaaca taccatcttt tcgcgtgtct ggattcgagg ttcctctgct gcttgtttat 480acccaagccg ctaatctgca ccttgctctc ttgagggata gcgtcgtgtt cggcgagcgg 540tggggtttga ccacgaccaa tgtgaatgat atttataatc ggcaggtgaa tcggatcggc 600gagtactcaa aacattgtgt cgacacgtat aaaacagagc tcgaacgcct tggattccgc 660agcatagctc agtggaggat ttacaatcag tttcggagag agctcacatt gaccgtcctt 720gatatagtgg ccgtctttcc caactatgat tctcggctct atcccatccg gactatatca 780cagcttacca gagagattta tacctcgcct gtctcagaat tttattatgg agtcattaac 840tctaacaaca taataggaac cctgactgaa cagcagatca gaaggcctca tctgatggac 900tttttcaact cgatgataat gtatacatcc gacaacaggc gcgagcacta ctggtcgggc 960ctcgaaatga cagctaccaa cactgaggga catcaacgga gctttccgct tgccggtacc 1020ataggcaaca gcgctccgcc ggttacagtc cggaacaacg gagaaggaat ataccgcatc 1080ctcagcgagc cgttctactc tgcacccttt ttggggactt cggtgctggg tagccgcggg 1140gaggaatttg cattcgcgtc gaatacgaca acgtccttgc catcaactat atacaggaac 1200cggggcaccg ttgatagctt ggtgtctata ccaccccagg attactcggt cccccctcat 1260cggggatatt cacacttgct cagccacgtc acgatgagga actcatcgcc catcttccat 1320tggactcacc ggtcagcaac acctaggaat acgatcgacc cagattcgat tacccagata 1380cctgctgtta agggggcata catcttcaac agcccagtca taaccggacc cggccacact 1440ggaggtgata ttatcaggtt caatcccaat acgcagaata acatcaggat accgttccaa

1500agcaatgctg tgcagagata tcgcatccgg atgcggtacg cagccgaggc tgactgtatc 1560cttgaatcgg gcgtgaacat tgttaccgga gccggtgtta cattccgccc gatcccgata 1620aaagctacga tgactcctgg ttctccattg acttactatt cttttcagta tgccgatctc 1680aatataaacc tcactgcccc catacgcccg aacaattttg tttccatacg caggtctaac 1740caacctggga acctctatat cgaccgcatc gaattcattc ctatagatcc catcagggaa 1800gccgaacacg accttgaacg cgctcagaaa gccgtcaatg cgctttttac gtcgtccaat 1860caaattggtc tcaaaaccga cgttaccgac taccacatcg atcaagtgtc aaaccttgtc 1920gcttgcttgt cagataagtt ctgcctcgat gaaaaaaggg agctttcgga aaaagttaag 1980catgctaaac gcctgtcgga cgaacgcaac ctgctgcagg atcagaattt caccgggatt 2040aataggcagg tggatagagg ctggcgcggc tccacagaca tcacaatcca aggggggaat 2100gatgtcttta aggaaaacta cgtcacgttg ccaggaacgt tcgatgagtg ctaccctacg 2160tatctttacc agaagattga tgaatcgaaa ttgaagccct atactaggta cgagcttcgc 2220gggtatattg aggatagcca ggaccttgag gtttacctta taaggtacaa cgcgaaacac 2280gagacgctta acgtgccagg gacaggttct ttgtggcccc tcgccgcaga gtcgtctata 2340ggccggtgcg gtgaaccaaa ccggtgcgct cctcacattg agtggaatcc ggagctggac 2400tgtagctgta gggacggaga aaagtgtgcc catcactcgc atcacttttc cctggacata 2460gacgttgggt gtactgatct taacgaagac cttggagttt gggtgatatt caagatcaag 2520actcaggatg gctatgctag gctcgggaac ttggagtttc tcgaggagaa acccctcctc 2580ggggaggctc ttgctagagt caagagagcg gaaaaaaaat ggcgcgataa acgcgacaag 2640ttggagtggg agacgaacat cgtctacaaa gaagctaaag aatcagtgga cgcccttttt 2700gtggactctc aatataaccg cctccaaact gacactaaca tcgctatgat tcacgtggcc 2760gataagcggg tgcataggat aagagaagcg tatttgcccg agctgtcagt tatccctggg 2820gtgaacgctg caattttcga ggaactggaa ggtctgatat ttaccgcatt ttctctctat 2880gacgcgcgga atgttattaa gaacggcgat ttcaaccacg gattgtcatg ttggaacgtc 2940aagggccatg tggatgtcga agaacagaac aaccacagaa gcgttcttgt cgttccggag 3000tgggaagctg aggtgagcca agaggtccgc gtctgccctg gacgggggta tattttgcgg 3060gtcaccgctt ataaagaggg gtacggagag gggtgtgtca cgatccacga gattgaagac 3120cacacagacg agcttaaatt cagaaattgt gaggaagaag aggtctaccc aaataacacc 3180gttacatgca acgattatcc cgctaatcag gaggaatatc gcgccgcaga aacgagcaga 3240aatcgcgggt acggcgaatc atacgagtcc aattcttcta tacccgctga atacgcccct 3300atttatgaga aggcttacac agacgggcgg aaggaaaata gctgcgagtc taataggggg 3360tacgggaatt acacaccact tccggcaggg tacgttacga aggaactgga gtattttccg 3420gaaaccgata aggtttggat cgagattggc gagacagaag gaacttttat cgttgactcc 3480gtcgaacttt tgcttatgga agag 3504203504DNAArtificial SequenceCodon optimized variant BT0002 20atggagatta acaatcagaa gcagtgcatc ccctacaact gcctgtccaa tccggaggag 60gtgctcctgg acggcgagcg catcctccct gacattgatc cgctggaggt ctcactctcc 120ctcctgcagt tcctcctgaa caatttcgtt cccggcgggg gcttcatttc ggggctggtg 180gacaagatct ggggcgcgct caggccatcc gagtgggatc tcttcctggc tcagatcgag 240aggctcattg accagaggat cgaggctacc gtgcgcgcta aggccatcgc tgagctggag 300gggctgggca ggtcctacca gctgtacggc gaggcgttca aggagtggga gaagaccccg 360gataacaccg cggccaggag cagggtcacg gagcgcttca ggatcattga cgcccagatt 420gaggcgaaca tccccagctt cagggtgtcg ggcttcgagg tcccactcct gctcgtttac 480acccaggctg ctaacctcca cctggctctg ctccgcgata gcgtggtctt cggcgagcgc 540tgggggctca ccacgacaaa cgtgaatgac atctacaacc ggcaggtcaa tcgcatcggc 600gagtactcta accactgcgt ggacacttac aataccgagc tggagaggct gggcttccgg 660tcaattgctc agtggcgcat ctacaaccag ttccgcaggg agctgaccct gacggtcctg 720gatatcgttg ccctcttccc caactacgac tcgcggctgt acccaattca gactttctct 780cagctcaccc gcgagatcgt gacgtctcct gtctcagagt tctactacgg cgtcattaac 840tccggcaaca tcaatgggac actgactgag cagcagattc ggcgcccgca cctcatggat 900ttcttcaact caatgatcat gtacacctcc gacaataggc gggagcatta ctggtcgggc 960ctcgagatga cggcgtactt cacgggcttc gcgggggcgc aggtgtcttt ccccctggtg 1020ggcacacgcg gggagtctgc gccgcccctc actgtgcggt cagtcaacga cggcatctac 1080cgcattctgt cggctccctt ctactctgcc ccattcctgg gcaccatcgt gctgggctcc 1140cgcggcgaga agttcgactt cgccctgaac aatatttctc cacctccgtc aaccatctac 1200cgccaccctg gcacggttga ttccctcgtg agcatcccgc cgcaggacaa ctcggtccct 1260ccgcataggg gctccagcca caggctgtct catgttacca tgagggcgtc gtctccgatc 1320ttccactgga cccatcggag cgccactacc acgaatacaa tcaaccctaa tgcgatcatt 1380cagatcccgc tggtcaaggc gttcaacctc cactccggcg ctacggttgt gagggggccc 1440ggcttcaccg gcggcgacat cctgcgcagg accaacacgg ggacattcgc ggacatgcgg 1500gtgaatatta ccggcccact gagccagcgc taccgcgtgc gcatccgcta cgctagcaca 1560actgacctcc agttcttcac acgcatcaac ggcacttccg tgaaccaggg gaatttccag 1620cgcacgatga acaggggcga caatctcgag tcagggaact tcaggaccgc cggcttctcc 1680acgcctttca gcttctcgaa tgctcagagc actttcaccc tgggcaccca ggccttctcg 1740aaccaggagg tctacatcga tcgcattgag ttcgtcccgg cggaggttac gttcgaggct 1800gagtctgacc tggagagggc ccagaaggcg gtgaacgctc tcttcacgtc aacaaatcag 1860ctcggcctga agacggacgt cacagattac cagatcgacc aggtgagcaa cctggtcgag 1920tgcctctcgg acgagttctg cctggatgag aagcgggagc tgtctgagaa ggtgaagcac 1980gcgaagcggc tgtcagacaa gcgcaacctg ctccaggacc cgaacttcac ctcaatcaat 2040aggcagctgg acaggggctg gagggggtcc actgatatca ccattcaggg cggcaacgac 2100gtcttcaagg agaattacgt tacgctgcct ggcacattcg atgagtgcta cccgacatac 2160ctctaccaga agatcgacga gtcaaagctg aaggcctaca ctcggtacga gctgcgcgga 2220tacatcgagg actcccagga tctggaggtg tacctcatcc gctacaacgc gaagcacgag 2280acagtgaatg tgccggggac tggctccctc tggccactgt cggttgagtc tccaattggc 2340cggtgcgggg agcctaacag gtgcgtgccc catatcgagt ggaatccaga cctggattgc 2400tcctgcaggg acggcgagaa gtgcgctcac cattcccacc atttcagcct cgacatcgat 2460gtcgggtgca cagacctgaa cgaggatctc ggcgtttggg tcatcttcaa gatcaagacc 2520caggacggcc acgctaggct ggggaacctg gagttcctgg aggagaagcc cctgctgggc 2580gaggctctgg ctagggtgaa gagggcggag aagaagtggc gcgacaagag ggagcagctc 2640cagttcgaga ccaacatcgt ctacaaggag gccaaggagt ccgttgacgc gctgttcgtg 2700gatagccact acaacaggct ccaggcggat acgaatatca caatgattca cgcggctgac 2760aagcgggtgc atcgcattag ggaggcctac ctgcctgagc tgtcggttat tccgggcgtg 2820aacgcggaca tcttcgagga gctggagggc ctcatcttca ccgctttctc tctgtacgat 2880gccaggaaca tcattaagaa tggcgacttc aacaatgggc tcagctgctg gaacgtcaag 2940ggccacgttg acatccagca gaatgatcat cgctcggtcc tcgtcgttcc tgagtgggag 3000tcagaggttt cccaggaggt cagggtttgc cccgggaggg gatacatcct gcgcgttacc 3060gcctacaagg aggggtacgg cgaggggtgc gtgacaatcc acgagattga ggacaacact 3120gatgagctga agttctccaa ttgcatcgag gaggaggtgt acccgactga caccggcaac 3180gattacaccg ctcatcaggg caccaccggg tgcgccgatg cttgcaactc caggaatgtc 3240ggctacgagg acgggtacga gatcaacaca actgcgagcg tgaattacaa gcccacatac 3300gaggaggaga tgtacactga cgtccggcgc gataaccact gcgagtacga ccgcggctac 3360gggaatcata ccccgctccc agcgggctac gtgaccaagg agctggagta cttcccagag 3420acggatacag tctggctcga gattggcgag actgagggga ccttcatcgt tgacagcgtg 3480gagctgatcc tgatggagga gtga 3504212133DNAArtificial SequenceCodon optimized variant BT0025 21atgaagtcca agaatcagaa catgcatcag tcactctcca acaatgcgac ggtcgacaag 60aatttcacag gcagcctcga gaacaatacc aacacggagc tgcagaattt caaccacgag 120ggcatcgagc cgttcgtcag cgtttcgaca attcagactg gcatcgggat tgccggcaag 180atcctcggca acctcggcgt gccgttcgcc ggccaggttg cttcgctcta ctctttcatc 240ctgggcgagc tgtggcccaa ggggaagtcg cagtgggaga ttttcatgga gcatgtcgag 300gagctgatca atcagaagat ttctacgtac gcccgcaaca aggccctggc tgacctcaag 360ggcctggggg atgctctggc cgtgtaccac gagtcactgg agtcctggat caagaacagg 420aacaatacaa ggactcgctc cgtggtcaag agccagtaca ttaccctcga gctgatgttc 480gtgcagtcgc tcccctcctt cgccgtttcc ggcgaggagg tgccgctcct gccaatctac 540gcccaggctg cgaatctcca tctcctgctc ctgcgcgacg cttctatctt cggcaaggag 600tgggggctgt ctgattcaga gatttcaacg ttctacaaca ggcaggtcga gcggacatct 660gactactcag atcactgcac aaagtggttc gacactggcc tcaataggct gaaggggtcc 720aacgcggaga tctgggtgaa gtacaaccag ttccgcaggg acatgacgct catggttctc 780gatctggtgg ccctgttcca gagctacgac acccacatgt accccatcaa gaccacggct 840cagctcaccc gggaggttta cacgaacgcc ctgggcacag tgcacccaca tccttccttc 900accagcacaa cttggtacaa caataacgct ccgtccttca gcgccatcga ggctgccgtc 960attaggagcc cccatctcct ggacttcctc gagcaggtta cgatctactc gctcctgtct 1020cggtggtcaa atacacaata catgaacatg tggggcgggc acaagctcga gttccgcaca 1080attggcggga ctctgaacac ttcgacgcag ggctctacaa atacttcaat caacccagtc 1140accctccctt tcacgtcacg cgacatctac aggactgagt ccctggcggg cctcaatctg 1200ttcctcacgc agcccgttaa cggggtgccc agggtcgact tccactggaa gttcgtgacc 1260catccaatcg cgagcgataa cttctactac cctggctacg ctggcattgg gacgcagctc 1320caggactcgg agaatgagct gccgcccgag gctacaggcc agccaaacta cgagtcgtac 1380tctcaccgcc tctcccatat cggcctgatt tcagcgtccc acgtcaaggc tctcgtttac 1440agctggaccc atcgctccgc cgaccgcacc aacacgatcc acagcgattc gatcactcag 1500attccgctcg tgaaggctca caccctgcag tcgggcacca ccgttgtgaa gggcccaggg 1560ttcactggcg gggacatcct gaggcgcacc tcgggcgggc ctttcgcgtt ctctaatgtt 1620aacctcgatt ggaatctgtc ccagcgctac cgcgcccgca tccgctacgc ctccacaact 1680aacctcagga tgtatgtgac catcgcgggc gagcggattt tcgctgggca gttcaataag 1740accatgaaca cgggcgaccc actgacgttc cagtctttct catacgctac aatcgatact 1800gccttcacct tccctaccaa ggcctccagc ctcactgtgg gcgccgacac cttctcgtct 1860gggaacgagg tctacgtgga taggttcgag ctgatcccgg tgacagccac gctggaggcc 1920gtcacggacc tggagcgggc tcagaaggcg gtgcatgagc tgttcacctc cacgaatcca 1980ggcggcctga agaccgacgt caaggattac cacctcgatc aggtgagcaa cctcgtcgag 2040tccctgagcg acaagttcta cctcgatgag aagcgcgagc tgttcgagct cgtgaagtac 2100gccaagcagc tgcacattga gaggaacatg tga 2133223507DNAArtificial SequenceCodon optimized variant BT0053 22atggcgatta acaatcagaa ccagtgcatc ccatacaact gcctgtccaa tcctgaggag 60gtgttcctgg acggcgagcg catcctcccg gacattgatc ccctggaggt gtctctctca 120ctcctgcagt tcctcctgaa caatttcgtc ccaggcgggg gcttcatttc gggcctcctg 180gacaagatct ggggcgccct caggccttcg gattgggagc tgttcctcga gcagatcgag 240cagctcattg acaggaggat cgagcgcacc gtcagggcta aggccatcgc tgagctggag 300gggctgggcc gctcttacca gctctacggc gaggcgttca aggagtggga gaagacgccc 360gacaacacgg cggccaggtc aagggtgacg gagcgcttca ggatcattga tgcccagatt 420gaggcgaaca tcccgtcctt ccgcgtgagc ggcttcgagg tccccctcct gctcgtttac 480acgcaggctg ccaacctcca tctggccctg ctccgggact cggtggtgtt cggcgagagg 540tgggggctca ccaccacaaa cgtcaatgat atctacaacc ggcaggttaa tcgcatcggc 600gagtactcaa agcactgcgt cgacacttac aagaccgagc tggagaggct gggcttccgg 660tccattgcgc agtggaggat ctacaaccag ttccggcgcg agctgacact gactgtgctc 720gacatcgtcg ctgttttccc aaactacgat tcccggctgt accctatccg cacgattagc 780cagctcacac gcgagatcta cacttcccca gttagcgagt tctactacgg cgtgatcaac 840tccaacaata tcattggcac cctcacggag cagcagatta ggcggcctca cctcatggac 900ttcttcaact cgatgatcat gtacacctct gataatcgca gggagcacta ctggagcggc 960ctggagatga cagccactaa caccgagggg catcagcgct ccttcccact ggccggcacc 1020atcgggaatt ctgctccgcc cgtgaccgtg cgcaacaatg gggagggcat ctacaggatt 1080ctgtccgagc cattctactc ggcccctttc ctgggcacgt cggtcctggg ctctcgcggg 1140gaggagttcg ctttcgcgtc gaacactacc acgtcgctgc catctacaat ctacaggaat 1200cgcggcactg tggactcact cgtctccatc ccacctcagg attactctgt tccgccccac 1260aggggctact cacacctgct ctcccatgtg acaatgcgca actccagccc gatcttccac 1320tggactcata ggagcgccac gccacggaat acaatcgacc ctgattcgat cacacagatt 1380cccgctgtga agggcgccta cattttcaac tcgccggtca tcaccgggcc cggccacacc 1440ggcggcgaca tcattcgctt caacccaaat acgcagaaca atatcaggat tcctttccag 1500tccaacgcgg tccagcgcta ccgcatccgc atgcgctacg cggctgaggc tgactgcatt 1560ctggagagcg gcgttaacat cgtgacaggg gctggcgtga ctttccgccc aatccctatt 1620aaggccacga tgacaccagg ctcacctctc acctactact ccttccagta cgccgacctg 1680aacattaatc tcacggcgcc gatccgcccc aacaatttcg tgagcatcag gaggtccaac 1740cagcccggca atctgtacat cgacaggatt gagttcatcc caattgatcc tatcagggag 1800gccgagcacg acctcgagcg cgcgcagaag gctgtcaacg ccctgttcac ctcgtctaat 1860cagattggcc tcaagacgga cgtgacagat taccatatcg accaggttag caacctggtg 1920gcctgcctct cggacaagtt ctgcctggat gagaagaggg agctgtcaga gaaggtcaag 1980cacgcgaagc gcctgtccga cgagaggaac ctgctccagg atcagaattt cacgggcatc 2040aacaggcagg tggatagggg ctggaggggg agcactgaca tcaccattca gggcggcaac 2100gatgtcttca aggagaatta cgttactctg ccgggcacct tcgacgagtg ctaccccaca 2160tacctctacc agaagatcga tgagtcgaag ctgaagccgt acactcgcta cgagctgagg 2220ggatacatcg aggactctca ggatctggag gtctacctca tccgctacaa cgccaagcat 2280gagaccctca atgtgcccgg gacgggcagc ctctggccgc tggcggccga gtcatccatc 2340ggcaggtgcg gggagccaaa caggtgcgcc cctcacatcg agtggaatcc ggagctggac 2400tgctcgtgca gggatggcga gaagtgcgcg caccattctc accatttctc actcgacatc 2460gatgtgggct gcaccgacct gaacgaggat ctcggggttt gggtcatctt caagatcaag 2520acccaggacg gctacgctag gctggggaac ctggagttcc tggaggagaa gccgctgctg 2580ggcgaggctc tggctagggt caagagggcg gagaagaagt ggcgcgacaa gagggataag 2640ctcgagtggg agaccaacat cgtgtacaag gaggccaagg agtctgtgga cgcgctgttc 2700gtcgattcac agtacaacag gctccagact gacaccaata tcgcgatgat tcacgttgct 2760gataagcggg tgcatcgcat ccgcgaggct tacctgcccg agctgtccgt cattcccggc 2820gttaacgctg ccatcttcga ggagctggag gggctcatct tcaccgcttt cagcctgtac 2880gacgccagga acgtcatcaa gaatggcgat ttcaaccacg ggctctcgtg ctggaacgtg 2940aagggccacg tcgacgttga ggagcagaac aatcatcgct ctgttctggt tgtgccggag 3000tgggaggctg aggtgtcaca ggaggtgcgg gtctgcccgg ggaggggata catcctcagg 3060gtcaccgcct acaaggaggg gtacggcgag gggtgcgtta ccatccacga gattgaggac 3120catacggatg agctgaagtt ccggaactgc gaggaggagg aggtgtaccc aaacaatacg 3180gtcacatgca atgactaccc ggccaaccag gaggagtaca gggccgctga gacatccagg 3240aacaggggct acggggagag ctacgagtcg aatagctcga ttccggcgga gtacgctccc 3300atctacgaga aggcctacac tgacggcagg aaggagaatt cttgcgagtc aaaccggggc 3360tacgggaatt acacaccgct gcccgcgggc tacgtcacta aggagctgga gtacttcccg 3420gagaccgaca aggtttggat cgagattggc gagacggagg ggacattcct cgtcgatagc 3480gttgagctgc tcctgatgga ggagtga 3507233504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 3 BT0002 23atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcac tgaattagaa 300ggtttaggga gaaattatca aatatatgca gaggcattta aagagtggga aagtgatcca 360gataacacag cggctcggtc tagagtaact gagagatttc gtataattga tgctcaaatt 420gaagcaaata tcccttcgtt tcgggtttcc ggatttgaag tgccacttct attggtttat 480acccaagcag ctaatttgca tctcgctcta ttaagagatt ctgttgtttt tggagagaga 540tggggattga cgactacaaa tgtcaatgat atctataata gacaagttaa tagaattggt 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa

3504243504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 4 BT0002 24atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcgc tgaattagaa 300ggtttaggga gaagttatca actatatgga gaggcattta aagagtggga aaaaactcca 360gataacgaag cggctaagtc tagagtaatt gatagatttc gtatattaga tggtttaatt 420gaagcaaata tcccttcgtt tcggattatc ggatttgaag tgccacttct attggtttat 480acccaagcag ctaatttgca tctcgctcta ttaagagatt ctgttgtttt tggagagaga 540tggggattga cgactacaaa tgtcaatgat atctataata gacaagttaa tagaattggt 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504253504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 5/6 BT0002 25atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcgc tgaattagaa 300ggtttaggga gaagttatca actatatgga gaggcattta aagagtggga aaaaactcca 360gataacacag cggctcggtc tagagtaact gagagatttc gtataattga tgctcaaatt 420gaagcaaata tcccttcgtt tcgggtttcc ggatttgaag tgccacttct atcagtttat 480gttcaagcag ctaatttgca tctcgctcta ttaagagatt ctgttatttt tggagagaga 540tggggattga cgactaaaaa tgtcaatgat atctataata gacaaataag agaaattcat 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504263504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 3-4 BT0002 26atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcac tgaattagaa 300ggtttaggga gaaattatca aatatatgca gaggcattta aagagtggga aagtgatcca 360gataacgaag cggctaagtc tagagtaatt gatagatttc gtatattaga tggtttaatt 420gaagcaaata tcccttcgtt tcggattatc ggatttgaag tgccacttct attggtttat 480acccaagcag ctaatttgca tctcgctcta ttaagagatt ctgttgtttt tggagagaga 540tggggattga cgactacaaa tgtcaatgat atctataata gacaagttaa tagaattggt 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504273504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 4/5/6 BT0002 27atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcgc tgaattagaa 300ggtttaggga gaagttatca actatatgga gaggcattta aagagtggga aaaaactcca 360gataacgaag cggctaagtc tagagtaatt gatagatttc gtatattaga tggtttaatt 420gaagcaaata tcccttcgtt tcggattatc ggatttgaag tgccacttct atcagtttat 480gttcaagcag ctaatttgca tctcgctcta ttaagagatt ctgttatttt tggagagaga 540tggggattga cgactaaaaa tgtcaatgat atctataata gacaaataag agaaattcat 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504283504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 3/5/6 BT0002 28atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcac tgaattagaa 300ggtttaggga gaaattatca aatatatgca gaggcattta aagagtggga aagtgatcca 360gataacacag cggctcggtc tagagtaact gagagatttc gtataattga tgctcaaatt 420gaagcaaata tcccttcgtt tcgggtttcc ggatttgaag tgccacttct

atcagtttat 480gttcaagcag ctaatttgca tctcgctcta ttaagagatt ctgttatttt tggagagaga 540tggggattga cgactaaaaa tgtcaatgat atctataata gacaaataag agaaattcat 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504293504DNAArtificial SequenceSynthetic sequence encoding modified alpha-helix 3/4/5/6 BT0002 29atggagataa ataatcagaa gcaatgcata ccatataatt gcttaagtaa tcctgaggaa 60gtacttttgg atggggagag gatattacct gatatcgatc cactcgaagt ttctttgtcg 120cttttgcaat ttcttttgaa taactttgtt ccagggggag gctttatttc aggattagtt 180gataaaatat ggggggcttt gagaccatct gaatgggact tatttcttgc acagattgaa 240cggttgattg atcaaagaat agaagcaaca gtaagagcaa aagcaatcac tgaattagaa 300ggtttaggga gaaattatca aatatatgca gaggcattta aagagtggga aagtgatcca 360gataacgaag cggctaagtc tagagtaatt gatagatttc gtatattaga tggtttaatt 420gaagcaaata tcccttcgtt tcggattatc ggatttgaag tgccacttct atcagtttat 480gttcaagcag ctaatttgca tctcgctcta ttaagagatt ctgttatttt tggagagaga 540tggggattga cgactaaaaa tgtcaatgat atctataata gacaaataag agaaattcat 600gaatatagca atcattgcgt agatacgtat aacacagaac tagaacgtct agggtttaga 660tctatagcgc agtggagaat atataatcag tttagaagag aactaacact aactgtatta 720gatattgtcg ctcttttccc gaactatgac agtagactgt atccgatcca aactttttct 780caattgacaa gagaaattgt tacatcccca gtaagcgaat tttattatgg tgttattaat 840agtggtaata taaatggtac tcttactgaa cagcagataa ggcgaccaca tcttatggac 900ttctttaact ccatgatcat gtatacatca gataatagac gggaacatta ttggtcagga 960cttgaaatga cggcttattt tacaggattt gcaggcgctc aagtgtcatt ccctttagtc 1020gggactagag gggagtcagc tccaccatta actgttagaa gtgttaatga tggaatttat 1080agaatattat cggcaccgtt ttattcagcg ccttttctag gcaccattgt attgggaagt 1140cgtggagaaa aatttgattt tgcgcttaat aatatttcac ctccgccatc tacaatatac 1200agacatcctg gaacagtaga ttcactagtc agtataccgc cacaggataa tagcgtacca 1260ccgcacaggg gatctagtca tcgattaagt catgttacaa tgcgcgcaag ttcccctata 1320ttccattgga cgcatcgcag cgcaaccact acaaatacaa ttaatccaaa tgctattatc 1380caaataccac tagtaaaagc atttaacctt cattcaggtg ccactgttgt tagaggacca 1440gggtttacag gtggtgatat ccttcgaaga acgaatactg gcacatttgc agatatgaga 1500gtaaatatta ctgggccatt atcccaaaga tatcgtgtaa gaattcgcta tgcttctacg 1560acagatttac aatttttcac gagaatcaat ggaacttctg taaatcaagg taatttccaa 1620agaactatga atagagggga taatttagaa tctggaaact ttaggactgc aggatttagt 1680acgcctttta gtttttcaaa tgcgcaaagt acattcacat tgggtactca ggctttttca 1740aatcaggaag tttatataga tcgaattgaa tttgtcccgg cagaagtaac attcgaggca 1800gaatctgatt tagaaagagc gcaaaaggcg gtgaatgccc tgtttacttc tacaaaccaa 1860ctagggctaa aaacagatgt gacggattat cagattgatc aagtgtccaa tttagtagaa 1920tgtttatcag atgaattttg tctggatgaa aagagagaat tgtccgagaa agtcaaacat 1980gcaaagcgac ttagtgataa gcggaaccta cttcaagatc caaacttcac atctatcaat 2040agacaactag accgtggatg gagaggaagt acggatatta ccatccaagg aggaaatgac 2100gtattcaaag agaattacgt cacactacca ggtacctttg atgagtgtta tccaacgtat 2160ttgtatcaaa aaatagatga gtcaaaatta aaagcctata ctcgctatga attaagaggg 2220tatattgaag atagtcaaga tttagaagtc tatttgattc gttacaatgc gaaacatgaa 2280acagtaaatg ttcccggtac agggtcctta tggccgcttt cagtcgaaag cccaatcgga 2340aggtgcggag aaccgaatcg atgtgtgcca catattgaat ggaatcctga tttagattgt 2400tcgtgtaggg atggggagaa gtgtgcccat cattcgcatc atttctctct agatattgat 2460gttggatgta cagacctaaa tgaggaccta ggtgtatggg tgatctttaa gattaaaacg 2520caggatggcc atgcaagatt aggaaatcta gagtttctcg aagagaaacc attgttagga 2580gaagcgttag ctcgtgtgaa aagagcggag aaaaaatgga gagacaaacg cgaacaattg 2640cagtttgaaa cgaatatcgt ttacaaagag gcaaaagaat ctgtagatgc tttattcgta 2700gattctcact ataatagatt acaagcggat acgaacatta cgatgattca tgcggcagat 2760aaacgcgttc atcgaatccg agaggcttat cttccggaat tatccgttat cccaggtgta 2820aatgcggaca tttttgaaga attagaaggt cttattttca ctgcattctc cctatatgat 2880gcgagaaata tcattaaaaa cggtgatttc aataatggtt tatcgtgttg gaacgtgaaa 2940gggcatgtag atatacaaca gaatgatcat cgttctgtcc tcgttgtccc ggaatgggaa 3000tcagaggtat cacaagaagt ccgcgtatgt ccaggtcgtg gctatattct tcgtgtcaca 3060gcgtacaaag agggctacgg agaaggatgc gtaacgatcc atgagatcga agacaataca 3120gacgaattga agtttagtaa ctgcatagaa gaggaagtct atccaacgga tacaggtaat 3180gattatactg cacaccaagg tacaacagga tgcgcagatg catgtaattc ccgtaatgtt 3240ggatatgagg atggatatga aataaatact acagcatctg ttaattacaa accgacttat 3300gaagaagaaa tgtatacaga tgtacgaaga gataatcatt gtgaatatga cagaggatat 3360gggaaccata caccgttacc agctggttat gtaacaaaag aattagagta cttccctgaa 3420acagatacag tatggataga gattggagaa acggaaggaa cattcatcgt agatagtgtg 3480gaattactcc tcatggagga ataa 3504309078DNAArtificial SequenceShuttle vector functional in E. coli and B. thuringiensis 30agaggccatc gtggcctata tatggcctgg gcgagaagta agtagattgt taacaccctg 60ggtcaaaaat tgatatttag taaaattagt tgcactttgt gcattttttc ataagatgag 120tcatatgttt taaattgtag taatgaaaaa cagtattata tcataatcaa ttggtatctt 180aataaaagag atggaggttt aaacggatcc atgaaatcta agaatcaaaa tatgcatcaa 240agcttgtcta acaatgcgac agttgataaa aactttacag gttcactaga aaataacaca 300aatacggaat tacaaaactt taatcatgaa ggtatagagc cgtttgttag tgtatcaaca 360attcaaacgg gtattggtat tgctggtaaa atccttggta acctaggcgt tccctttgct 420gggcaagtag ctagcctcta tagttttatc ctaggtgagc tttggcccaa agggaaaagc 480caatgggaaa tttttatgga acatgtagaa gagcttatta atcaaaagat atcgacttat 540gcaagaaaca aagcacttgc agatttaaaa ggattaggag atgctttggc tgtctaccat 600gaatcgctgg aaagttggat taaaaatcgc aataacacaa gaactagaag tgttgtcaag 660agccaataca ttaccttgga acttatgttc gtacaatcat taccttcttt tgcagtgtct 720ggagaggaag taccactatt accaatatat gctcaagctg caaatttaca cttgttgcta 780ttaagagatg cgtctatttt tggaaaagaa tggggattat cagactcaga aatttcgaca 840ttctataatc gtcaagtgga aagaacatca gattattccg atcattgcac gaaatggttt 900gatacgggct tgaatagatt aaagggctca aatgctgaaa tctgggtaaa gtataatcaa 960ttccgtagag acatgacttt aatggtacta gatttagtgg cactattcca aagctatgat 1020acacatatgt acccaattaa aactacagcc caacttacta gagaagtata tacaaacgca 1080ttggggacag tacatccgca cccaagtttt acaagtacga cttggtataa taataatgca 1140ccttcgtttt ctgccataga ggctgccgtt atccgaagcc cgcacctact cgattttcta 1200gaacaagtta caatttacag cttattaagc cgatggagta acactcagta tatgaatatg 1260tggggaggac ataaactaga attccgaaca ataggaggaa cgttaaatac ctcaacacaa 1320ggatctacta atacttctat taatcctgta acattaccgt tcacgtctcg agacatctat 1380aggactgaat cattggcagg gctgaatcta tttttaactc aacctgttaa tggagtacct 1440agggttgatt ttcattggaa attcgtcaca catccgatcg catctgataa tttctattat 1500ccagggtatg ctggaattgg gacgcaatta caggattcag aaaatgaatt accacctgaa 1560gcaacaggac agccaaatta tgaatcttat agtcatagat tatctcatat aggactcatt 1620tcagcatcac atgtgaaagc attggtatat tcttggacgc atcgtagtgc agatcgtacg 1680aatacaattc attcagatag tataacacaa ataccactgg taaaagcaca tacccttcag 1740tcaggtacta ctgttgtaaa agggccaggg tttacaggtg gagatatcct ccgacgaact 1800agtggaggac catttgcttt tagtaatgtt aatttagact ggaacttgtc acaaagatat 1860cgtgctagaa tacgctatgc ttctactact aatctaagaa tgtacgtaac gattgcaggg 1920gaacgaattt ttgctggtca atttaataaa acaatgaata ctggtgatcc attaacattc 1980caatctttta gttacgcaac tattgataca gcatttacat tcccaacgaa agcgagcagc 2040ttgactgtag gtgctgatac ttttagctca ggtaatgaag tttatgtaga tagatttgaa 2100ttgatcccag ttactgcaac acttgaggca gtaactgatt tagaaagagc gcagaaggcg 2160gttcatgaac tgtttacatc tacgaatccg ggaggattaa aaacggatgt aaaggattat 2220catattgacc aggtatcaaa tttagtagag tctctatcag ataaattcta tcttgatgaa 2280aagagagaat tattcgagat agttaaatac gcgaagcaac tccatattga gcgtaacatg 2340taggagctcg aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc 2400acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 2460gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 2520tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 2580cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 2640gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 2700aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 2760gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 2820aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 2880gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 2940ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 3000cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 3060ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 3120actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 3180tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 3240gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 3300ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 3360cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 3420ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 3480tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 3540agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3600gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 3660ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 3720gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 3780cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 3840acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 3900cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 3960cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 4020ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 4080tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 4140atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 4200tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 4260actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 4320aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 4380ctcatactct tcctttttca atgtacccca aattccccgt aggcgctagg gacctcttta 4440gcttcttgga agctgtcagt agtatatcta ataatttatc tccattccct ttagtaacgt 4500gtaactttcc aaatttaaaa aagcgactca tagaattatt tcctcccgtt aaataataga 4560taactattaa aaatagacaa tacttgctca taagtaatgg tacttaaatt gtttactttg 4620gcgtgtttca ttgcttgatg aaactgattt ttagtaaaca gttgacgata ttctcgattg 4680acccattttg aaacaaagta cgtatatagc ttccaatatt tatctggaac atctgtggta 4740tggcgggtaa gttttattaa gacactgttt acttttggtt taggatgaaa gcattccgct 4800ggcagcttaa gcaattgctg aatcgagact tgagtgtgca agagcaaccc tagtgttcgg 4860tgaatatcca aggtacgctt gtagaatcct tcttcaacaa tcagatagat gtcagacgca 4920tggctttcaa aaaccacttt tttaataatt tgtgtgctta aatggtaagg aatactccca 4980acaattttat acctctgttt gttagggaat tgaaactgta gaatatcttg gtgaattaaa 5040gtgacacgaa tgttcagttt taatttttct gacgataagt tgaatagatg actgtctaat 5100tcaatagacg ttacctgttt acttatttta gccagtttcg tcgttaaatg ccctttacct 5160gttccaattt cgtaaacggt atcggtttct tttaaattca attgttttat tatttggttg 5220agtacttttt cactcgttaa aaagttttga gaatatttta tatttttgtt catgtaatta 5280ctcctgaagt gattacatct gtaaataaat acagaagtta aacgatttgt ttgtaatttt 5340agttatctgt ttaaaaagtc ataagattag tcactggtag gaattaatct aacgtattta 5400tttatctgcg taatcactgt ttttagtctg tttcaaaaca gtagatgttt tatctacatt 5460acgcatttgg aataccaaca tgacgaatcc ctccttctta attacaaatt tttagcatct 5520aatttaactt caattcctat tatacacaaa attttaagat actgcactat caacacactc 5580ttaagtttgc ttcggggatc gatcccacat tagaaataga attggcatca gctaagagtt 5640gttcagcttc aagaatcttc ctctccaatt gagcgctttt ctcctctagc acagaaactt 5700tactttctgc ttcttctaat tttctactag cctcgtccat attcttttta gcacgcttat 5760tagaatatgt aacataagca aacgctgctg taaaaaccgc cgttataatt gccactccaa 5820tacctactgc tgttaacata ttagatatcg tggtgttcaa aaacgctacc tcatgatcct 5880gcaatgatat aactttatct tgcaaatcct ttacgacatc agtagatggc gccgctgtaa 5940atagtatccc cagcatcaat agttccccct gattatataa attcagacga atactattat 6000ccattatttt tttattaagg taaattaata ttttacctat taaaggaggg acgatttaag 6060tatggacgtg atgtttggtc aaggtgggaa cataaatttt aattacattg gaacatattg 6120tggacaagca tctaattcat aatccccact ttccaagcat gaaagtccct tataccatca 6180attattcaat aaaatatccc tatcataaac ctgtcccgcg cgacggttca aatactcctt 6240tatgataaaa taaagatata aagagacgag atgagggaga aatttgacgg ctaataaaaa 6300aaagattgta aataaaccaa ctgttgtggc aaaacttcca caaatcatta tagatacata 6360taacttgact tgcacttcta acgatgtaaa aatgtttccc ggatttataa agcacgtaaa 6420gaaacaacat cctggtattt atgaaaaata ttcatcgcac ataaaagata tcgttgaaca 6480ccccgactat gtcgggcaaa atcctaaaga acctaatagc gttgaattgg taaagatttt 6540aaatgaccat atactaattg ctataaagtt ggacccaagc ggttatttgt tcttatcaac 6600tatgttcgat ctaaaaaacg gtcctgcgaa aatccaaaga cgattgaata gtggacgttt 6660aattgcttac aaagacctgt taagctaacg gggagtttac taaaatataa gaacatggta 6720tactacctgt agaaagtaac ataataatga tataggatct ttgaggtggg aaaggttccc 6780cacgcgctta tctaaccgcc gttctcaagc atttgattgg ctaggtaagt taccagagat 6840gtaggatacg ccgccctacc aacgatccta tgtacggtaa gccatccgtt ttttacggat 6900ggctttttat attgactttt tcatcgctaa tggttacttt tctatcaaac gacaccgaaa 6960ggcgatgatt attgtgttaa aaaacccaat attataacat ccactaatgg ataatcttac 7020ttttagtaag taggttgcct agtgaaatac aataattcca acattgggcc ttaatacata 7080ttaattagtt ttaaccctct cactataaaa gagttaaata tgactccgca acctctttaa 7140tatcactatt acattttgta acaacattat tactttcatc ctctgtacca ttaacaaata 7200aatgaggatt ctttaccata aagtctaaca atagatcaat aaaccgctgc ctttgttctc 7260tagccatttc ctttctcata gagaaccact cccctatctt tcactattct tttaatttca 7320tcttgtcttt cacttgattt aaacagctct ggattcttcc tcaatccccc actatcttag 7380ttaatggtag ttgcttcagt actctacatt ttttgctaat cgaggttaaa atccttcaaa 7440tcccttgact ttaaggtctt aaggttttca tactggcgag tttgttagga aatctgaatg 7500tagttttggg catagtaacc cctgttgaaa atttacgagg ttaaaacgcc cactcattcg 7560tactggctta gatgccgagg tctcgaattg agattggttg cattgacgac aaacggttat 7620agcttacaaa actactatac cgtccgtata tagcttattg gcattgtatt gcgccgtacg 7680gtgcgtttct cacgcccaac accgttccct ttcgggtgat tcgctatata acccgcatat 7740ccttgtaata aagcttgtac aagaatagcc gtgaatcagt gtcatgatcg ccgcttacga 7800gtaactgttt aactccagtc gcacaatcat gtcttatttt gaacccccaa cactttttgc 7860cgctaacgtc ttggttatca tcattattag gatttacatc ccgtaggctt gttttatgca 7920aggcttgtgt cctttacctc gcttacgtca gacagttgaa tgcaacgaca actgatatcc 7980gagtgtactc tcctgcattg gctacactag accatctttt taaagtggta gcttccactg 8040gaacggaaca caaaaagggc gttctctata tctaaacgcc ctgtacattt acaagacttc 8100taggtataga aaacgccctt ttatatccgt tttattatgt atatgacaat gctagtaatt 8160actagttgaa atattcgtag agtaacggta caataggtgt atctaataag ccttgttcgc 8220gaaaacaagg caaataactt atttgaggtt ggcgcctctt ataagtcgaa gtcctgtatg

8280gctgtacagg attaagtcat tctcgctaaa ggttggtagc caatagcata tgagagtggc 8340ttttttcatt tccgttctat taagttactg ctaattttac cgctatgttt ctattaaatc 8400aacgaaaatt tgataaggtt caccagaaac attaatattt gcgacataaa actcactctc 8460caactttgga ggatggatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 8520atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 8580gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 8640atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 8700cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 8760cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 8820cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 8880aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 8940gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 9000agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag 9060cttgcatgcc tgcagtct 90783116384DNAArtificial SequenceBinary vector for plant expression of Cry proteins 31attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 60taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 120tgtcaaacac tgatagttta aactggcact agcctaacgg tgttgactaa ctaggccgct 180tccctaatta gctaacccgg gggcgcgccg ggacccctgc agtgcagcgt gacccggtcg 240tgcccctctc tagagataat gagcattgca tgtctaagtt ataaaaaatt accacatatt 300ttttttgtca cacttgtttg aagtgcagtt tatctatctt tatacatata tttaaacttt 360actctacgaa taatataatc tatagtacta caataatatc agtgttttag agaatcatat 420aaatgaacag ttagacatgg tctaaaggac aattgagtat tttgacaaca ggactctaca 480gttttatctt tttagtgtgc atgtgttctc cttttttttt gcaaatagct tcacctatat 540aatacttcat ccattttatt agtacatcca tttagggttt agggttaatg gtttttatag 600actaattttt ttagtacatc tattttattc tattttagcc tctaaattaa gaaaactaaa 660actctatttt agttttttta tttaataatt tagatataaa atagaataaa ataaagtgac 720taaaaattaa acaaataccc tttaagaaat taaaaaaact aaggaaacat ttttcttgtt 780tcgagtagat aatgccagcc tgttaaacgc cgccgacgag tctaacggac accaaccagc 840gaaccagcag cgtcgcgtcg ggccaagcga agcagacggc acggcatctc tgtcgctgcc 900tctggacccc tctcgagagt tccgctccac cgttggactt gctccgctgt cggcatccag 960aaattgcgtg gcggagcggc agacgtgagc cggcacggca ggcggcctcc tcctcctctc 1020acggcaccgg cagctacggg ggattccttt cccaccgctc cttcgctttc ccttcctcgc 1080ccgccgtaat aaatagacac cccctccaca ccctctttcc ccaacctcgt gttgttcgga 1140gcgcacacac acacaaccag atctccccca aatccacccg tcggcacctc cgcttcaagg 1200tacgccgctc gtcctccccc cccccccctc tctaccttct ctagatcggc gttccggtcc 1260atagttaggg cccggtagtt ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt 1320agatccgtgc tgttagcgtt cgtacacgga tgcgacctgt acgtcagaca cgttctgatt 1380gctaacttgc cagtgtttct ctttggggaa tcctgggatg gctctagccg ttccgcagac 1440gggatcgatt tcatgatttt ttttgtttcg ttgcataggg tttggtttgc ccttttcctt 1500tatttcaata tatgccgtgc acttgtttgt cgggtcatct tttcatgctt ttttttgtct 1560tggttgtgat gatgtggtct ggttgggcgg tcgttctaga tcggagtaga attctgtttc 1620aaactacctg gtggatttat taattttgga tctgtatgtg tgtgccatac atattcatag 1680ttacgaattg aagatgatgg atggaaatat cgatctagga taggtataca tgttgatgcg 1740ggttttactg atgcatatac agagatgctt tttgttcgct tggttgtgat gatgtggtgt 1800ggttgggcgg tcgttcattc gttctagatc ggagtagaat actgtttcaa actacctggt 1860gtatttatta attttggaac tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa 1920gatggatgga aatatcgatc taggataggt atacatgttg atgtgggttt tactgatgca 1980tatacatgat ggcatatgca gcatctattc atatgctcta accttgagta cctatctatt 2040ataataaaca agtatgtttt ataattattt tgatcttgat atacttggat gatggcatat 2100gcagcagcta tatgtggatt tttttagccc tgccttcata cgctatttat ttgcttggta 2160ctgtttcttt tgtcgatgct caccctgttg tttggtgtta cttctgcagg gatcctaaac 2220catggcgatt aacaatcaga accagtgcat cccatacaac tgcctgtcca atcctgagga 2280ggtgttcctg gacggcgagc gcatcctccc ggacattgat cccctggagg tgtctctctc 2340actcctgcag ttcctcctga acaatttcgt cccaggcggg ggcttcattt cgggcctcct 2400ggacaagatc tggggcgccc tcaggccttc ggattgggag ctgttcctcg agcagatcga 2460gcagctcatt gacaggagga tcgagcgcac cgtcagggct aaggccatcg ctgagctgga 2520ggggctgggc cgctcttacc agctctacgg cgaggcgttc aaggagtggg agaagacgcc 2580cgacaacacg gcggccaggt caagggtgac ggagcgcttc aggatcattg atgcccagat 2640tgaggcgaac atcccgtcct tccgcgtgag cggcttcgag gtccccctcc tgctcgttta 2700cacgcaggct gccaacctcc atctggccct gctccgggac tcggtggtgt tcggcgagag 2760gtgggggctc accaccacaa acgtcaatga tatctacaac cggcaggtta atcgcatcgg 2820cgagtactca aagcactgcg tcgacactta caagaccgag ctggagaggc tgggcttccg 2880gtccattgcg cagtggagga tctacaacca gttccggcgc gagctgacac tgactgtgct 2940cgacatcgtc gctgttttcc caaactacga ttcccggctg taccctatcc gcacgattag 3000ccagctcaca cgcgagatct acacttcccc agttagcgag ttctactacg gcgtgatcaa 3060ctccaacaat atcattggca ccctcacgga gcagcagatt aggcggcctc acctcatgga 3120cttcttcaac tcgatgatca tgtacacctc tgataatcgc agggagcact actggagcgg 3180cctggagatg acagccacta acaccgaggg gcatcagcgc tccttcccac tggccggcac 3240catcgggaat tctgctccgc ccgtgaccgt gcgcaacaat ggggagggca tctacaggat 3300tctgtccgag ccattctact cggccccttt cctgggcacg tcggtcctgg gctctcgcgg 3360ggaggagttc gctttcgcgt cgaacactac cacgtcgctg ccatctacaa tctacaggaa 3420tcgcggcact gtggactcac tcgtctccat cccacctcag gattactctg ttccgcccca 3480caggggctac tcacacctgc tctcccatgt gacaatgcgc aactccagcc cgatcttcca 3540ctggactcat aggagcgcca cgccacggaa tacaatcgac cctgattcga tcacacagat 3600tcccgctgtg aagggcgcct acattttcaa ctcgccggtc atcaccgggc ccggccacac 3660cggcggcgac atcattcgct tcaacccaaa tacgcagaac aatatcagga ttcctttcca 3720gtccaacgcg gtccagcgct accgcatccg catgcgctac gcggctgagg ctgactgcat 3780tctggagagc ggcgttaaca tcgtgacagg ggctggcgtg actttccgcc caatccctat 3840taaggccacg atgacaccag gctcacctct cacctactac tccttccagt acgccgacct 3900gaacattaat ctcacggcgc cgatccgccc caacaatttc gtgagcatca ggaggtccaa 3960ccagcccggc aatctgtaca tcgacaggat tgagttcatc ccaattgatc ctatcaggga 4020ggccgagcac gacctcgagc gcgcgcagaa ggctgtcaac gccctgttca cctcgtctaa 4080tcagattggc ctcaagacgg acgtgacaga ttaccatatc gaccaggtta gcaacctggt 4140ggcctgcctc tcggacaagt tctgcctgga tgagaagagg gagctgtcag agaaggtcaa 4200gcacgcgaag cgcctgtccg acgagaggaa cctgctccag gatcagaatt tcacgggcat 4260caacaggcag gtggataggg gctggagggg gagcactgac atcaccattc agggcggcaa 4320cgatgtcttc aaggagaatt acgttactct gccgggcacc ttcgacgagt gctaccccac 4380atacctctac cagaagatcg atgagtcgaa gctgaagccg tacactcgct acgagctgag 4440gggatacatc gaggactctc aggatctgga ggtctacctc atccgctaca acgccaagca 4500tgagaccctc aatgtgcccg ggacgggcag cctctggccg ctggcggccg agtcatccat 4560cggcaggtgc ggggagccaa acaggtgcgc ccctcacatc gagtggaatc cggagctgga 4620ctgctcgtgc agggatggcg agaagtgcgc gcaccattct caccatttct cactcgacat 4680cgatgtgggc tgcaccgacc tgaacgagga tctcggggtt tgggtcatct tcaagatcaa 4740gacccaggac ggctacgcta ggctggggaa cctggagttc ctggaggaga agccgctgct 4800gggcgaggct ctggctaggg tcaagagggc ggagaagaag tggcgcgaca agagggataa 4860gctcgagtgg gagaccaaca tcgtgtacaa ggaggccaag gagtctgtgg acgcgctgtt 4920cgtcgattca cagtacaaca ggctccagac tgacaccaat atcgcgatga ttcacgttgc 4980tgataagcgg gtgcatcgca tccgcgaggc ttacctgccc gagctgtccg tcattcccgg 5040cgttaacgct gccatcttcg aggagctgga ggggctcatc ttcaccgctt tcagcctgta 5100cgacgccagg aacgtcatca agaatggcga tttcaaccac gggctctcgt gctggaacgt 5160gaagggccac gtcgacgttg aggagcagaa caatcatcgc tctgttctgg ttgtgccgga 5220gtgggaggct gaggtgtcac aggaggtgcg ggtctgcccg gggaggggat acatcctcag 5280ggtcaccgcc tacaaggagg ggtacggcga ggggtgcgtt accatccacg agattgagga 5340ccatacggat gagctgaagt tccggaactg cgaggaggag gaggtgtacc caaacaatac 5400ggtcacatgc aatgactacc cggccaacca ggaggagtac agggccgctg agacatccag 5460gaacaggggc tacggggaga gctacgagtc gaatagctcg attccggcgg agtacgctcc 5520catctacgag aaggcctaca ctgacggcag gaaggagaat tcttgcgagt caaaccgggg 5580ctacgggaat tacacaccgc tgcccgcggg ctacgtcact aaggagctgg agtacttccc 5640ggagaccgac aaggtttgga tcgagattgg cgagacggag gggacattcc tcgtcgatag 5700cgttgagctg ctcctgatgg aggagtgaga gctcgccatc agtcgttgaa gctgctgctg 5760tatctgggtt atctagtgtc tctgccattg cccaaggatg gtgctgtctt tcaaagtatt 5820tgtatggttt gtgtcgtgag tcgtgactga gctggtttca tggaccagtt gtgttctcgt 5880tacccaaaac tatcgtgcga ccgcatatgg cttaatcatg aataaatgtt gtttgaattt 5940aaactattcg ctgaatattg ttgttttttg tcatgtcagt taatgttact aaattggttg 6000ccttctaatt tttgtttact ggtgtttgtc gcaccttatc tttttactgt atgtttactt 6060caggttctgg cagtctcatt ttttgtgact agttaaaact tacagctaaa aaaatgcagt 6120ttttcatttt catttgaagt ttgattagag ctattgatac ccggaccatc aggttaggtt 6180agttgtgcat agaatcataa atattaatca tgttttctat gaattaagtc aaacttgaaa 6240gtctggctga atatagtttc tatgaatcat attgatatac atgtttgatt atttgttttg 6300ctattagcta tttactttgg tgaatctata taggcttatg cagaaccttt ttttttgttc 6360tatatatcca tatcctagta ctcagtagct ctatgttttc tggagactag tggcttgctt 6420tttcgtatgt ctaatttttt gcttgaccat tgcaaaacaa aaattaccta gtgtaatctc 6480tttttataat aatcttgtaa tgcgtctacc tataggtcaa agtaggtttt gtttggaacc 6540cttagagcta actgttagct agttgataaa ttattagctg agttaagcta gctaatgaac 6600tagttttgat attagctgag gatgtttgaa acctaataat tattttttat tagctaacta 6660tactaaattt tagtagagag attccaaaca ggagttaaca tgggatcaga ttggctatgc 6720gtttgcaatc ccatactaat tagctaacgg accgcgatcg cttaattaag cttgcatgcc 6780tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat aatgagcatt gcatgtctaa 6840gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 6900ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 6960atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 7020tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 7080tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 7140tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 7200gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat 7260aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 7320actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgccgac 7380gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 7440ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 7500cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 7560gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 7620ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 7680tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagatctccc ccaaatccac 7740ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 7800tctctagatc ggcgttccgg tccatagtta gggcccggta gttctacttc tgttcatgtt 7860tgtgttagat ccgtgtttgt gttagatccg tgctgttagc gttcgtacac ggatgcgacc 7920tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 7980atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 8040gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 8100tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttct 8160agatcggagt agaattctgt ttcaaactac ctggtggatt tattaatttt ggatctgtat 8220gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa tatcgatcta 8280ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg ctttttgttc 8340gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttctag atcggagtag 8400aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt gtgtgtcata 8460catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata ggtatacatg 8520ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta ttcatatgct 8580ctaaccttga gtacctatct attataataa acaagtatgt tttataatta ttttgatctt 8640gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag ccctgccttc 8700atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg ttgtttggtg 8760ttacttctgc agggatctcc gatcatgcaa aaactcatta actcagtgca aaactatgcc 8820tggggcagca aaacggcgtt gactgaactt tatggtatgg aaaatccgtc cagccagccg 8880atggccgagc tgtggatggg cgcacatccg aaaagcagtt cacgagtgca gaatgccgcc 8940ggagatatcg tttcactgcg tgatgtgatt gagagtgata aatcgactct gctcggagag 9000gccgttgcca aacgctttgg cgaactgcct ttcctgttca aagtattatg cgcagcacag 9060ccactctcca ttcaggttca tccaaacaaa cacaattctg aaatcggttt tgccaaagaa 9120aatgccgcag gtatcccgat ggatgccgcc gagcgtaact ataaagatcc taaccacaag 9180ccggagctgg tttttgcgct gacgcctttc cttgcgatga acgcgtttcg tgaattttcc 9240gagattgtct ccctactcca gccggtcgca ggtgcacatc cggcgattgc tcacttttta 9300caacagcctg atgccgaacg tttaagcgaa ctgttcgcca gcctgttgaa tatgcagggt 9360gaagaaaaat cccgcgcgct ggcgatttta aaatcggccc tcgatagcca gcagggtgaa 9420ccgtggcaaa cgattcgttt aatttctgaa ttttacccgg aagacagcgg tctgttctcc 9480ccgctattgc tgaatgtggt gaaattgaac cctggcgaag cgatgttcct gttcgctgaa 9540acaccgcacg cttacctgca aggcgtggcg ctggaagtga tggcaaactc cgataacgtg 9600ctgcgtgcgg gtctgacgcc taaatacatt gatattccgg aactggttgc caatgtgaaa 9660ttcgaagcca aaccggctaa ccagttgttg acccagccgg tgaaacaagg tgcagaactg 9720gacttcccga ttccagtgga tgattttgcc ttctcgctgc atgaccttag tgataaagaa 9780accaccatta gccagcagag tgccgccatt ttgttctgcg tcgaaggcga tgcaacgttg 9840tggaaaggtt ctcagcagtt acagcttaaa ccgggtgaat cagcgtttat tgccgccaac 9900gaatcaccgg tgactgtcaa aggccacggc cgtttagcgc gtgtttacaa caagctgtaa 9960gagcttactg aaaaaattaa catctcttgc taagctgggt catgggtcgt ttaagctgcc 10020gatgtgcctg cgtcgtctgg tgccctctct ccatatggag gttgtcaaag tatctgctgt 10080tcgtgtcatg agtcgtgtca gtgttggttt aataatggac cggttgtgtt gtgtgtgcgt 10140actacccaga actatgacaa atcatgaata agtttgatgt ttgaaattaa agcctgtgct 10200cattatgttc tgtctttcag ttgtctccta atatttgcct ccaggtactg gctatctacc 10260gtttcttact taggaggtgt ttgaatgcac taaaactaat agttagtggc taaaattagt 10320taaaacatcc aaacaccata gctaatagtt gaactattag ctatttttgg aaaattagtt 10380aatagtgagg tagttatttg ttagctagct aattcaacta acaattttta gccaactaac 10440aattagtttc agtgcattca aacaccccct taatgttaac gtggttctat ctaccgtctc 10500ctaatatatg gttgattgtt cggtttgttg ctatgctatt gggttctgat tgctgctagt 10560tcttgctgaa tccagaagtt ctcgtagtat agctcagatt catattattt atttgagtga 10620taagtgatcc aggttattac tatgttagct aggttttttt tacaaggata aattatctgt 10680gatcataatt cttatgaaag ctttatgttt cctggaggca gtggcatgca atgcatgaca 10740gcaacttgat cacaccagct gaggtagata cggtaacaag gttcttaaat ctgttcacca 10800aatcattgga gaacacacat acacattctt gccagtcttg gttagagaaa tttcatgaca 10860aaatgccaaa gctgtcttga ctcttcactt ttggccatga gtcgtgactt agtttggttt 10920aatggaccgg ttctcctagc ttgttctact caaaactgtt gttgatgcga ataagttgtg 10980atggttgatc tctggatttt gttttgctct caatagtgga cgagattaga tagcctgcag 11040gcccgggggc gcgccctaat tagctaacgg ccaggatcgc cgcgtgagcc tttagcaact 11100agctagatta attaacgcaa tctgttatta agttgtctaa gcgtcaattt gtttacacca 11160caatatatcc tgccaccagc cagccaacag ctccccgacc ggcagctcgg cacaaaatca 11220ccactcgata caggcagccc atcagaatta attctcatgt ttgacagctt atcatcgact 11280gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt atggctgtgc 11340aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc tggataatgt 11400tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg ttgacaatta 11460atcatccggc tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca 11520gaccatgagg gaagcgttga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt 11580catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga 11640tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga 11700tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga 11760gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg 11820gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc 11880aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag 11940agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga 12000acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg 12060ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac 12120cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca 12180gtatcagccc gtcatacttg aagctaggca ggcttatctt ggacaagaag atcgcttggc 12240ctcgcgcgca gatcagttgg aagaatttgt tcactacgtg aaaggcgaga tcaccaaagt 12300agtcggcaaa taaagctcta gtggatctcc gtacccaggg atctggctcg cggcggacgc 12360acgacgccgg ggcgagacca taggcgatct cctaaatcaa tagtagctgt aacctcgaag 12420cgtttcactt gtaacaacga ttgagaattt ttgtcataaa attgaaatac ttggttcgca 12480tttttgtcat ccgcggtcag ccgcaattct gacgaactgc ccatttagct ggagatgatt 12540gtacatcctt cacgtgaaaa tttctcaagc gctgtgaaca agggttcaga ttttagattg 12600aaaggtgagc cgttgaaaca cgttcttctt gtcgatgacg acgtcgctat gcggcatctt 12660attattgaat accttacgat ccacgccttc aaagtgaccg cggtagccga cagcacccag 12720ttcacaagag tactctcttc cgcgacggtc gatgtcgtgg ttgttgatct agatttaggt 12780cgtgaagatg ggctcgagat cgttcgtaat ctggcggcaa agtctgatat tccaatcata 12840attatcagtg gcgaccgcct tgaggagacg gataaagttg ttgcactcga gctaggagca 12900agtgatttta tcgctaagcc gttcagtatc agagagtttc tagcacgcat tcgggttgcc 12960ttgcgcgtgc gccccaacgt tgtccgctcc aaagaccgac ggtctttttg ttttactgac 13020tggacactta atctcaggca acgtcgcttg atgtccgaag ctggcggtga ggtgaaactt 13080acggcaggtg agttcaatct tctcctcgcg tttttagaga aaccccgcga cgttctatcg 13140cgcgagcaac ttctcattgc cagtcgagta cgcgacgagg aggtttatga caggagtata 13200gatgttctca ttttgaggct gcgccgcaaa cttgaggcag atccgtcaag ccctcaactg 13260ataaaaacag caagaggtgc cggttatttc tttgacgcgg acgtgcaggt ttcgcacggg 13320gggacgatgg cagcctgagc caattcccag atccccgagg aatcggcgtg agcggtcgca 13380aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg gagaagttga 13440aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc ggtgaatcgt 13500ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca gccggtgcgc 13560cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt ccgatgctct 13620atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc cgtctgtcga 13680agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg cacgtagagg 13740tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta ctgatggcgg 13800tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac aagcccggcc 13860gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc gatggcggaa 13920agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac gttgccatgc 13980agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt gaagccttga 14040ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc gagatcgagc 14100tggctgattg gatgtaccgc gagatcacag aaggcaagaa

cccggacgtg ctgacggttc 14160accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc ctggcacgcc 14220gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa cgcagtggca 14280gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg tcaaatgacc 14340tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta gtcatgcgct 14400accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag cagatgctag 14460ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg gatagcacgt 14520acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg aacccaaagc 14580cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa aaaggcgatt 14640tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg gcctgtgcat 14700aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt cggtcgctgc 14760gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc tcaaaaatgg 14820ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg ccactcgacc 14880gccggcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag gcctgaatcg 14940ccccatcatc cagccagaaa gtgagggagc cacggttgat gagagctttg ttgtaggtgg 15000accagttggt gattttgaac ttttgctttg ccacggaacg gtctgcgttg tcgggaagat 15060gcgtgatctg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg 15120tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa 15180ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt 15240ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc 15300aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt 15360cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg 15420tgagaatggc aaaagctctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 15480gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 15540ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 15600acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 15660cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 15720caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 15780gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 15840tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 15900aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 15960ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 16020cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 16080tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 16140tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 16200ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 16260aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 16320aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttgatccgga 16380atta 16384

Claims

1. A recombinant Cry protein that is toxic to a lepidopteran pest, wherein the recombinant Cry protein comprises (a) an amino acid sequence that has at least 99% sequence identity with an amino acid sequence of SEQ ID NO: 3.

2. (canceled)

3. The recombinant Cry protein of claim 2, wherein the recombinant Cry protein comprises SEQ ID NO:3, SEQ ID NO:6, or a toxin fragment of SEQ ID NO:3 or SEQ ID NO:6.

4. The recombinant Cry protein of claim 3, wherein the lepidopteran pest is selected from the group consisting of European corn borer (Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), sugarcane borer (Diatraea saccharalis), velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), southwest corn borer (Diatraea grandiosella), western bean cutworm (Richia albicosta), tobacco budworm (Heliothis virescens), Asian corn borer (Ostrinia furnacalis), cotton bollworm (Helicoverpa armigera), striped stem borer (Chilo suppressalis), pink stem borer (Sesamia calamistis) and rice leaffolder (Cnaphalocrocis medinalis).

5. A polynucleotide comprising a nucleotide sequence that a) encodes a recombinant Cry protein of claim 1; or b) comprises any of SEQ ID NOs:14-29, or toxin-encoding fragments thereof.

6. A chimeric gene comprising a heterologous promoter that functions in a plant or bacteria operably linked to the polynucleotide of claim 5.

7. The chimeric gene of claim 6, wherein a) the plant expressible promoter is a ubiquitin, cestrum yellow virus, corn TrpA, OsMADS 6, maize H3 histone, bacteriophage T3 gene 9 5' UTR, corn sucrose synthetase 1, corn alcohol dehydrogenase 1, corn light harvesting complex, corn heat shock protein, maize mtl, pea small subunit RuBP carboxylase, rice actin, rice cyclophilin, Ti plasmid mannopine synthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, bean glycine rich protein 1, potato patatin, lectin, CaMV 35S or a S-E9 small subunit RuBP carboxylase promoter; or b) the bacteria expressible promoter comprises nucleotides 12-197 of SEQ ID NO:30.

8. An insecticidal composition comprising the recombinant Cry protein of claim 1 wherein the composition comprises a bacterium or a plant comprising the recombinant Cry protein.

9. A recombinant vector comprising the polynucleotide of claim 5.

10. A transgenic bacterial cell or plant cell comprising the recombinant vector of claim 9.

11. The transgenic plant cell of claim 10, wherein the plant cell is a) a dicot plant cell; or b) a monocot plant cell; or c) a dicot plant cell selected from the group consisting of a soybean cell, sunflower cell, tomato cell, cole crop cell, cotton cell, sugar beet cell and tobacco cell; or d) a monocot plant cell selected from the group consisting of a barley cell, maize cell, oat cell, rice cell, sorghum cell, sugar cane cell and wheat cell.

12. A transgenic plant comprising the transgenic plant cell of claim 11.

13. A transgenic seed of the transgenic plant of claim 12, wherein the transgenic seed comprises the polynucleotide.

14. A harvested product derived from seed of claim 13, wherein the harvested product comprises the recombinant Cry protein and the recombinant Cry protein has the same function it had in the transgenic plant.

15. A processed product derived from the harvested product of claim 14, wherein the processed product is selected from the group consisting of flour, meal, oil, and starch, or a product derived therefrom, and wherein the processed product comprises the recombinant Cry protein and the recombinant Cry protein has the same function it had in the transgenic plant.

16. A method of producing a recombinant Cry protein that is toxic to a lepidopteran pest comprising: culturing the transgenic cell of claim 10 under conditions in which the transgenic cell produces the recombinant Cry protein.

17. A method of producing an insect-resistant transgenic plant comprising: introducing into a plant the chimeric gene of claim 6, wherein the recombinant Cry protein is expressed in the plant, thereby producing an insect-resistant transgenic plant.

18. The method of claim 17, wherein the introducing step is achieved by a) transforming the plant with the chimeric gene; or b) crossing a first plant comprising the chimeric gene with a different second plant; or c) genome editing a chimeric gene preexisting in a transgenic plant.

19. A method of controlling a lepidopteran pest, comprising delivering to the lepidopteran pest or an environment thereof a composition comprising an effective amount of the recombinant Cry protein of claim 1.

20. (canceled)