SYNTHETIC GENES

Abstract

The invention provides synthetic nucleic acid sequences encoding proteins of interest that are particularly adapted to express well in plants. The claimed synthetic sequences utilize plant-optimized codons roughly in the same frequency at which they are utilized, on average, in genes naturally occurring in the plant species. The invention further includes synthetic DNA sequence for herbicide tolerance, water and/or heat stress tolerance, healthy oil modifications and for transformation marker genes and selectable marker genes are used. DNA construct and transgenic plants containing the synthetic sequences are taught as are methods and compositions for using the plants in agriculture.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to provisional application Ser. No. 61/475,921 filed Apr. 15, 2011, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] To achieve desired expression levels of heterologous proteins in transgenic plants it has been found beneficial to alter the native, sometimes referred to as wild-type or original, DNA coding sequence in various ways, e.g. so that codon usage more closely matches the codon usage of the host plant species, and/or so the G+C content of the coding sequence more closely matches the G+C level typically found in coding sequences of the host plant species, and/or so that certain sequences that destabilize mRNA are removed. Expression in plants of Bacillus thuringiensis (B.t.) crystal protein insect toxins, for example, has been improved using one or more of these approaches. See, for example, U.S. Pat. No. 5,380,301, U.S. Pat. No. 5,625,136, U.S. Pat. No. 6,218,188, U.S. Pat. No. 6,340,593, U.S. Pat. No. 6,673,990, U.S. Pat. No. 7,741,118. Codon degeneracy allows one to make synthetic DNA sequences that encode a protein of interest using codons that differ from those used in the original DNA coding sequence.

[0003] In regard to removing sequences that may destabilize mRNA, U.S. Pat. No. 7,741,118 discloses a list of 16 polyadenylation signal sequences (column 15, Table II) and calls for reducing the number of such sequences in synthetic coding sequences that are intended for expression in plants. The polyadenylation signal sequences listed in U.S. Pat. No. 7,741,118, Table II are listed below in Table 1:

TABLE-US-00001 TABLE 1 Polyadenylation signal sequences listed in US 7741118, Table II. 1 AATAAA 6 ATACTA 11 ATACAT 16 CATAAA 2 AATAAT 7 ATAAAA 12 AAAATA 3 AACCAA 8 ATGAAA 13 ATTAAA 4 ATATAA 9 AAGCAT 14 AATTAA 5 AATCAA 10 ATTAAT 15 AATACA

[0004] U.S. Pat. No. 7,741,118 also calls for preferably removing the sequence ATTTA (known as the Shaw-Kamen sequence), because it has been identified as potentially destabilizing mRNA.

[0005] Contrary to the teaching of U.S. Pat. No. 7,741,118, we have found that reduction in the number of the polyadenylation signal sequences identified in Table 1 above is neither necessary nor sufficient to enable enhanced expression of synthetic genes in plants.

SUMMARY OF THE INVENTION

[0006] Table 2 below identifies 20 potential polyadenylation signal sequences that occur frequently in maize genes.

TABLE-US-00002 TABLE 2 Potential polyadenylation signal sequences found in maize genes. 1 ATATAT 6 TATTTT 11 TAATAA 16 TATTAT 2 TTGTTT 7 TTTTTT 12 ATTTAT 17 TGTTTG 3 TTTTGT 8 ATTTTT 13 TATATT 18 TTATAT 4 TGTTTT 9 TTATTT 14 TTTTAT 19 TGTAAT 5 TATATA 10 TTTATT 15 ATATTT 20 AAATAA

[0007] Table 3 below identifies 20 potential polyadenylation signal sequences that occur frequently in soybean genes.

TABLE-US-00003 TABLE 3 Potential polyadenylation signal sequences found in soybean genes. 1 ATTTTT 6 TTTTAT 11 AAATTT 16 ATATAT 2 TATTTT 7 AATTTT 12 AAATAA 17 ATTATT 3 TTATTT 8 TTTTTA 13 ATATTT 18 ATTTTA 4 TTTATT 9 TAATTT 14 TTTGTT 19 TTTAAT 5 TTTTTT 10 TTAATT 15 TTGTTT 20 TTTTAA

[0008] The present invention provides a synthetic DNA sequence for expressing a protein of interest in maize cells which comprises:

[0009] a) a codon-optimized DNA sequence encoding the protein of interest,

[0010] b) at least one polyadenylation signal sequence chosen from the group consisting of

[0011] Class I and Class II wherein:

[0012] Class I is chosen from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA. AATTAA, AATACA, and CATAAA; and

[0013] Class II is chosen from the group consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA. TATTTT, TTTTTT, ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT, TGTTTG, TTATAT, TGTAAT, and AAATAA; and

[0014] wherein said codon-optimized DNA sequence contains-at least one polyadenylation signal sequence from Class II and wherein said synthetic DNA sequence contains fewer Class II polyadenylation signal sequences than the protein's native DNA sequence and contains the same number of Class I polyadenylation signal sequences compared to the native DNA sequence.

[0015] The present invention also provides a synthetic DNA sequence for expressing a protein of interest in soybean cells which comprises:

[0016] a) a codon-optimized DNA sequence encoding the protein of interest,

[0017] b) at least one polyadenylation signal sequence chosen from the group consisting of

[0018] Class I and Class III wherein:

[0019] Class I is chosen from the group consisting of AATAAA. AATAAT, AACCAA, ATATAA, AATCAA, ATACTA. ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA. AATACA, and CATAAA; and

[0020] Class III is chosen from the group consisting of ATTTTT, TATTTT, TTATTT, TTTATT, TTTTTT, TTTTAT, AATTTT, TTTTTA, ATATAT, TAATTT, TTAATT, AAATTT, AAATAA, ATATTT, TTTGTT TTGTTT, ATTATT, ATTTTA, TTTAAT, and TTTTAA, and

[0021] wherein said codon-optimized DNA sequence contains at least one polyadenylation signal sequence from Class III and wherein said synthetic DNA sequence contains fewer Class III polyadenylation signal sequences than the protein's native DNA sequence and contains the same number of Class I polyadenylation signal sequences compared to the native DNA sequence.

[0022] The invention also provides a method of making a synthetic DNA sequence that encodes a protein of interest which comprises (a) starting with an amino acid sequence of a protein of interest derived from naturally occurring polypeptide(s) encoded by native sequence(s) that comprise at least one polyadenylation signal sequence listed in Table 2, and (b) making a synthetic DNA sequence that encodes said amino acid sequence and contains fewer polyadenylation signal sequences listed in Table 2 compared to the corresponding coding sequence of the native sequence(s) and contains the same number of polyadenylation signal sequences listed in Table 1.

[0023] In another embodiment the invention provides a method of making a synthetic DNA sequence that encodes a protein of interest which comprises (a) starting with an amino acid sequence of a protein of interest derived from naturally occurring polypeptide(s) encoded by native sequence(s) that comprise at least one polyadenylation signal sequence-listed in Table 3, and (b) making a synthetic DNA sequence that encodes said amino acid sequence and contains fewer polyadenylation signal sequences listed in Table 3 compared to the corresponding coding sequence of the native sequence(s) and contains the same number of polyadenylation signal sequences listed in Table 1.

[0024] In some embodiments the synthetic DNA sequences provided by the invention are devoid of the polyadenylation signal sequences listed in Table 2 and/or Table 3, or the number of polyadenylation signal sequences identified in Table 2 and/or Table 3 is reduced as much as possible consistent with maintaining the same number of polyadenylation signal sequences identified in Table 1 and maintaining the Table 1 sequences in their original positions in the sequence.

[0025] In some embodiments the synthetic DNA sequences provided by the invention encode an insecticidal protein, optionally derived from Bacillus thuringiensis, as well as DNA sequences useful for herbicide tolerance, water and/or heat stress tolerance, healthy oil modifications and for transformation marker genes and selectable marker genes.

[0026] The synthetic DNA sequences of the invention may be used in a DNA construct for expression of a protein of interest, where the construct comprises a 5' non-translated sequence, a synthetic DNA sequence of the invention, and a 3' non-translated region, and said 5' non-translated sequence contains a promoter that functions in plants, and said 3' non-translated sequence comprises a transcription termination and polyadenylation signal.

[0027] The invention also provides a transgenic plant containing the synthetic DNA sequences of the invention.

[0028] Also provided is a method of controlling pests in a plant which comprises expressing a synthetic DNA sequence of the invention in the plant where the synthetic DNA sequence encodes an insect toxin, for example a Bacillus thuringiensis Cry protein.

[0029] Also provided is a method for herbicide tolerance in a plant which comprises expressing a synthetic DNA sequence of the invention in the plant where the synthetic DNA sequence encodes a known herbicide tolerance enzyme, for example the ryloxylkanoate gioxygenase (AAD1) see WO/2005/107437, or phosphinothricin acetytransferase, or 5-enolpyruvylshikimate-3-phosphate synthase enzymes.

[0030] Also provided is a method for modifying oil profiles in a plant which comprises expressing one or more synthetic DNA sequences of the invention in the plant where the synthetic DNA sequence encodes one or more known enzymes for modifying oil profiles in plants, for example fatty acid desaturase.

[0031] Also provided is a method for stress tolerance in a plant which comprises expressing a synthetic DNA sequence of the invention in the plant where the synthetic DNA sequence encodes known stress tolerance genes for water and/or heat stress, for example the stress associated protein (SAP1); US Patent Publication No: 2010/0275327, and 1-Cys peroxiredoxin (Perl) proteins (Mowla et al., 2002, Planta 215:716-726).

[0032] Also provided is a method adding reporter genes in a plant which comprises expressing a synthetic DNA sequence of the invention in the plant where the synthetic DNA sequence encodes a known transformation marker protein functional in plants, for example green fluorescence protein (GFP) or beta glucuronidase enzyme.

[0033] Also provided is a method of controlling pests in grain or seed which comprises obtaining said grain or seed from plants containing a synthetic gene of the invention that expresses an insect toxin, and a method of controlling pests in meal or flour which comprises obtaining said meal or flour from grain containing a synthetic gene of the invention that expresses an insect toxin.

[0034] Also provided is a composition derived from transgenic plants containing synthetic DNA of the invention wherein said composition is a commodity product selected from the group consisting of meal, flour, protein concentrate, or oil.

[0035] In some cases the number of polyadenylation signals listed in Table 1 can be maintained in synthetic DNA sequences of the invention by deleting occurrences of AATAAA and substituting another polyadenylation signal sequence listed in Table 1. This is exemplified in Example 1, SEQ ID NO:5.

DESCRIPTION OF THE SEQUENCES

[0036] SEQ ID NO:1 is the native DNA sequence encoding Bacillus thuringiensis Cry1Fa core toxin.

[0037] SEQ ID NO:2 is Bacillus thuringiensis Cry1Fa core toxin sequence.

[0038] SEQ ID NO:3 is a synthetic DNA sequence encoding Bacillus thuringiensis Cry1Fa core toxin using codons optimized for maize and Table 1 sequences are maintained.

[0039] SEQ ID NO:4 is Bacillus thuringiensis Cry1Fa core toxin sequence.

[0040] SEQ ID NO:5 is a synthetic DNA sequence in accordance with the invention encoding-Bacillus thuringiensis Cry1Fa core toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0041] SEQ ID NO 6 is Bacillus thuringiensis Cry1Fa core toxin sequence.

[0042] SEQ ID NO:7 is the native DNA sequence encoding Bacillus thuringiensis Cry34Ab1 toxin.

[0043] SEQ ID NO:8 is Bacillus thuringiensis Cry34Ab1 toxin sequence.

[0044] SEQ ID NO:9 is a synthetic DNA sequence encoding Bacillus thuringiensis Cry34Ab1 toxin using codons optimized for maize and Table 1 sequences are maintained.

[0045] SEQ ID NO: 10 is Bacillus thuringiensis Cry34Ab1 toxin sequence.

[0046] SEQ ID NO: 11 is a synthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry34Ab1 toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0047] SEQ ID NO: 12 is Bacillus thuringiensis Cry34Ab1 toxin sequence.

[0048] SEQ ID NO: 13 is the native DNA sequence encoding Bacillus thuringiensis Cry35Ab1 toxin.

[0049] SEQ ID NO:14 is Bacillus thuringiensis Cry35Ab1 toxin sequence.

[0050] SEQ ID NO: 15 is a synthetic DNA sequence encoding Bacillus thuringiensis Cry35Ab1 toxin using codons optimized for maize and Table 1 sequences are maintained

[0051] SEQ ID NO:16 is Bacillus thuringiensis Cry35Ab1 toxin sequence.

[0052] SEQ ID NO: 17 is a synthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry35Ab1 toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0053] SEQ ID NO:18 is Bacillus thuringiensis Cry35Ab1 toxin sequence.

[0054] SEQ ID NO:19 is the native DNA sequence encoding Bacillus thuringiensis Cry1Ab1 core toxin.

[0055] SEQ ID NO:20 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.

[0056] SEQ ID NO:21 is a synthetic DNA sequence encoding Bacillus thuringiensis Cry1Ab1 core toxin using codons optimized for maize and Table 1 sequences are maintained.

[0057] SEQ ID NO:22 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.

[0058] SEQ ID NO:23 is a synthetic DNA sequence in accordance with the invention encoding-Bacillus thuringiensis Cry1Ab1 core toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0059] SEQ ID NO:24 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.

[0060] SEQ ID NO:25 is the native DNA sequence encoding Bacillus thuringiensis CryICa core toxin.

[0061] SEQ ID NO:26 is encoding Bacillus thuringiensis CryICa core toxin sequence.

[0062] SEQ ID NO:27 is a synthetic DNA sequence encoding Bacillus thuringiensis CryICa core toxin using codons optimized for maize and Table 1 sequences are maintained.

[0063] SEQ ID NO:28 is encoding Bacillus thuringiensis CryICa core toxin sequence.

[0064] SEQ ID NO:29 is a synthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis CryICa core toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0065] SEQ ID NO:30 is encoding Bacillus thuringiensis CryICa core toxin sequence.

[0066] SEQ ID NO:31 is the native DNA sequence encoding Bacillus thuringiensis Cry6Aa toxin.

[0067] SEQ ID NO:32 is Bacillus thuringiensis Cry6Aa toxin sequence.

[0068] SEQ ID NO:33 is a synthetic DNA sequence encoding Bacillus thuringiensis Cry6Aa toxin using codons optimized for maize and Table 1 sequences are maintained.

[0069] SEQ ID NO:34 is Bacillus thuringiensis Cry6Aa toxin sequence.

[0070] SEQ ID NO:35 is a synthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry6Aa toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0071] SEQ ID NO:36 is Bacillus thuringiensis Cry6Aa toxin sequence.

[0072] SEQ ID NO:37 is the native DNA sequence encoding Sphingobiurn herbicidovorans AAD1 protein.

[0073] SEQ ID NO:38 is Sphingobiurn herbicidovorans AADI protein sequence.

[0074] SEQ ID NO:39 is a synthetic DNA sequence encoding Sphingobiurn herbicidovorans AAD1 protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained.

[0075] SEQ ID NO:40 is Sphingobiurn herbicidovorans AADI protein sequence.

[0076] SEQ ID NO:41 is a synthetic DNA sequence in accordance with the invention encoding Sphingobiurn herbicidovorans AAD1 protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0077] SEQ ID NO:42 is Sphingobiurn herbicidovorans AADI protein sequence.

[0078] SEQ ID NO:43 is the native DNA sequence encoding Aspergillus nidulans delta-9 fatty acid desaturase protein.

[0079] SEQ ID NO:44 is Aspergillus nidulans delta-9 fatty acid desaturase protein sequence.

[0080] SEQ ID NO:45 is a synthetic DNA sequence encoding Aspergillus nidulans delta-9 fatty acid desaturase protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained.

[0081] SEQ ID NO:46 is Aspergillus nidulans delta-9 fatty acid desaturase protein sequence.

[0082] SEQ ID NO:47 is a synthetic DNA sequence in accordance with the invention encoding Aspergillus nidulans delta-9 fatty acid desaturase protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained.

[0083] SEQ ID NO:48 is Aspergillus nidulans delta-9 fatty acid desaturase protein.

[0084] SEQ ID NO:49 is the native DNA sequence encoding Xerophyta viscosa SAPI protein.

[0085] SEQ ID NO:50 is Xerophyta viscosa SAPI protein sequence.

[0086] SEQ ID NO:51 is a synthetic DNA sequence encoding Xerophyta viscosa SAPI protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained

[0087] SEQ ID NO:52 is Xerophyta viscosa SAPI protein sequence.

[0088] SEQ ID NO:53 is a synthetic DNA sequence in accordance with the invention encoding Xerophyta viscosa SAP1 protein using codons optimized for maize and with sequences identified in Table 2 removed and Table I sequences are maintained

[0089] SEQ ID NO:54 is Xerophyta viscosa SAPI protein sequence.

[0090] SEQ ID NO:55 is the native DNA sequence encoding Aequorea victoria GFPI protein.

[0091] SEQ ID NO:56 is Aequorea victoria GFPI protein sequence.

[0092] SEQ ID NO:57 is a synthetic DNA sequence encoding Aequorea victoria GFPI protein using codons optimized for maize and Table I & Table 2 sequences are maintained.

[0093] SEQ ID NO:58 is Aequorea victoria GFPI protein sequence.

[0094] SEQ ID NO:59 is a synthetic DNA sequence in accordance with the invention encoding Aequorea victoria GFPI protein using codons optimized for maize and with sequences identified in Table 2 removed and Table I sequences are maintained.

[0095] SEQ ID NO:60 is Aequorea victoria GFPI protein sequence.

[0096] SEQ ID NO:6I is the native DNA sequence encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein.

[0097] SEQ ID NO:62 is Leptosphaeria nodorum delta-9 fatty acid desaturase protein sequence.

[0098] SEQ ID NO:63 is a synthetic DNA sequence encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein using codons optimized for maize and Table I & Table 2 sequences are maintained

[0099] SEQ ID NO:64 is Leptosphaeria nodorum delta-9 fatty acid desaturase protein sequence.

[0100] SEQ ID NO:65 is a synthetic DNA sequence in accordance with the invention encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained

[0101] SEQ ID NO:66 is Leptosphaeria nodorum delta-9 fatty acid desaturase protein sequence.

[0102] SEQ ID NO:67 is the native DNA sequence encoding Xerophyta viscosa PERI protein.

[0103] SEQ ID NO:68 is Xerophyta viscosa PERI protein sequence.

[0104] SEQ ID NO:69 is a synthetic DNA sequence encoding Xerophyta viscosa PERI protein using codons optimized for maize and Table I & Table 2 sequences are maintained.

[0105] SEQ ID NO:70 is Xerophyta viscosa PERI protein sequence.

[0106] SEQ ID NO:7I is a synthetic DNA sequence in accordance with the invention encoding-Xerophyta viscosa PERI protein using codons optimized for maize and with sequences identified in Table 2 removed and Table I sequences are maintained.

[0107] SEQ ID NO:72 is Xerophyta viscosa PERI protein sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0108] The invention provides synthetic nucleic acid sequences encoding proteins of interest. The synthetic coding sequences are particularly adapted for use in expressing the proteins of interest in transgenic plants.

[0109] A protein of interest is any protein or polypeptide that occurs in nature, or any naturally occurring variant including but not limited to processed forms of such proteins. The protein of interest also may be a protein formed by combining portions or fragments of more than one naturally occurring protein such as by mixing and matching functional protein domains.

[0110] A preferred group of proteins of interest is one in which the resulting phenotype is an agronomic trait or reporter protein useful for creating agronomic traits. These include but are not limited to resistance to insects, tolerance to herbicides, tolerance to water and/or heat stress, and oil profile modification.

[0111] A more preferred group of proteins of interest is one in which the resulting phenotype is an agronomic trait. Another preferred group is one in which the resulting phenotype provides herbicide tolerance. Another preferred group is one in which the resulting phenotype provides stress tolerance. Another preferred group is one in which the resulting phenotype provides a modified oil profile for healthier food. A more highly preferred group is one in which the protein of interest is a Cry protein that provides insect resistance.

[0112] The native/wild-type DNA sequences encoding the protein of interest must be identified and analyzed to determine whether polyadenylation signal sequences listed in Tables I and 2 and/or 3 are present. In accordance with the invention, for coding sequences intended for use in maize, the number of polyadenylation signal sequences listed in Table 2 is reduced compared to the number present in the native sequence. For coding sequences intended for use in soybean, the number of polyadenylation signal sequences listed in Table 3 is reduced. It is very important to remove the polyadenylation signal sequences listed in Tables 2 and 3, particularly where they occur in nested multimeric form.

[0113] In addition to removing polyadenylation signal sequences listed in Tables 2 and 3, it may be desirable to remove occurrences of the Shaw-Kamen sequence, ATTTA.

[0114] In addition to removing polyadenylation signal sequences and Shaw-Kamen sequences, we prefer to build synthetic DNA coding sequences that utilize codons roughly in the same frequency at which they are utilized, on average, in genes naturally occurring in the plant species in which the synthetic DNA sequence will be used. Table 4 gives suitable target percentages for codon usage in synthetic genes intended for use in various specific crops as well as for use in dicots generally or plants generally.

TABLE-US-00004 TABLE 4 Target rescaled codon compositions of synthetic plant genes. Maize Soybean Maize Soybean Amino Acid Codon % % Amino Acid Codon % % ALA (A) GCA 18.0 33.1 LEU (L) CTA 0 0 GCC 34.0 24.5 CTC 29.9 22.4 GCG 24.0 0 CTG 33.3 16.3 GCT 24.0 42.3 CTT 19.5 31.5 ARG (R) AGA 18.8 36.0 TTA 0 0 AGG 32.5 32.2 TTG 17.2 29.9 CGA 0 0 LYS (K) AAA 22.0 42.5 CGC 30.0 15 AAG 78.0 57.5 CGG 18.8 0 MET (M) ATG 100 100 CGT 0 16.9 PHE (F) TTC 71.0 49.2 ASN (N) AAC 68.0 50.0 TTT 29.0 50.8 AAT 32.0 50.0 PRO (P) CCA 26.0 39.8 ASP (D) GAC 63.0 38.1 CCC 24.0 20.9 GAT 37.0 61.9 CCG 28.0 0.0 CYC (C) TGC 68.0 50.0 CCT 22.0 39.3 TGT 32.0 50.0 SER (S) AGC 25.3 16.0 END TAA 0 0 AGT 0.0 18.2 TAG 0 0 TCA 17.6 21.9 TGA 100 100 TCC 25.3 18.0 GLN (Q) CAA 38.0 55.5 TCG 15.4 0 CAG 62.0 44.5 TCT 16.5 25.8 GLU (E) GAA 29.0 50.5 THR (T) ACA 21.0 32.4 GAG 71.0 49.5 ACC 37.0 30.2 GLY (G) GGA 19.0 31.9 ACG 22.0 0.0 GGC 42.0 19.3 ACT 20.0 37.4 GGG 19.0 18.4 TRP (W) TGG 100 100 GGT 20.0 30.4 TYR (Y) TAC 73.0 48.2 HIS (H) CAC 62.0 44.8 TAT 27.0 51.8 CAT 38.0 55.2 VAL (V) GTA 0 11.5 ILE (I) ATA 14.0 23.4 GTC 34.8 17.8 ATC 58.0 29.9 GTG 42.4 32.0 ATT 28.0 46.7 TTT 22.8 38.7

Transgenic Plants

[0115] A preferred embodiment of the subject invention is the transformation of plants with genes encoding insect toxins. The transformed plants that express insect toxin genes are resistant to attack by an insect target pest by virtue of the presence of controlling amounts of the subject insecticidal protein or its variants in the cells of the transformed plant. By incorporating genetic material that encodes the insecticidal properties of the B.t. insecticidal toxins into the genome of a plant eaten by a particular insect pest, the adult or larvae die after consuming the food plant. Numerous members of the monocotyledonous and dicotyledonous classifications have been transformed. Transgenic agronomic crops as well as fruits and vegetables are of commercial interest. Such crops include but are not limited to maize, rice, soybeans, canola sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. Several techniques exist for introducing foreign genetic material into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (U.S. Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may be transformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010, European Patent No. EPI31624B1, European Patent No. EP159418B1, European Patent No. EP17611281, U.S. Pat. No. 5,149,645, EP120516B1, U.S. Pat. No. 5,464,763, U.S. Pat. No. 4,693,976, European Patent No. EP116718B1, European Patent No. EP290799B1, European Patent No. EP320500B1, European Patent No. EP604662B1, U.S. Pat. No. 7,060,876, U.S. Pat. No. 6,037,526, U.S. Pat. No. 6,376,234, European Patent No. EP292435B1, U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No. 4,762,785, U.S. Pat. No. 5,608,142, and U.S. Pat. No. 5,159,135. Other transformation technology includes WHISKERS.TM. technology, see U.S. Pat. No. 5,302,523 and U.S. Pat. No. 5,464,765. Electroporation technology has also been used to transform plants, see WO1987006614, U.S. Pat. No. 5,472,869, U.S. Pat. No. 5,384,253, WO199209696, U.S. Pat. No. 6,074,877, WO1993021335, and U.S. Pat. No. 5,679,558. In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and type II, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques within the skill of an artisan.

[0116] Known techniques of inserting DNA into plants include transformation with T-DNA delivered by Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent. The use of T-DNA-containing vectors for the transformation of plant cells has been intensively researched and sufficiently described in European Patent No. EP12051681; Lee and Gelvin (2008) Plant Physiol. 146:325-332; Fraley et al (1986) Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J. 4:277-284; and is well established in the field. Additionally, fusion of plant protoplasts with liposomes containing the DNA to be delivered, direct injection of the DNA, biolistics transformation (microparticle bombardment), or electroporation, as well as other possible methods, may be employed.

[0117] Once the inserted DNA has been integrated into the plant genome, it is relatively stable throughout subsequent generations. The vector used to transform the plant cell normally contains a selectable marker gene encoding a protein that confers on the transformed plant cells resistance to a herbicide or an antibiotic, such as Bialaphos, Kanamycin. G418, Bleomycin, or Hygromycin, inter alia. The individually employed selectable marker gene should accordingly permit the selection of transformed cells while the growth of cells that do not contain the inserted DNA is suppressed by the selective compound.

[0118] In a preferred embodiment of the subject invention, plants are transformed with genes wherein the codon usage of the protein coding region has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831. Also, advantageously, plants encoding a truncated toxin, e.g. a functional protein domain, maybe used. The truncated toxin typically encodes about 55% to about 80% of the native full length toxin. Methods for creating synthetic B.t. genes for use in plants are known in the art (Stewart 2007, Frontiers in Drug Design and Discovery 1:297-341).

[0119] Regardless of transformation technique, the gene is preferably incorporated into a gene transfer vector adapted to express the protein of interest in the plant cell by including in the vector a plant promoter. In addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoters of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter, promoters of viral origin, such as the 35S and 19S promoters of cauliflower mosaic virus (CaMV), and the like may be used. Plant-derived promoters include, but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat-shock promoters, ADF (actin depolymerization factor) promoter, and tissue specific promoters. Promoters may also contain certain enhancer sequence elements that may improve the transcription efficiency. Typical enhancers include but are not limited to ADHI-intron 1 and ADHI-intron 6. Constitutive promoters may be used. Constitutive promoters direct continuous gene expression in nearly all cells types and at nearly all times (e.g. actin, ubiquitin, CaMV 35S). Tissue specific promoters are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g. zein, oleosin, napin, ACP (Acyl Carrier Protein)), and these promoters may also be used. Promoters may also be used that are active during a certain stage of the plants' development as well as active in specific plant tissues and organs. Examples of such promoters include but are not limited to promoters that are root specific, pollen-specific, embryo specific, com silk specific, cotton fiber specific, seed endosperm specific, phloem specific, and the like.

[0120] Under certain circumstances it may be desirable to use an inducible promoter. An inducible promoter is responsible for expression of genes in response to a specific signal, such as: physical stimulus (e.g. heat shock genes); light (e.g. RUBP carboxylase); hormone (e.g. glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress (e.g. drought). Other desirable transcription and translation elements that function in plants may be used, such as 5' untranslated leader sequences, RNA transcription termination sequences and poly-adenylate addition signal sequences. Numerous plant-specific gene transfer vectors are known to the art.

[0121] Transgenic crops containing insect resistance (IR) traits are prevalent in com and cotton plants throughout North America, and usage of these traits is expanding globally. Commercial transgenic crops combining IR and herbicide tolerance (HT) traits have been developed by numerous seed companies. These include combinations of IR traits conferred by B.t. insecticidal proteins and HT traits such as tolerance to Acetolactate Synthase (ALS) inhibitors such as sulfonylureas, imidazolinones, triazolopyrimidine, sulfonanilides, and the like, Glutamine Synthetase (GS) inhibitors such as Bialaphos, glufosinate, and the like, 4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such as mesotrione, isoxaflutole, and the like, 5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such as glyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase) inhibitors such as haloxyfop, quizalofop, diclofop, and the like. Other examples are known in which transgenically provided proteins provide plant tolerance to herbicide chemical classes such as phenoxy acids herbicides and pyridyloxyacetates auxin herbicides (see WO2007053482), or phenoxy acids herbicides and aryloxyphenoxypropionates herbicides (see US Patent Application No. 20090093366). The ability to control multiple pest problems through IR traits is a valuable commercial product concept, and the convenience of this product concept is enhanced if insect control traits and weed control traits are combined in the same plant. Further, improved value may be obtained via single plant combinations of IR traits conferred by a B.t. insecticidal protein such as that of the subject invention with one or more additional HT traits such as those mentioned above, plus one or more additional input traits (e.g. other insect resistance conferred by B.t.-derived or other insecticidal proteins, insect resistance conferred by mechanisms such as RNAi and the like, nematode resistance, disease resistance, stress tolerance, improved nitrogen utilization, and the like), or output traits (e.g. high oils content, healthy oil composition, nutritional improvement, and the like). Such combinations may be obtained either through conventional breeding (breeding stack) or jointly as a novel transformation event involving the simultaneous introduction of multiple genes (molecular stack or co-transformation). Benefits include the ability to manage insect pests and improved weed control in a crop plant that provides secondary benefits to the producer and/or the consumer. Thus, the subject invention can be used in connection with a variety of traits to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic issues.

[0122] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification. Unless specifically indicated or implied, the terms "a", "an", and "the" signify "at least one" as used herein. By "isolated" applicants mean that the nucleotide or polypeptide molecules have been removed from their native environment and have been placed in a different environment by the hand of man.

[0123] Embodiments of the present invention are further defined in the following Examples. It should be understood that these Examples are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0124] All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

Example 1

Synthetic Coding Region Encoding Bacillus thuringiensis Cry1Fa Core Toxin

[0125] Comparative Sequence. The native DNA sequence encoding the Cry1Fa core toxin is given in SEQ ID NO: 1. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO: 1 and their locations. The amino acid sequence encoded by SEQ ID NO: 1 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction sites, and restore sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO: 1 was preserved. The resulting DNA sequence is given in SEQ ID NO:3.

[0126] SEQ ID NO:3 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the same number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:5. Table 5 shows that the number and locations of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:5, with the exception that the two occurrences of AATAAA, one at nt 426 and one at nt 582, in SEQ ID NO:1 were replaced with AATCAA, which maintains the number and location of polyadenylation signal sequences identified in Table 1, but substitutes a less problematic sequence for each of the two AATAAA sequences. Table 6 shows that the number of polyadenylation signal sequences identified in Table 2 are reduced in SEQ ID NO:5. Because there is overlap in the sequences identified in Tables 2 and 3 (sequences 1, 2, 6, 7, 8, 9, 10, 14, 13, and 20 in Table 2 correspond to sequences 16, 15, 2, 5, 1, 3, 4, 6, 13, and 12, respectively, in Table 3) it is also true that the number of polyadenylation signal sequences identified in Table 3 are reduced in SEQ ID NO:5.

[0127] The synthetic coding region of SEQ ID NO:5 was optimized for expression in maize.

[0128] A construct for use in expressing the synthetic coding region of SEQ ID NO:5 is made by combining the synthetic coding region of SEQ ID NO:5 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription termination and polyadenylation sequence.

[0129] In one embodiment of such a construct, production of the primary mRNA transcript comprising SEQ ID NO:5 was driven by a copy of a maize ubiquitin1 promoter with its native intron1 (U.S. Pat. No. 5,510,474). A fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (ZmPer5 3'UTR v2; U.S. Pat. No. 6,699,984) was used to terminate transcription. A binary plant transformation plasmid, pDAB1 11440, containing the aforementioned gene expression cassette, was constructed and utilized in the production of transgenic maize plants. Plasmid pDAB1 11440 further comprises a herbicide resistance gene comprising a coding region for aryloxyalknoate dioxygenase (AAD-1 v3; U.S. Pat. No. 7,838,733(82), and Wright et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:20240-5) under the transcriptional control of a sugarcane bacilliform badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol. 39:1221-30). A fragment comprising a 3' untranslated region from a maize lipase gene (ZmLip 3'UTR; U.S. Pat. No. 7,179,902) was used to terminate transcription.

TABLE-US-00005 TABLE 5 Table 1 sequences found in the native Cry1Fa core toxin coding region (SEQ ID NO: 1) and in the redesigned version (SEQ ID NO: 5) No. Sites in nt Location in No. Sites in nt Location in Native Cry1Fa Native Cry1Fa redesigned Cry1Fa redesigned Cry1Fa Table 1 core sequence core sequence core sequence core sequence Sequence (SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 5) (SEQ ID NO: 5) 1 AATAAA 2 426; 582 0 NA* 2 AATAAT 5 7; 46; 358; 5 7; 46; 358; 430; 430; 562 562 3 AACCAA 0 NA 0 NA 4 ATATAA 1 1520 1 1520 5 AATCAA 2 19; 628 4 19; 426; 582; 628 6 ATACTA 1 1508 1 1508 7 ATAAAA 0 NA 0 NA 8 ATGAAA 2 314; 1211 2 314; 1211 9 AAGCAT 0 NA 0 NA 10 ATTAAT 2 579; 1690 2 579; 1690 11 ATACAT 0 NA 0 NA 12 AAAATA 0 NA 0 NA 13 ATTAAA 2 66; 1266 2 66; 1266 14 AATTAA 2 368; 779 2 368; 779 15 AATACA 3 400; 1369; 1693 3 400; 1369; 1693 16 CATAAA 0 NA 0 NA Total 22 22 *NA = Not Applicable

TABLE-US-00006 TABLE 6 Table 2 sequences found in the native Cry1Fa core toxin coding region (SEQ ID NO: 1) and in the redesigned version (SEQ ID NO: 2) No. Sites in nt Location in No. Sites in nt Location in Native Cry1Fa Native Cry1Fa redesigned Cry1Fa redesigned Cry1Fa Table 2 core sequence core sequence core Sequence core Sequence Sequence (SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 5) (SEQ ID NO: 5) 1 ATATAT 1 104 0 NA* 2 TTGTTT 3 39; 612; 907 0 NA 3 TTTTGT 1 1089 0 NA 4 TGTTTT 2 1086; 1334 0 NA 5 TATATA 1 1771 0 NA 6 TATTTT 0 NA 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 1 1615 0 NA 9 TTATTT 2 172; 217 0 NA 10 TTTATT 0 NA 0 NA 11 TAATAA 4 357; 416; 561; 581 0 NA 12 ATTTAT 3 319; 497; 793 0 NA 13 TATATT 1 322 0 NA 14 TTTTAT 3 192; 464; 1063 0 NA 15 ATATTT 0 NA 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 2 613; 908 0 NA 18 TTATAT 2 321; 1770 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 2 45; 429 0 NA Total 28 0 NA *NA = Not Applicable

Example 2

Synthetic Coding Region Encoding Bacillus thuringiensis Cry34A Toxin

[0130] Comparative Sequences. The native DNA sequence encoding the Cry34A toxin is given in SEQ ID NO:7. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:7 and their locations. The native DNA sequence was translated into the corresponding amino acid sequence using the standard genetic code. The amino acid sequence encoded by SEQ ID NO:7 was then reverse translated using the target codon frequencies given in the column of Table 7 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction sites, and restore all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:7 was preserved. The resulting DNA sequence is given in SEQ ID NO:9. DNA having the sequence of SEQ ID NO:9 is synthesized.

[0131] SEQ ID NO:9 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the same number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO: 11. Table 7 shows that the number and locations of polyadenylation signals sequences identified in Table 1 are maintained in SEQ ID NO: 11. Table 8 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:5.

[0132] DNA of SEQ ID NO:5 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:1 and SEQ ID NO:3.

[0133] The synthetic coding region of SEQ ID NO:5 was optimized for expression in maize.

[0134] A construct for use in expressing the synthetic coding region of SEQ ID NO:5 is made by combining the synthetic coding region of SEQ ID NO:5 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription termination and polyadenylation sequence.

TABLE-US-00007 TABLE 7 Table 1 sequences found in the native Cry34Ab1 coding region (SEQ ID NO: 7) and the redesigned version (SEQ ID NO: 11) nt Location No. Sites nt Location No. Sites in native in native in redesigned in redesigned Table 1 Cry34Ab1 sequence Cry34Ab1 sequence Cry34Ab1 sequence Cry34Ab1 sequence Sequence (SEQ ID NO: 7) (SEQ ID NO: 7) (SEQ ID NO: 11) (SEQ ID NO: 11) 1 AATAAA 2 247; 268 2 247; 268 2 AATAAT 1 31 1 31 3 AACCAA 0 NA* 0 NA 4 ATATAA 0 NA 0 NA 5 AATCAA 2 146; 310 2 146; 310 6 ATACTA 1 329 1 329 7 ATAAAA 1 65 1 65 8 ATGAAA 1 281 1 281 9 AAGCAT 0 NA 0 NA 10 ATTAAT 0 NA 0 NA 11 ATACAT 1 47 1 47 12 AAAATA 0 NA 0 NA 13 ATTAAA 1 127 1 127 14 AATTAA 1 126 1 126 15 AATACA 0 NA 0 NA 16 CATAAA 1 361 1 361 Total 12 12 *NA = Not Applicable

TABLE-US-00008 TABLE 8 Table 2 sequences found in the native Cry34Ab1 coding region (SEQ ID NO: 7) and in the redesigned version (SEQ ID NO: 11) nt Location No. Sites nt Location No. Sites in native in native in redesigned in redesigned Table 2 Cry34Ab1 sequence Cry34Ab1 sequence Cry34Ab1 sequence Cry34Ab1 sequence Sequence (SEQ ID NO: 7) (SEQ ID NO: 7) (SEQ ID NO: 11) (SEQ ID NO: 11) 1 ATATAT 1 181 0 NA* 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 1 180 0 NA 6 TATTTT 1 220 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 0 NA 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 0 NA 0 NA 11 TAATAA 2 33; 246 2 33; 246 12 ATTTAT 0 NA 0 NA 13 TATATT 2 182; 218 0 NA 14 TTTTAT 1 159 0 NA 15 ATATTT 1 219 0 NA 16 TATTAT 1 184 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 1 217 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 1 30 1 30 Total 12 3 *NA = Not Applicable

Example 3

Synthetic Coding Region Encoding Bacillus thuringiensis Cry35Ab1 Toxin

[0135] Comparative Sequences. The native DNA sequence encoding the Cry35Ab1 toxin is given in SEQ ID NO: 13. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO: 13 and their locations. The amino acid sequence encoded by SEQ ID NO: 13 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO: 13 was preserved. The resulting DNA sequence is given in SEQ ID NO: 15. This sequence will be synthesized and used for comparison with a synthetic coding region designed in accordance with the invention.

[0136] SEQ ID NO:15 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the same number of sequences identified in Table 1, except that two of the occurrences of AATAAA, one at nt 228 and one at nt 276 of SEQ ID NO:8 were changed to AATCAA. The resulting sequence, which embodies the present invention, is given in SEQ ID NO: 17. Table 9 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO: 17. Table 10 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:17 compared to SEQ ID NO:13.

[0137] DNA of SEQ ID NO:17 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:13 and SEQ ID NO:15.

[0138] The synthetic coding region of SEQ ID NO: 17 was optimized for expression in maize.

[0139] A construct for use in expressing the synthetic coding region of SEQ ID NO: 17 is made by combining the synthetic coding region of SEQ ID NO: 17 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription termination and polyadenylation sequence.

TABLE-US-00009 TABLE 9 Table 1 sequences found in the native Cry35Ab1 coding regin (SEQ ID NO: 1) and in the redesigned version (SEQ ID NO: 17) No. Sites nt Location No. Sites nt Location in native in native in redesigned in redesigned Table 1 Cry 35Ab1 sequence Cry35Ab1 sequence Cry35Ab1 sequence Cry35Ab1 sequence Sequence (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 17) (SEQ ID NO: 17) 1 AATAAA 5 13; 100; 228; 3 13; 100; 810 276; 810 2 AATAAT 4 193; 217; 385; 4 193; 217; 385; 864 864 3 AACCAA 0 NA* 0 NA 4 ATATAA 1 966 1 966 5 AATCAA 3 394; 750; 914 5 228; 276; 394; 750; 914 6 ATACTA 1 8 1 8 7 ATAAAA 5 101; 224; 277; 5 101; 224; 277; 575; 811 575; 811 8 ATGAAA 5 23; 671; 769; 5 23; 671; 769; 806; 854 806; 854 9 AAGCAT 0 NA 0 NA 10 ATTAAT 1 522 1 522 11 ATACAT 1 734 1 734 12 AAAATA 7 226; 578; 618; 7 226; 578; 618; 838; 862; 873; 838; 862; 873; 1137 1137 13 ATTAAA 4 462; 589; 834; 4 462; 589; 834; 1131 1131 14 AATTAA 5 461; 521; 588; 5 461; 521; 588; 833; 1130 833; 1130 15 AATACA 3 261; 303; 733 3 261; 303; 733 16 CATAAA 0 NA 0 NA Total 45 45 *NA = Not Applicable

TABLE-US-00010 TABLE 10 Table 2 sequences found in the native Cry35Ab1 coding region (SEQ ID NO: 13) and in the redesigned version (SEQ ID NO: 17) No. Sites nt Location No. Sites nt Location in native in native in redesigned in redesigned Table 2 Cry 35Ab1 sequence Cry35Ab1 sequence Cry35Ab1 sequence Cry35Ab1 sequence Sequence (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 17) (SEQ ID NO: 17) 1 ATATAT 1 168 0 NA* 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 1 959 0 NA 6 TATTTT 2 609; 1144 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 1 1145 0 NA 9 TTATTT 3 63; 145; 1143 1 1143 10 TTTATT 2 144; 1056 0 NA 11 TAATAA 2 12; 216 1 12 12 ATTTAT 0 NA 0 NA 13 TATATT 2 169; 607 0 NA 14 TTTTAT 1 143 0 NA 15 ATATTT 1 608 0 NA 16 TATTAT 4 171; 549; 604; 1 1141 1141 17 TGTTTG 0 NA 0 NA 18 TTATAT 1 606; 958 0 NA 19 TGTAAT 1 300 0 NA 20 AAATAA 8 26; 192; 227; 275; 2 809; 863 384; 809; 863; 1097 Total 31 5 *NA = Not Applicable

Example 4

Synthetic Coding Region Encoding Bacillus thuringiensis Cry1Ab Core Toxin

[0140] Comparative Sequences. The native DNA sequence encoding Cry1Ab core toxin is given in SEQ ID NO: 19. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO: 19 and their locations. The amino acid sequence encoded by SEQ ID NO:19 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:19 was preserved. The resulting DNA sequence is given in SEQ ID NO:21.

[0141] SEQ ID NO:21 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the same number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:23. Table 11 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:23. Table 12 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:23 compared to SEQ ID NO:19.

[0142] The synthetic coding region of SEQ ID NO:23 was optimized for expression in maize.

[0143] A construct for use in expressing the synthetic coding region of SEQ ID NO:23 was made by combining the synthetic coding region of SEQ ID NO:23 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription termination and polyadenylation sequence.

[0144] In one embodiment of such a construct, production of the primary mRNA transcript comprising SEQ ID NO:23 was driven by a copy of a maize ubiquitin1 promoter with its native intron1 (U.S. Pat. No. 5,510,474). A fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (ZmPer5 3'UTR v2; U.S. Pat. No. 6,699,984) was used to terminate transcription. A binary plant transformation plasmid, pDAB1 11449, containing the aforementioned gene expression cassette, was constructed and utilized in the production of transgenic maize plants. Plasmid pDAB1 11449 further comprises a herbicide resistance gene comprising a coding region for aryloxyalknoate dioxygenase (AAD-1 v3; U.S. Pat. No. 7,838,733(82), and Wright et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:20240-5) under the transcriptional control of a sugarcane bacilliform badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol. 39:1221-30). A fragment comprising a 3' untranslated region from a maize lipase gene (ZmLip 3'UTR; U.S. Pat. No. 7,179,902) was used to terminate transcription.

TABLE-US-00011 TABLE 11 Table 1 sequences found in the native Cry35Ab1 coding region (SEQ ID NO: 13) and in the redesigned version (SEQ ID NO: 17) No. Sites in nt Location in No. Sites in nt Location in Native Cry1Ab Native Cry1Ab redesigned Cry1Ab redesigned Cry1Ab Table 1 core sequence core sequence core sequence core sequence Sequence (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 17) (SEQ ID NO: 17) 1 AATAAA 0 NA* 0 NA 2 AATAAT 3 960, 1126, 1387 3 960, 1126, 1387 3 AACCAA 2 253, 280 2 253, 280 4 ATATAA 2 185, 1391 2 185, 1391 5 AATCAA 2 688, 1129 3 688, 1129, 1639 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 1 1232 1 1232 9 AAGCAT 1 NA 0 NA 10 ATTAAT 0 1636 1 1636 11 ATACAT 2 1366, 1613 2 1366, 1613 12 AAAATA 0 NA 0 NA 13 ATTAAA 3 249, 704, 785 3 249, 704, 785 14 AATTAA 0 NA 0 NA 15 AATACA 0 NA 0 NA 16 CATAAA 0 NA 0 NA Total 16 NA 17 NA *NA = Not Applicable

TABLE-US-00012 TABLE 12 Table 2 sequences found in the native Cry1Ab coding region (SEQ ID NO: 19) and in the redesigned version (SEQ ID NO: 23) No. Sites in nt Location in No. Sites in nt Location in Native Cry1Ab Native Cry1Ab redesigned Cry1Ab redesigned Cry1Ab Table 2 core sequence core sequence core sequence core sequence Sequence (SEQ ID NO: 19) (SEQ ID NO: 19) (SEQ ID NO: 23) (SEQ ID NO: 23) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 1 42 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 2 1097, 1792 0 NA 6 TATTTT 0 NA 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 2 199, 1649 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 1 470 0 NA 11 TAATAA 2 1340, 1386 0 NA 12 ATTTAT 2 503, 799 0 NA 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 1 110 0 NA 16 TATTAT 2 937, 940 0 NA 17 TGTTTG 1 530 0 NA 18 TTATAT 2 1096, 1791 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 2 959, 1125 1 959 Total 18 1 *NA = Not Applicable

EXAMPLES

Synthetic Coding Region Encoding Bacillus thuringiensis CryICa Core Toxin

[0145] Comparative Sequences. The native DNA sequence encoding the Cry35A core toxin is given in SEQ ID NO:25. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:25 and their locations. The amino acid sequence encoded by SEQ ID NO:25 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames, and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:25 was preserved. The resulting DNA sequence is given in SEQ ID NO:27. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0146] SEQ ID NO:27 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the same number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:29. Table 13 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:29. Table 14 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:29 compared to SEQ ID NO:25.

[0147] DNA of SEQ ID NO:29 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:25 and SEQ ID NO:27.

[0148] The synthetic gene of SEQ ID NO:29 was optimized for expression in maize.

[0149] A construct for use in expressing the synthetic gene of SEQ ID NO:29 is made by combining the synthetic gene of SEQ ID NO:29 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00013 TABLE 13 Table 1 sequences found in the native Cry1Ca core toxin coding region (SEQ ID NO: 25) and in the redesigned version (SEQ ID NO: 29) No. Sites in nt Location in No. Sites in nt Location in Native Cry1Ca Native Cry1Ca redesigned Cry1Ca redesigned Cry1Ca Table 1 core sequence core sequence core sequence core sequence Sequence (SEQ ID NO: 25) (SEQ ID NO: 25) (SEQ ID NO: 29) (SEQ ID NO: 29) 1 AATAAA 0 NA* 0 NA 2 AATAAT 2 646, 916 2 646, 916 3 AACCAA 0 NA 1 1042 4 ATATAA 2 684, 1757 2 684, 1757 5 AATCAA 1 1405 1 1405 6 ATACTA 0 NA 0 7 ATAAAA 1 1826 1 8 ATGAAA 2 254, 569 2 9 AAGCAT 1 335 1 10 ATTAAT 7 177, 246, 250, 7 177, 246, 250, 813, 817, 1402, 813, 817, 1402, 1534 1534 11 ATACAT 0 NA 0 12 AAAATA 0 NA 0 13 ATTAAA 4 245, 249, 816, 4 245, 249, 816, 1401 1401 13 AATTAA 1 642 1 642 15 AATACA 1 1381 1 1381 16 CATAAA 0 NA 0 NA Total 22 23 *NA = Not Applicable

TABLE-US-00014 TABLE 14 Table 2 sequences found in the native CryICa core toxin coding region (SEQ ID NO: 25) and in the redesigned version (SEQ ID NO: 29) No. Sites in nt Location in No. Sites in Native nt Location in Native redesigned redesigned Cry1Ca core Cry1Ca core Cry1Ca core Cry1Ca core Table 2 sequence (SEQ ID sequence (SEQ ID sequence (SEQ sequence (SEQ Sequence NO: 25) NO: 25) ID NO: 29) ID NO: 29) 1 ATATAT 4 323, 325, 908, 1024 0 NA* 2 TTGTTT NA 0 NA 3 TTTTGT 3 186, 1302, 1512 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 3 324, 1023, 1819 0 NA 6 TATTTT 1 1346 0 NA 7 TTTTTT 1 1326 0 NA 8 ATTTTT 2 529, 959 0 NA 9 TTATTT 1 901 0 NA 10 TTTATT 2 900, 962 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 1 899 0 NA 13 TATATT 2 510, 909 0 NA 14 TTTTAT 2 470, 961 0 NA 15 ATATTT 1 110 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 1 1818 0 NA 19 TGTAAT 1 525 0 NA 20 AAATAA 1 645 1 645 Total 26 1 *NA = Not Applicable

Example 6

Synthetic Coding Region Encoding Bacillus thuringiensis Cry6Aa Toxin

[0150] Comparative Sequences. The native DNA sequence encoding the Cry6Aa toxin is given in SEQ ID NO:31. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:31 and their locations. The amino acid sequence encoded by SEQ ID NO:31 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames, and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:31 was preserved. The resulting DNA sequence is given in SEQ ID NO:33. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0151] SEQ ID NO:33 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:35. Table 15 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:35. Table 16 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:35 compared to SEQ ID NO:31.

[0152] DNA of SEQ ID NO:35 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:31 and SEQ ID NO:33.

[0153] The synthetic coding region of SEQ ID NO:35 was optimized for expression in maize.

[0154] A construct for use in expressing the synthetic coding region of SEQ ID NO:35 is made by combining the synthetic coding region of SEQ ID NO:35 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00015 TABLE 15 Table 1 sequences found in the native Cry6Aa coding region (SEQ ID NO: 31) and in the redesigned version (SEQ ID NO: 35) No. Sites in No. Sites in nt Location in Native Cry6Aa nt Location in Native redesigned redesigned Cry6Aa Table 1 sequence (SEQ Cry6Aa sequence Cry6Aa sequence sequence (SEQ ID Sequence ID NO: 31) (SEQ ID NO: 31) (SEQ ID NO: 35) NO: 35) 1 AATAAA 1 292 1 292 2 AATAAT 6 430, 1309, 1360, 1384, 6 430, 1309, 1360, 1384, 1402, 1420 1402, 1420 3 AACCAA 0 NA* 0 NA 4 ATATAA 2 824, 1344 2 824, 1344 5 AATCAA 5 103, 634, 832, 1234, 5 103, 634, 832, 1234, 1270 1270 6 ATACTA 0 NA 0 NA 7 ATAAAA 3 269, 293, 826 3 269, 293, 826 8 ATGAAA 1 794 1 794 9 AAGCAT 0 NA 0 NA 10 ATTAAT 2 919, 1183 2 919, 1183 11 ATACAT 0 NA 1 1275 12 AAAATA 3 530, 806, 1358 3 530, 806, 1358 13 ATTAAA 5 51, 56, 188, 495, 963 5 51, 56, 188, 495, 963 13 AATTAA 7 52, 57, 316, 463, 496, 7 52, 57, 316, 463, 496, 718, 964 718, 964 15 AATACA 2 922, 1238 3 922, 1238, 1274 16 CATAAA 1 664 1 664 Total 38 40 *NA = Not Applicable

TABLE-US-00016 TABLE 16 Table 2 sequences found in the native Cry6Aa coding region (SEQ ID NO: 31) and in the redesigned version (SEQ ID NO: 35) nt Location in No. Sites in nt Location in No. Sites in redesigned Native Cry6Aa Native Cry6Aa redesigned Cry6Aa Table 2 sequence (SEQ sequence (SEQ ID Cry6Aa sequence sequence (SEQ Sequence ID NO: 31) NO: 31) (SEQ ID NO: 35) ID NO: 35) 1 ATATAT 4 147, 218, 1275, 1372 0 NA* 2 TTGTTT 1 788 0 NA 3 TTTTGT NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 1 941 0 NA 6 TATTTT 2 388, 489 0 NA 7 TTTTTT NA 0 NA 8 ATTTTT 2 236, 555 0 NA 9 TTATTT 1 113 0 NA 10 TTTATT 1 109, 257 0 NA 11 TAATAA 5 66, 429, 1383, 1401, 0 NA 1419 12 ATTTAT 3 108, 299, 938 0 NA 13 TATATT 2 148, 1373 0 NA 14 TTTTAT 2 1314, 1365 0 NA 15 ATATTT 1 387 0 NA 16 TATTAT 1 111 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 4 247, 301, 940, 1190 0 NA 19 TGTAAT 1 1204 0 NA 20 AAATAA 2 1308, 1359 1 1359 Total 33 1 *NA = Not Applicable

Example 7

Synthetic Coding Region Encoding Sphingobiurn herbicidovorans AAD1

[0155] Comparative Sequences. The native DNA sequence encoding the AAD1 protein is given in SEQ ID NO:37. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:37 and their locations. The amino acid sequence encoded by SEQ ID NO:37 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames, and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:37 was preserved. The resulting DNA sequence is given in SEQ ID NO:39. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0156] SEQ ID NO:39 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:41. Table 17 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:41. Table 18 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:41 compared to SEQ ID NO:37.

[0157] DNA of SEQ ID NO:41 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:37 and SEQ ID NO:39.

[0158] The synthetic coding region of SEQ ID NO:41 was optimized for expression in maize.

[0159] A construct for use in expressing the synthetic coding region of SEQ ID NO:41 is made by combining the synthetic coding region of SEQ ID NO:41 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00017 TABLE 17 Table 1 sequences found in the native AADI coding region (SEQ ID NO: 37) and in the redesigned version (SEQ ID NO: 41) No. Sites in Nt Location in No. Sites in nt Location in NativeAAD1 NativeAAD1 redesigned redesigned AAD1 Table 1 sequence (SEQ sequence (SEQ ID AAD1 sequence sequence (SEQ ID Sequence ID NO: 37) NO: 37) (SEQ ID NO: 41) NO: 41) 1 AATAAA 0 NA* 0 NA 2 AATAAT 0 NA 0 NA 3 AACCAA 0 NA 1 652 4 ATATAA 0 NA 0 NA 5 AATCAA 0 NA 0 NA 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 0 NA 0 NA 9 AAGCAT 0 NA 0 NA 10 ATTAAT 0 NA 0 NA 11 ATACAT 0 NA 0 NA 12 AAAATA 0 NA 0 NA 13 ATTAAA 0 NA 0 NA 14 AATTAA 0 NA 0 NA 15 AATACA 0 NA 0 NA 16 CATAAA 0 NA 0 NA Total 0 1 *NA = Not Applicable

TABLE-US-00018 TABLE 18 Table 2 sequences found in the native AADI coding region (SEQ ID NO: 37) and in the redesigned version (SEQ ID NO: 41) No. Sites in nt Location in No. Sites in nt Location in NativeAAD1 NativeAAD1 redesigned redesigned AAD1 Table 2 sequence (SEQ sequence (SEQ ID AAD1 sequence sequence (SEQ ID Sequence ID NO: 37) NO: 37) (SEQ. ID NO: 41) NO: 41) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 0 NA 0 NA 3 TTTATT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 0 NA 0 NA 6 TATTTT 1 166 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 0 NA 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 0 NA 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 0 NA 0 NA 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 0 NA 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 0 NA 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 0 NA 0 NA Total 1 0 *NA = Not Applicable

Example 8

Synthetic Coding Region Encoding Aspergillus nidulans Delta-9 Desaturase

[0160] Comparative Sequences. The native DNA sequence encoding the Aspergillus nidulans Delta-9 Desaturase protein is given in SEQ ID NO:43. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:43 and their locations. The amino acid sequence encoded by SEQ ID NO:43 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:43 was preserved. The resulting DNA sequence is given in SEQ ID NO:45. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0161] SEQ ID NO:45 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:47. Table 1 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:47. Table 20 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:47 compared to SEQ ID NO:43.

[0162] DNA of SEQ ID NO:47 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:43 and SEQ ID NO:45.

[0163] The synthetic coding region of SEQ ID NO:47 was optimized for expression in maize.

[0164] A construct for use in expressing the synthetic coding region of SEQ ID NO:47 is made by combining the synthetic coding region of SEQ ID NO:47 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription termination and polyadenylation sequence.

TABLE-US-00019 TABLE 19 Table 1 sequences found in the native Aspergillus nidulans Delta-9 Desaturase coding region (SEQ ID NO: 43) and in the redesigned version (SEQ ID NO: 47) No. Sites in nt Location in No. Sites in nt Location in Native Asp-L19 Native Asp-L19 redesigned Asp- redesigned Asp- Table 1 sequence (SEQ sequence (SEQ ID L19 sequence (SEQ L19 Sequence (SEQ Sequence ID NO: 43) NO: 43) ID NO: 47) ID NO: 47) 1 AATAAA 0 NA* 0 NA 2 AATAAT 0 NA 0 NA 3 AACCAA 1 1326 1 1326 4 ATATAA 0 NA 0 NA 5 AATCAA 0 NA 0 NA 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 0 NA 0 NA 9 AAGCAT 1 94 1 94 10 ATTAAT 0 NA 0 NA 11 ATACAT 0 NA 0 NA 12 AAAATA 0 NA 0 NA 13 ATTAAA 0 NA 0 NA 14 AATTAA 0 NA 0 NA 15 AATACA 0 NA 0 NA 16 CATAAA 0 NA 0 NA Total 2 2 *NA = Not Applicable

TABLE-US-00020 TABLE 20 Table 2 sequences found in the native Aspergillus nidulans Delta-9 Desaturase coding region (SEQ ID NO: 43) and in the redesigned version (SEQ ID NO: 47) nt Location in No. Sites in nt Location in No. Sites in Native Native Asp-L19 redesigned Asp- redesigned Asp-L19 Table 2 Asp-L19 sequence Sequence (SEQ L19 Sequence Sequence (SEQ ID Sequence (SEQ ID NO 43) ID NO: 43) (SEQ ID NO: 47) NO: 47) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 0 NA 0 NA 6 TATTTT 1 166 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 0 NA 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 0 NA 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 0 NA 0 NA 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 1 479 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 0 NA 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 0 NA 0 NA Total 1 0 *NA = Not Applicable

Example 9

Synthetic Coding Region Encoding Xerophyta viscosa SAP1

[0165] Comparative Sequences. The native DNA sequence encoding the Xerophyta viscosa SAPI protein is given in SEQ ID NO:49. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:49 and their locations. The amino acid sequence encoded by SEQ ID NO:49 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:49 was preserved. The resulting DNA sequence is given in SEQ ID NO:51. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0166] SEQ ID NO:52 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:53. Table 1 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:53. Table 21 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:53 compared to SEQ ID NO:49.

[0167] DNA of SEQ ID NO:53 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:49 and SEQ ID NO:51.

[0168] The synthetic coding region of SEQ ID NO:53 was optimized for expression in maize.

[0169] A construct for use in expressing the synthetic coding region of SEQ ID NO:53 is made by combining the synthetic coding region of SEQ ID NO:53 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00021 TABLE 21 Table 1 sequences found in the native Xerophyta viscosa SAPI coding region (SEQ ID NO: 49) and in the redesigned version (SEQ ID NO: 53) No. Sites in Native nt Location in No. Sites in nt Location in XvSAP1 Native XvSAP1 redesigned redesigned Table 1 sequence (SEQ ID sequence (SEQ ID XvSAP1 sequence XvSAP1 sequence Sequence NO: 49) NO: 49) (SEQ ID NO: 53) (SEQ ID NO: 53) 1 AATAAA 0 NA* 0 NA 2 AATAAT 0 NA 0 NA 3 AACCAA 0 NA 0 NA 4 ATATAA 0 NA 0 NA 5 AATCAA 0 NA 0 NA 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 0 NA 1 25 9 AAGCAT 0 NA 0 NA 10 ATTAAT 0 NA 0 NA 11 ATACAT 0 NA 0 NA 12 AAAATA 0 NA 0 NA 13 ATTAAA 0 NA 0 NA 14 AATTAA 0 NA 0 NA 15 AATACA 0 NA 0 NA 16 CATAAA 0 NA 0 NA Total 0 1 *NA = Not Applicable

TABLE-US-00022 TABLE 22 Table 2 sequences found in native the Native Xerophyta viscosa SAPI coding region (SEQ ID NO: 49) and in the redesigned version (SEQ ID NO: 53) No. Sites in Native nt Location in No. Sites in nt Location in XvSAP1 Native XvSAP1 redesigned XvSAP1 redesigned Table 2 sequence (SEQ ID sequence (SEQ sequence (SEQ ID XvSAP1 sequence Sequence NO: 49) ID NO: 49) NO: 53) (SEQ ID NO: 53) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 0 NA 0 NA 6 TATTTT 1 755 0 NA 7 TTTTTT 0 NA 0 NA 8 AITTTT 1 756 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 0 NA 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 0 NA 0 NA 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 1 754 0 NA 16 TATTAT 1 665 0 NA 17 TGTTTG 1 696 0 NA 18 TTATAT 0 NA 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 0 NA 0 NA Total 5 0 *NA = Not Applicable

Example 10

Synthetic Coding Region Encoding Aequorea victoria GFP 1

[0170] Comparative Sequences. The native DNA sequence encoding the Aequorea victoria GFPI is given in SEQ ID NO:55. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:55 and their locations. The amino acid sequence encoded by SEQ ID NO:55 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:55 was preserved. The resulting DNA sequence is given in SEQ ID NO:57. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0171] SEQ ID NO:57 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:59. Table 1 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:59. Table 23 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:59 compared to SEQ ID NO:55.

[0172] DNA of SEQ ID NO:59 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:55 and SEQ ID NO:57.

[0173] The synthetic coding region of SEQ ID NO:59 was optimized for expression in maize.

[0174] A construct for use in expressing the synthetic coding region of SEQ ID NO:59 is made by combining the synthetic coding region of SEQ ID NO:59 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00023 TABLE 23 Table 1 sequences found in the native Aequorea victoria GFP1 coding region (SEQ ID NO: 55) and in the redesigned version (SEQ ID NO: 59) nt Location in No. Sites in nt Location in No. Sites in Native Native GFP1 redesigned redesigned GFP1 Table 1 GFP1 sequence sequence (SEQ ID GFP1 sequence sequence (SEQ ID Sequence (SEQ ID NO: 55) NO: 55) (SEQ ID NO: 59) NO: 59) 1 AATAAA 0 NA* 0 NA 2 AATAAT 0 NA 0 NA 3 AACCAA 1 467 1 467 4 ATATAA 0 NA 0 NA 5 AATCAA 0 NA 0 NA 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 1 237 1 237 9 AAGCAT 0 NA 0 NA 10 ATTAAT 0 NA 0 NA 11 ATACAT 1 450 1 450 12 AAAATA 1 551 1 551 13 ATTAAA 1 511 1 511 14 AATTAA 0 NA 0 NA 15 AATACA 1 425 1 425 16 CATAAA 0 NA 1 480 Total 6 7 *NA = Not Applicable

TABLE-US-00024 TABLE 24 Table 2 sequences found in the native the Aequorea victoria GFPI coding region (SEQ ID NO: 55) and in the redesigned version (SEQ ID NO: 59) No. Sites in nt Location in No. Sites in nt Location in Native GFP1 Native GFP1 redesigned GFP1 redesigned GFP1 Table 2 sequence (SEQ sequence (SEQ sequence (SEQ ID sequence (SEQ ID Sequence ID NO: 55) ID NO: 55) NO: 59) NO: 59) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 0 NA 0 NA 6 TATTTT 1 293 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 0 NA 0 NA 9 TTATTT 1 137 0 NA 10 TTTATT 1 136 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 0 NA 0 NA 13 TATATT 1 291 0 NA 14 TTTTAT 1 135 0 NA 15 ATATTT 1 292 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 0 NA 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 0 NA 0 NA Total 6 0 *NA = Not Applicable

Example 11

Synthetic Coding Region Encoding Leptosphaeria nodorum FAD9

[0175] Comparative Sequences. The native DNA sequence encoding the Leptosphaeria nodorum FAD9 protein is given in SEQ ID NO:61. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:61 and their locations. The amino acid sequence encoded by SEQ ID NO:61 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:61 was preserved. The resulting DNA sequence is given in SEQ ID NO:63. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0176] SEQ ID NO:63 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:65. Table 1 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:65. Table 25 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:65 compared to SEQ ID NO:61.

[0177] DNA of SEQ ID NO:65 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:61 and SEQ ID NO:63.

[0178] The synthetic coding region of SEQ ID NO:65 was optimized for expression in maize.

[0179] A construct for use in expressing the synthetic coding region of SEQ ID NO:65 is made by combining the synthetic coding region of SEQ ID NO:65 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00025 TABLE 25 Table 1 sequences found in the native Leptosphaeria nodorum FAD9 coding region (SEQ ID NO: 61) and in the redesigned version (SEQ ID NO: 65) No. Sites in Native nt Location in No. Sites in nt Location in LnFAD9 Native Ln FAD9 redesigned Ln redesigned Ln Table 1 sequence (SEQ ID sequence (SEQ ID FAD9 sequence FAD9 sequence Sequence NO: 61) NO: 61) (SEQ ID NO: 65) (SEQ ID NO: 65) 1 AATAAA 0 NA* 0 NA 2 AATAAT 0 NA 0 NA 3 AACCAA 0 NA 0 NA 4 ATATAA 0 NA 0 NA 5 AATCAA 0 NA 0 NA 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 0 NA 0 NA 9 AAGCAT 0 NA 0 NA 10 ATTAAT 0 NA 0 NA 11 ATACAT 0 NA 0 NA 12 AAAATA 0 NA 0 NA 13 ATTAAA 0 NA 0 NA 14 AATTAA 0 NA 0 NA 15 AATACA 0 NA 0 NA 16 CATAAA. 0 NA 0 NA Total 0 0 *NA = Not Applicable

TABLE-US-00026 TABLE 26 Table 2 sequences found in the native Leptosphaeria nodorum FAD9 coding region (SEQ ID NO: 61) and redesigned version (SEQ ID NO: 65) No. Sites in Native nt Location in No. Sites in nt Location in LnFAD9 Native Ln FAD9 redesigned Ln FAD9 redesigned Ln Table 2 sequence (SEQ ID sequence (SEQ sequence (SEQ ID FAD9 sequence Sequence NO: 61) ID NO: 61) NO: 65) (SEQ ID NO: 65) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 1 1275 0 NA 5 TATATA 0 NA 0 NA 6 TATTTT 0 NA 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 0 NA 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 1 1090 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 0 NA 0 NA 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 0 NA 0 NA 16 TATTAT 1 416 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 0 NA 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 0 NA 0 NA Total 3 0 *NA = Not Applicable

Example 12

Synthetic Coding Region Encoding Xerophyta viscosa PERJ

[0180] Comparative Sequences. The native DNA sequence encoding the Xerophyta viscosa PERI protein is given in SEQ ID NO:67. This sequence was analyzed to determine which sequences identified in Table 1 are present in SEQ ID NO:67 and their locations. The amino acid sequence encoded by SEQ ID NO:67 was then reverse translated using the target codon frequencies given in the column of Table 4 for synthetic genes to be used in maize. The resulting DNA sequence was analyzed and codons were changed where necessary to remove unwanted open reading frames and remove unwanted restriction enzyme recognition sites, while maintaining all sequences identified in Table 1. The amino acid sequence encoded by SEQ ID NO:67 was preserved. The resulting DNA sequence is given in SEQ ID NO:69. This sequence will be synthesized and used for comparison with a synthetic gene designed in accordance with the invention.

[0181] SEQ ID NO:69 was analyzed and codons were changed to remove potential polyadenylation signal sequences identified in Table 2, while maintaining the number of sequences identified in Table 1. The resulting sequence, which embodies the present invention, is given in SEQ ID NO:71. Table 1 shows that the number and location of polyadenylation signal sequences identified in Table 1 are maintained in SEQ ID NO:71. Table 27 shows that the number of polyadenylation signal sequences identified in Tables 2 and 3 are reduced in SEQ ID NO:71 compared to SEQ ID NO:67.

[0182] DNA of SEQ ID NO:71 is synthesized, and expression levels observed in plant cells transformed to express this sequence are compared with expression levels observed in plant cells transformed to express SEQ ID NO:67 and SEQ ID NO:69.

[0183] The synthetic coding region of SEQ ID NO:71 was optimized for expression in maize.

[0184] A construct for use in expressing the synthetic coding region of SEQ ID NO:71 is made by combining the synthetic coding region of SEQ ID NO:71 with a 5' non-translated region comprising a promoter that functions in plant cells and a 3' non-translated region comprising a transcription terminator and polyadenylation sequence.

TABLE-US-00027 TABLE 27 Table 1 sequences found in the native Xerophyta viscosa PERI coding region (SEQ ID NO: 67) and in the redesigned version (SEQ ID NO: 71) No. Sites in Native nt Location in No. Sites in nt Location in XvPER1 Native XvPER1 redesigned XvPER1 redesigned Table 1 sequence (SEQ ID sequence (SEQ ID sequence (SEQ ID XvPER1 sequence Sequence NO: 67) NO: 67) NO: 71) (SEQ ID NO: 71) 1 AATAAA 0 NA* 0 NA 2 AATAAT 0 NA 0 NA 3 AACCAA 0 NA 0 NA 4 ATATAA 0 NA 0 NA 5 AATCAA 0 NA 0 NA 6 ATACTA 0 NA 0 NA 7 ATAAAA 1 605 1 605 8 ATGAAA 0 NA 0 NA 9 AAGCAT 0 NA 0 NA 10 ATTAAT 0 NA 0 NA 11 ATACAT 0 NA 0 NA 12 AAAATA 1 282 1 282 13 ATTAAA 0 NA 0 NA 14 AATTAA 0 NA 0 NA 15 AATACA 0 NA 0 NA 16 CATAAA 0 NA 0 NA Total 2 2 *NA = Not Applicable

TABLE-US-00028 TABLE 28 Table 2 sequences found in the native the Xerophyta viscosa PERI coding region (SEQ ID NO: 67 and in the redesigned version (SEQ ID NO: 71) No. Sites in No. Sites in Native nt Location in redesigned nt Location in XvPer1 Native XvPER1 XvPER1 redesigned Table 2 sequence (SEQ ID sequence (SEQ sequence (SEQ XvPER1 sequence Sequence NO: 67) ID NO: 67) ID NO: 71) (SEQ ID NO: 71) 1 ATATAT 0 NA* 0 NA 2 TTGTTT 0 NA 0 NA 3 TTTTGT 0 NA 0 NA 4 TGTTTT 0 NA 0 NA 5 TATATA 0 NA 0 NA 6 TATTTT 0 NA 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 0 NA 0 NA 9 TTATTT 0 NA 0 NA 10 TTTATT 0 NA 0 NA 11 TAATAA 0 NA 0 NA 12 ATTTAT 0 NA 0 NA 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 0 NA 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 0 NA 0 NA 18 TTATAT 0 NA 0 NA 19 TGTAAT 0 NA 0 NA 20 AAATAA 0 NA 0 NA Total 0 0 *NA = Not Applicable

Example 13

WHISKERS.RTM. Transformation of Maize with Xv SAPI

[0185] A standard WHISKERS transformation vector was constructed in which the Arabidopsis thaliana promoter. Rd29A, was placed 5' to the XvSAP1 redesigned coding region sequence of the invention (SEQ ID NO:53). These sequences were flanked by Zea maize PERS, 3' and 5' untranslated regions to stabilize expression of the redesigned coding region. Apat selection cassette (See, for example, U.S. Pat. No. 5,648,477) driven by the rice actin1 promoter was placed 3' to the XvSAP1 expression cassette.

[0186] Vector DNA was digested with appropriate restriction enzymes to release a fragment containing the bacterial ampicillin resistance gene present in the vector backbone, and to produce a linear DNA fragment suitable for WHISKERS.TM.-mediated transformation. Purification of the linear fragment containing the XvSAP1 and pat expression cassettes was accomplished on a preparative scale by high pressure liquid chromatography (HPLC). This plant transformation DNA was delivered into maize Hi-II suspension cell cultures via WHISKERS.TM.-mediated transformation (essentially as described in U.S. Pat. Nos. 5,302,523 and 5,464,765; US Patent Publication No. 2008/0182332; and Petolino and Arnold (2009) (Methods Molec. Biol. 526:59-67).

[0187] Transformants were placed in selective medium after which transformed isolates were obtained over the course of approximately 8 weeks. The selection medium was an LS based medium (LS Basal medium, N6 vitamins, 1.5 mg/L 2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate; PhytoTechnologies Labr.), 30.0 gm/L sucrose, 6 mM L-proline, 1.0 mg/L AgNO.sub.3. 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) containing Bialaphos (Gold BioTechnology). The embryos were transferred to selection media containing 3 mg/L Bialaphos until embryogenic isolates were obtained. Recovered isolates were bulked up by transferring to fresh selection medium at 2-week intervals for regeneration and further analysis.

[0188] For regeneration, the cultures were transferred to "28" induction medium (MS salts and vitamins, 30 gm/L sucrose, 5 mg/L Benzylaminopurine, 0.25 mg/L 2, 4-D, 3 mg/L Bialaphos, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week under low-light conditions (14 .mu.Em-.sup.2s-.sup.1) then 1 week under high-light conditions (approximately 89 .mu.Em-.sup.2s-1)_ Tissues were subsequently transferred to "36" regeneration medium (same as induction medium except lacking plant growth regulators). When plantlets reached 3-5 cm in length, they were transferred to glass culture tubes containing SHGA medium (Schenk and Hildebrandt salts and vitamins (1972); PhytoTechnologies Labr.), 1.0 gm/L myo-inositol, 10 gm/L sucrose and 2.0 gm/L Gellan gum, pH 5.8) to allow for further growth and development of the shoot and roots. Plants were transplanted to the same soil mixture as described earlier herein and grown to flowering in the greenhouse. Controlled pollinations for seed production were conducted.

Example 14

Agrobacterium Transformation

[0189] Standard cloning methods are used in the construction of binary plant transformation and expression plasmids. Restriction endonucleases and T4 DNA Ligase are obtained from NEB. Plasmid preparations are performed using the NucleoSpin.RTM. Plasmid Preparation kit or the NucleoBond.RTM. AX Xtra Midi kit (both from Macherey-Nagel), following the instructions of the manufacturers. DNA fragments are purified using the QIAquick.RTM. PCR Purification Kit or the QIAEX II.RTM. Gel Extraction Kit (both from Qiagen) after gel isolation.

[0190] Synthetic genes in accordance with the invention may be synthesized by a commercial vendor (e.g. DNA2.0, Menlo Park, Calif.) and supplied as cloned fragments in standard plasmid vectors, or may be obtained by standard molecular biology manipulation of other constructs containing appropriate nucleotide sequences.

[0191] In a non-limiting example, a basic cloning strategy may be to subclone full length coding sequences (CDS) into a plant expression plasmid at Neal and Sac! restriction sites. The resulting plant expression cassettes containing the appropriate coding region under the control of plant expression elements, (e.g., plant expressible promoters, 3' terminal transcription termination and polyadenylate addition determinants, and the like) are subcloned into a binary vector plasmid, utilizing, for example, Gateway.RTM. technology or standard restriction enzyme fragment cloning procedures. LR Clonase.TM. (Invitrogen) for example, may be used to recombine the full length and modified gene plant expression cassettes into a binary plant transformation plasmid if the Gateway.RTM. technology is utilized. It is convenient to employ a binary plant transformation vector that harbors a bacterial gene that confers resistance to the antibiotic spectinomycin when the plasmid is present in E. coli and Agrobacterium cells. It is also convenient to employ a binary vector plasmid that contains a plant-expressible selectable marker gene that is functional in the desired host plants. Examples of plant-expressible selectable marker genes include but are not limited those that encode the aminoglycoside phosphotransferase gene (aph!I) of transposon Tn5, which confers resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which code for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos), imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorosulfuron, bromoxynil, dalapon and the like.

[0192] Electro-competent cells of Agrobacterium tumefacicens strain Z707S (a streptomycin-resistant derivative of Z707; Hepburn et al., 1985, J. Gen. Microbiol. 131:2961-2969.) are prepared and transformed using electroporation (Weigel and Glazebrook, 2002, Arabidopsis: A Laboratory Manual). After electroporation, 1 mL of YEP broth (gm/L: yeast extract, 10; peptone, 10; NaCl, 5) are added to the cuvette and the cell-YEP suspension is transferred to a 15 mL culture tube for incubation at 28.degree. in a water bath with constant agitation for 4 hours. The cells are plated on YEP plus agar (25 gm/L) with spectinomycin (200 .mu.g/mL) and streptomycin (250 .mu.g/mL) and the plates are incubated for 2-4 days at 280. Well separated single colonies are selected and streaked onto fresh YEP+agar plates with spectinomycin and streptomycin as before, and incubated at 28.degree. for 1-3 days.

[0193] The presence of the synthetic gene insert in the binary plant transformation vector is performed by PCR analysis using vector-specific primers with template plasmid DNA prepared from selected Agrobacterium colonies. The cell pellet from a 4 mL aliquot of a 15 mL overnight culture grown in YEP with spectinomycin and streptomycin as before is extracted using Qiagen Spin.RTM. Mini Preps, performed per manufacturer's instructions. Plasmid DNA from the binary vector used in the Agrobacterium electroporation transformation is included as a control. The PCR reaction is completed using Taq DNA polymerase from Invitrogen per manufacture's instructions at 0.5.times. concentrations. PCR reactions are carried out in a MJ Research Peltier Thermal Cycler programmed with the following conditions: Step 1) 940 for 3 minutes; Step 2) 94.degree. for 45 seconds; Step 3) 55.degree. for 30 seconds; Step 4) 72.degree. for 1 minute per kb of expected product length; Step 5) 29 times to Step 2; Step 6) 72.degree. for 10 minutes. The reaction is maintained at 40 after cycling. The amplification products are analyzed by agarose gel electrophoresis (e.g. 0.7% to 1% agarose, w/v) and visualized by ethidium bromide staining. A colony is selected whose PCR product is identical to the plasmid control.

[0194] Alternatively, the plasmid structure of the binary plant transformation vector containing the synthetic gene insert is performed by restriction digest fingerprint mapping of plasmid DNA prepared from candidate Agrobacterium isolates by standard molecular biology methods well known to those skilled in the art of Agrobacterium manipulation.

[0195] Those skilled in the art of obtaining transformed plants via Agrobacterium-mediated transformation methods will understand that other Agrobacterium strains besides Z707S may be used to advantage, and the choice of strain may depend upon the identity of the host plant species to be transformed.

Example 15

Production of Insecticidal Proteins in Dicot Plants

[0196] Arabidopsis Transformation.

[0197] Arabidopsis thaliana Col-01 is transformed using the floral dip method (Weigel and Glazebrook, supra). The selected Agrobacterium colony is used to inoculate 1 mL to 15 mL cultures of YEP broth containing appropriate antibiotics for selection. The culture is incubated overnight at 28.degree. with constant agitation at 220 rpm. Each culture is used to inoculate two 500 mL cultures of YEP broth containing appropriate antibiotics for selection and the new cultures are incubated overnight at 280 with constant agitation. The cells are pelleted at approximately 8700.times.g for 10 minutes at room temperature, and the resulting supernatant is discarded. The cell pellet is gently resuspended in 500 mL of infiltration media containing: 1/2.times. Murashige and Skoog salts (Sigma-Aldrich)/Gamborg's BS vitamins (Gold BioTechnology. St. Louis, Mo.), 10% (w/v) sucrose, 0.044 .mu.M benzylaminopurine (10 .mu.L/liter of 1 mg/mL stock in DMSO) and 300 .mu.L/liter Silwet L-77. Plants approximately 1 month old are dipped into the media for 15 seconds, with care taken to assure submergence of the newest inflorescence. The plants are then laid on their sides and covered (transparent or opaque) for 24 hours, washed with water, and placed upright. The plants are grown at 22.degree., with a 16-hour light/8-hour dark photoperiod. Approximately 4 weeks after dipping, the seeds are harvested.

[0198] Arabidopsis Growth and Selection.

[0199] Freshly harvested T1 seed is allowed to dry for at least 7 days at room temperature in the presence of desiccant. Seed is suspended in a 0.1% agar/water (Sigma-Aldrich) solution and then stratified at 4.degree. for 2 days. To prepare for planting, Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) in 10.5 inch.times.21 inch germination trays (T.O. Plastics Inc., Clearwater, Minn.) is covered with fine vermiculite, sub-irrigated with Hoagland's solution (Hoagland and Amon, 1950) until wet, then allowed to drain for 24 hours. Stratified seed is sown onto the vermiculite and covered with humidity domes (KORD Products, Bramalea, Ontario, Canada) for 7 days. Seeds are germinated and plants are grown in a Conviron (Models CMP4030 or CMP3244; Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long day conditions (16 hours light/8 hours dark) at a light intensity of 120-150 .mu.mol/nr'sec under constant temperature (22.degree.) and humidity (40-50%). Plants are initially watered with Hoagland's solution and subsequently with deionized water to keep the soil moist but not wet.

[0200] The domes are removed 5-6 days post sowing and plants are sprayed with a chemical selection agent to kill plants germinated from nontransformed seeds. For example, if the plant expressible selectable marker gene provided by the binary plant transformation vector is a pat or bar gene (Wehrmann et al., 1996, Nat. Biotech. 14:1274-1278), transformed plants may be selected by spraying with a 1000.times. solution of Finale (5.78% glufosinate ammonium, Farnam Companies Inc., Phoenix, Ariz.). Two subsequent sprays are performed at 5-7 day intervals. Survivors (plants actively growing) are identified 7-10 days after the final spraying and transplanted into pots prepared with Sunshine Mix LP5. Transplanted plants are covered with a humidity dome for 3-4 days and placed in a Conviron under the above-mentioned growth conditions.

[0201] Those skilled in the art of dicot plant transformation will understand that other methods of selection of transformed plants are available when other plant expressible selectable marker genes (e.g. herbicide tolerance genes) are used.

[0202] Insect Bioassays of Transgenic Arabidopsis.

[0203] Transgenic Arabidopsis lines expressing Cry proteins are demonstrated to be active against sensitive insect species in artificial diet overlay assays. Protein extracted from transgenic and non-transgenic Arabidopsis lines is quantified by appropriate methods and sample volumes are adjusted to normalize protein concentration. Bioassays are conducted on artificial diet as described above. Non-transgenic Arabidopsis and/or buffer and water are included in assays as background check treatments.

Example 16

Agrobacterium Transformation for Generation of Superbinary Vectors

[0204] The Agrobacteriumn superbinary system is conveniently used for transformation of monocot plant hosts. Methodologies for constructing and validating superbinary vectors are well disclosed and incorporated herein by reference (Operating Manual for Plasmid pSB 1, Version 3.1, available from Japan Tobacco, Inc., Tokyo, Japan). Standard molecular biological and microbiological methods are used to generate superbinary plasmids. Verification/validation of the structure of the superbinary plasmid is done using methodologies as described above for binary vectors, and may be modified as suggested in the Operating Manual for Plasmid pSB1.

Example 17

Production of Insecticidal Proteins in Monocot Plants

[0205] Agrobacterium-Mediated Transformation of Maize.

[0206] Seeds from a High II F.sub.1 cross (Armstrong et al., 1991, Maize Genet. Coop. Newsletter 65:92-93) are planted into 5-gallon-pots containing a mixture of 95% Metro-Mix 360 soilless growing medium (Sun Gro Horticulture, Bellevue, Wash.) and 5% clay/loam soil. The plants are grown in a greenhouse using a combination of high pressure sodium and metal halide lamps with a 16:8 hour Light:Dark photoperiod. For obtaining immature F.sub.2 embryos for transformation, controlled sib-pollinations are performed. Immature embryos are isolated at 8-10 days post-pollination when embryos are approximately 1.0 to 2.0 mm in size.

[0207] Infection and Co-Cultivation.

[0208] Maize ears are surface sterilized by scrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, and then immersing in 20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes before being rinsed with sterile water. A suspension of Agrobacterium cells containing a superbinary vector is prepared by transferring 1-2 loops of bacteria grown on YEP solid medium containing 100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 mg/L streptomycin at 280 for 2-3 days into 5 mL of liquid infection medium (LS Basal Medium (Linsmaier and Skoog, 1965, Physiol. Plant. 18:100-127), N6 vitamins (Chu et al., 1975, Scientia Sinica 18:659-668), 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5 gm/L sucrose, 36.0 gm/L glucose, 6 mM L-proline, pH 5.2) containing 100 .mu.M acetosyringone. The solution was vortexed until a uniform suspension was achieved, and the concentration is adjusted to a final density of about 200 Klett units, using a Klett-Summerson colorimeter with a purple filter, or an optical density of approximately 0.4 at 550 nm. Immature embryos are isolated directly into a micro centrifuge tube containing 2 mL of the infection medium. The medium is removed and replaced with 1 mL of the Agrobacterium solution with a density of 200 Klett units, and the Agrobacterium and embryo solution is incubated for 5 minutes at room temperature and then transferred to co-cultivation medium (LS Basal Medium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mM L-proline, 0.85 mg/L AgNO.sub.3, 100 .mu.M acetosyringone, 3.0 gm/L Gellan gum (PhytoTechnology Laboratories., Lenexa, Kans.), pH 5.8) for 5 days at 250 under dark conditions.

[0209] After co-cultivation, the embryos are transferred to selective medium after which transformed isolates are obtained over the course of approximately 8 weeks. For selection of maize tissues transformed with a superbinary plasmid containing a plant expressible pat or bar selectable marker gene, an LS based medium (LS Basal medium, N6 vitamins, 1.5 mg/L 2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate; PhytoTechnologies Labr.), 30.0 gm/L sucrose, 6 mM L-proline, 1.0 mg/L AgNO.sub.3, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) is used with Bialaphos (Gold BioTechnology). The embryos are transferred to selection media containing 3 mg/L Bialaphos until embryogenic isolates were obtained. Recovered isolates are bulked up by transferring to fresh selection medium at 2-week intervals for regeneration and further analysis.

[0210] Those skilled in the art of maize transformation will understand that other methods of selection of transformed plants are available when other plant expressible selectable marker genes (e.g. herbicide tolerance genes) are used.

[0211] Regeneration and Seed Production.

[0212] For regeneration, the cultures are transferred to "28" induction medium (MS salts and vitamins, 30 gm/L sucrose, 5 mg/L Benzylaminopurine, 0.25 mg/L 2, 4-D, 3 mg/L Bialaphos, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week under low-light conditions (14 .mu.Em-.sup.2s-.sup.1) then 1 week under high-light conditions (approximately 89 .mu.Em-.sup.2s-1).sub.-- Tissues are subsequently transferred to "36" regeneration medium (same as induction medium except lacking plant growth regulators). When plantlets grow to 3-5 cm in length, they were transferred to glass culture tubes containing SHGA medium (Schenk and Hildebrandt salts and vitamins (1972); PhytoTechnologies Labr.), 1.0 gm/L myo-inositol, 10 gm/L sucrose and 2.0 gm/L Gellan gum, pH 5.8) to allow for further growth and development of the shoot and roots. Plants are transplanted to the same soil mixture as described earlier herein and grown to flowering in the greenhouse. Controlled pollinations for seed production are conducted.

[0213] Alternatively, binary vectors may be used to produce transgenic maize plants that contain one or more chimeric genes stably-integrated into the plant genome and comprising a coding region disclosed herein. For example, plants comprising at least one coding region of SEQ ID NOs:5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, or 71 are produced following Agrobacterium-mediated transformation. Maize transformation methods employing binary transformation vectors are known in the art. In one embodiment, transformed tissues are selected by their ability to grow on haloxyfop-containing medium and are screened for protein production, as appropriate.

[0214] Ear Sterilization and Embryo Isolation.

[0215] Maize immature embryos were obtained from plants of Zea mays inbred line B104 grown in the greenhouse and self- or sib-pollinated to produce ears. The ears were harvested approximately 9 to 12 days post-pollination. On the experimental day, de-husked ears were surface-sterilized by immersion in a 20% solution of sodium hypochlorite (6.15%) and shaken for 20 to 30 min, followed by three rinses in sterile water. After sterilization, immature zygotic embryos (1.5 to 2.4 mm) were aseptically dissected from each ear and randomly distributed into microcentrifuge tubes containing liquid Inoculation Medium. Inoculation Medium contains: 2.2 gm/L MS salts (Frame et al., 2011, Genetic Transformation Using Maize Immature Zygotic Embryos. IN Plant Embryo Culture Methods and Protocols: Methods in Molecular Biology. T. A. Thorpe and E. C. Yeung, (Eds), SPRINGER SCIENCE AND BUSINESS MEDIA, LLC. pp 327-341); IX ISU Modified MS Vitamins (Frame et al, 2011 supra); 68.4 gm/L sucrose; 36 gm/L glucose; 115 mg/L L-proline; 100 mg/L myo-inositol; and 200 .mu.M acetosyringone (prepared in DMSO); at pH 5.4. For a given set of experiments, embryos from pooled ears were used for each transformation.

[0216] Agrobacterium Culture Initiation.

[0217] Glycerol stocks of Agrobacterium strain DAt13192 (International PCT Publication No. WO2012016222(A2)) containing the binary transformation vector pDAB111440 (Example 1) were streaked on AB minimal medium plates (Watson, et al., (1975) J. Bacteriol. 123:255-264) containing appropriate antibiotics and were grown at 20.degree. C. for 3 to 4 days. A single colony was picked and streaked onto YEP plates (gm/L: yeast extract, 10; Peptone, 10; NaCl 5) containing the same antibiotics and was incubated at 20.degree. C. for 1-2 days.

[0218] Agrobacterium Culture and Co-Cultivation.

[0219] Agrobacterium colonies were taken from a YEP plate, suspended in 10 mL of Inoculation Medium in a 50 mL disposable tube, and the cell density was adjusted to an OO.sub.550 of 0.2 to 0.4 (Optical Density measured at 550 nm; a measure of cell growth) using a spectrophotometer. The Agrobacterium cultures were incubated on a rotary shaker at 125 rpm (room temperature) while embryo dissection was performed. Immature zygotic embryos (previously isolated from the sterilized maize kernels and placed in 1 mL of Inoculation Medium) were washed once in the same medium. Two ml of the Agrobacterium suspension was added to each tube of embryos and the tubes were placed on a shaker platform for 10 to 15 minutes. The embryos were transferred onto Co-cultivation Medium, oriented with the scutellum facing up, and incubated at 25.degree. C., under 24-hour light at 50 .mu.Em-.sup.2 sec'' light intensity for 3 days. Co-cultivation Medium, contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/l sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH (3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid); 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 100 .mu.M acetosyringone in DMSO; and 3 gm/L GELZAN.TM. (SIGMA-ALDRICH); at pH 5.8.

[0220] Callus Selection and Regeneration of Putative Events.

[0221] Following the co-cultivation period, embryos were transferred to Resting Medium and incubated under 24-hour light at 50 .mu.Em-.sup.2 sec-.sup.1 light intensity and at 25.degree. C. for 3 days. Resting Medium contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIES LABR.; Lenexa, Kans.); 250 mg/L Carbenicillin; and 2.3 gm/L GELZAN.TM.; at pH 5.8. Embryos were transferred onto Selection Medium 1 (which consists of the Resting Medium (above) with 100 nM R-Haloxyfop acid (0.0362 mg/L)), and incubated in either dark and/or under 24-hour light at 50 .mu.Em-.sup.2 sec-.sup.1 light intensity for 7 to 14 days at 28.degree. C. Proliferating embryogenic calli were transferred onto Selection Medium 2 (which consists of Resting Medium (above), with 500 nM R-Haloxyfop acid (0.1810 mg/L)), and were incubated in 24-hour light at 50 .mu.Em-.sup.2 sec-.sup.1 light intensity for 14 to 21 days at 28.degree. C. This selection step allowed transgenic callus to further proliferate and differentiate.

[0222] Proliferating, embryogenic calli were transferred onto PreRegeneration Medium and cultured under 24-hour light at 50 .mu.Em-.sup.2 sec-.sup.1 light intensity for 7 days at 28.degree. C. PreRegeneration Medium contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 45 gm/L sucrose; 350 mg/L L-proline; 100 mg/L myo-inositol; 50 mg/L Casein Enzymatic Hydrolysate; 1.0 mg/L AgNO.sub.3; 0.25 gm/L MES; 0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid in ethanol; 1 mg/L 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L GELZAN.TM.; and 500 nM R-Haloxyfop acid; at pH 5.8. Embryogenic calli with shoot-like buds were transferred onto Regeneration Medium and cultured under 24-hour light at 50 .mu.Em-.sup.2 sec'' light intensity for 7 days. Regeneration Medium I contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 60 gm/L sucrose; 100 mg/L myo-inositol; 125 mg/L Carbenicillin; 3.0 gm/L GELZAN.TM.; and 500 nM R-Haloxyfop acid; at pH 5.8. Small shoots with primary roots were transferred to Shoot/Root medium in PHYTATRAYS (PHYTOTECHNOLOGIES LABR; Lenexa, Kans.) and were incubated under 16:8 hr. light:dark at 140 to 190 .mu.Em-.sup.2 sec'' light intensity for 7 days at 27.degree. C. Shoot/Root Medium contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 100 mg/L myo-inositol; 3.5 gm/L GELZAN.TM.; at pH 5.8. Putative transgenic plantlets were analyzed for transgene copy number by quantitative real-time PCR or other standard molecular analysis techniques, and were transferred to soil.

[0223] Transfer and establishment of T.sub.0 plants in the greenhouse for seed production. Transformed plant tissues selected by their ability to grow on medium containing 500 nM R-Haloxyfop acid were transplanted into METRO-MIX 360 soilless growing medium (SUN GRO HORTICULTURE) and hardened-off in a growth room. Plants were then transplanted into SUNSHINE CUSTOM BLEND 160 soil mixture and grown to flowering in the greenhouse. Controlled pollinations for seed production are conducted.

[0224] Leaf tissues of selected T.sub.0 plants were sampled at the V-3 to V-5 stage. Two 6 mm diameter leaf samples were stored in a 96 well cluster tube rack at -80.degree. C. until the day of analysis. Two DAISY.TM. steel BB's and 200 .mu.L of extraction buffer (PBS solution containing 0.05% of Tween 20 and 5 .mu.L/ml of SIGMA protease inhibitor cocktail (catalog number 9599)) were added to each tube. The samples were milled in a KLECO bead mill (Visalia, Calif.) for 3 minutes, on maximum setting. Samples were centrifuged at 3,000.times.g for 5 minutes, then 100 .mu.L of the supernatant were transferred to an empty sample tube. Another 100 .mu.L of extraction buffer was added to the plant sample and bead-milled an 3 additional minutes. After centrifuging again, 100 .mu.L of this extract was combined with the first 100 .mu.L. The combined supernatants were mixed and analyzed on the same day as the extraction.

[0225] Proteins extracted from measured areas of leaf tissue were analyzed for expression of CryIFa protein and AAD-1 protein by standard ELISA (Enzyme-Linked Immunosorbant Assay) or protein immunoblots (western blots). For CryIFa ELSA detection, reagents from an ENVIROLOGIX ELISA kit (Cat. No. AP 016 NW VIO; Portland, Me.) were used according to the manufacturer's instructions. AAD-1 detection was performed by standard ELISA methodologies (for example, as taught in Ausubel et al. (1995 and updates) Current Protocols in Molecular Biology, John Wiley and Sons, New York) using rabbit antibodies prepared against purified AAD-1 protein.

[0226] The ELISA results obtained from extracts of pDAB1 11440-transformed plants are disclosed in Table 29. Protein levels are expressed as ng of the subject protein detected per square centimeter of leaf area harvested.

TABLE-US-00029 TABLE 29 Expression levels of Cry1Fa and AAD-1 proteins extracted from maize plants transformed with plasmid pDAB111440, as detected by ELISA methods. Sample ID Cry1Fa n2/cm.sup.2 AAD-1 n2/cm.sup.2 111440[3]-001.001 2.30 14.0 111440[3]-015.001 3.80 0.0 111440[3]-023.001 3.80 320.0 111440[3]-020.001 5.40 190.0 111440[3]-011.001 17.00 0.0

[0227] Protein extracts of the five pDAB1 11440-transformed plants listed in Table 29 (as well as extract from a non-transformed negative control plant) were prepared as above and probed with Cry1Fa antibody on immunoblots (western blots). Immunoblot procedures were essentially as described by Gallagher et al. (2008; Immunoblotting and Immunodetection. Current Protocols in Immunology 8.10.1-8.10.28). Protein samples (80 .mu.L) were mixed with 20 .mu.L of INVITROGEN NuPAGE LDS Sample Buffer, heated at >90.degree. C. for five min, loaded on an INVITROGEN NuPAGE 4-12% Bis-Tris gel, and run in MOPS SDS Running Buffer (200 Volts for 45 minutes). BIORAD PRECISION PLUS Dual Color Standards were loaded in a separate lane. Proteins were transferred to 0.2 .mu.M nitrocellulose membrane by means of an INVITROGEN iBLOT Gel Transfer system according to the manufacturer's instructions. The membrane was blocked with INVITROGEN WESTERN BREEZE BLOCKING MIX, then reacted with Primary antibody (anti-Cry1F Purified Rabbit Antibody No. D0609RA07-A0; Strategic Diagnostics Inc., Newark, Del.), followed by Secondary antibody (INVITROGEN Biotinylated goat anti-rabbit antibody.) This was followed by INVITROGEN HRP-Streptavidin conjugate and reacted bands were detected by PIERCE SUPERSIGNAL WEST PICO LUMINOL ENHANCER AND STABLE PEROXIDE (No. 34080).

[0228] Positive control lanes contained 0.5 ng or 1.0 ng of purified Cry1Fa core toxin protein produced by expression of a full length Cry1Fa coding region in a Pseudomonas j luorescens expression system (See, for example, US Patent Application No. 20100269223A1). The full-length Cry1Fa protein was trypsin treated to release the Cry1Fa core toxin segment of calculated molecular size 68 kDa, which was used as the positive control standard on the immunoblot. No antibody-reacting bands were detected in the extract from the negative control plant, while all five transgenic plant extracts contained a single predominant band (roughly equal in intensity to the control Cry1Fa proteins) of estimated size somewhat larger than 75 kDa.

[0229] Methods of Controlling Insect Pests.

[0230] When an insect comes into contact with an effective amount of toxin delivered via transgenic plant expression the results are typically death of the insect, or the insects do not feed upon the source which makes the toxins available to the insects.

Sequence CWU 1

1

7211818DNABacillus thuringiensisCDS(1)..(1818)Native DNA sequence encoding Bacillus thuringiensis Cry1Fa core toxin 1atg gag aat aat att caa aat caa tgc gta cct tac aat tgt tta aat 48Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 aat cct gaa gta gaa ata tta aat gaa gaa aga agt act ggc aga tta 96Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 ccg tta gat ata tcc tta tcg ctt aca cgt ttc ctt ttg agt gaa ttt 144Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 gtt cca ggt gtg gga gtt gcg ttt gga tta ttt gat tta ata tgg ggt 192Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 ttt ata act cct tct gat tgg agc tta ttt ctt tta cag att gaa caa 240Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 ttg att gag caa aga ata gaa aca ttg gaa agg aac cgg gca att act 288Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 aca tta cga ggg tta gca gat agc tat gaa att tat att gaa gca cta 336Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 aga gag tgg gaa gca aat cct aat aat gca caa tta agg gaa gat gtg 384Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 cgt att cga ttt gct aat aca gac gac gct tta ata aca gca ata aat 432Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 aat ttt aca ctt aca agt ttt gaa atc cct ctt tta tcg gtc tat gtt 480Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 caa gcg gcg aat tta cat tta tca cta tta aga gac gct gta tcg ttt 528Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 ggg cag ggt tgg gga ctg gat ata gct act gtt aat aat cat tat aat 576Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 aga tta ata aat ctt att cat aga tat acg aaa cat tgt ttg gac aca 624Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 tac aat caa gga tta gaa aac tta aga ggt act aat act cga caa tgg 672Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 gca aga ttc aat cag ttt agg aga gat tta aca ctt act gta tta gat 720Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 atc gtt gct ctt ttt ccg aac tac gat gtt aga aca tat cca att caa 768Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 acg tca tcc caa tta aca agg gaa att tat aca agt tca gta att gag 816Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 gat tct cca gtt tct gct aat ata cct aat ggt ttt aat agg gcg gaa 864Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 ttt gga gtt aga ccg ccc cat ctt atg gac ttt atg aat tct ttg ttt 912Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 gta act gca gag act gtt aga agt caa act gtg tgg gga gga cac tta 960Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 gtt agt tca cga aat acg gct ggt aac cgt ata aat ttc cct agt tac 1008Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 ggg gtc ttc aat cct ggt ggc gcc att tgg att gca gat gag gat cca 1056Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 cgt cct ttt tat cgg aca tta tca gat cct gtt ttt gtc cga gga gga 1104Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 ttt ggg aat cct cat tat gta ctg ggg ctt agg gga gta gca ttt caa 1152Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 caa act ggt acg aac cac acc cga aca ttt aga aat agt ggg acc ata 1200Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 gat tct cta gat gaa atc cca cct cag gat aat agt ggg gca cct tgg 1248Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 aat gat tat agt cat gta tta aat cat gtt aca ttt gta cga tgg cca 1296Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 ggt gag att tca gga agt gat tca tgg aga gct cca atg ttt tct tgg 1344Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 acg cac cgt agt gca acc cct aca aat aca att gat ccg gag agg att 1392Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 act caa ata cca ttg gta aaa gca cat aca ctt cag tca ggt act act 1440Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 gtt gta aga ggg ccc ggg ttt acg gga gga gat att ctt cga cga aca 1488Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 agt gga gga cca ttt gct tat act att gtt aat ata aat ggg caa tta 1536Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 ccc caa agg tat cgt gca aga ata cgc tat gcc tct act aca aat cta 1584Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 aga att tac gta acg gtt gca ggt gaa cgg att ttt gct ggt caa ttt 1632Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 aac aaa aca atg gat acc ggt gac cca tta aca ttc caa tct ttt agt 1680Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 tac gca act att aat aca gct ttt aca ttc cca atg agc cag agt agt 1728Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 ttc aca gta ggt gct gat act ttt agt tca ggg aat gaa gtt tat ata 1776Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 gac aga ttt gaa ttg att cca gtt act gca aca ttt gaa tag 1818Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600 605 2605PRTBacillus thuringiensis 2Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600 605 31818DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus thuringiensis Cry1Fa core toxin using codons optimized for maize and Table 1 sequences are maintained 3atg gag aat aat atc cag aat caa tgc gtg cct tac aat tgt tta aat 48Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 aat ccc gag gtg gag ata tta aac gag gag aga tcc act ggc aga ctg 96Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 cca ctc gat ata tcc ttg tcc ctt acc cgt ttc ctt ttg agc gaa ttt 144Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 gtt cct ggt gtg gga gtg gct ttc gga tta ttt gat ctg ata tgg ggt 192Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 ttt atc act cct tct gat tgg agc tta ttt ctt ctc cag att gag caa 240Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 ttg att gag cag aga ata gaa acc ttg gaa agg aac cgt gca atc acg 288Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 acc ttg cgc ggt ctc gcc gat agc tat gaa att tat att gaa gca ctg 336Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 agg gag tgg gag gcc aac cct aat aat gct caa tta agg gaa gat gtg 384Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 cgt att cgt ttt gct aat aca gac gac gct tta ata aca gca ata aat 432Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 aat ttc aca ctt aca tcc ttt gaa atc ccg ctt tta tca gtg tac gtt 480Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 caa gcc gcc aat ctc cat tta tca ctt ctg agg gac gct gtc tcc ttt 528Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 ggg caa ggt tgg gga ctg gat atc gct act gtt aat aat cac tac aat 576Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 aga tta ata aac ctg att cat aga tat acg aag cat tgt ttg gac aca 624Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 tac aat caa gga ctg gag aac ctt agg gga act aac act agg cag tgg 672Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 gca agg ttc aac cag ttc aga cgt gat ctc aca ctt act gtg ctg gat 720Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 atc gtt gct ctc ttt ccg aac tac gat gtt cgc acc tac cca atc cag 768Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 acg tca tcc caa tta aca agg gaa att tat acc tcc tca gtg att gag 816Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 gac tct ccc gtt tct gct aac ata cct aac ggc ttc aac cgc gcc gag 864Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 ttc gga gtt aga ccg ccc cac ctt atg gac ttt atg aat agc ttg ttt 912Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 gtg act gct gag act gtt aga agc caa act gtg tgg ggc ggc cac ttg 960Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 gtc agc tca cgc aac acg gct ggc aac cgt atc aac ttc ccg tct tac 1008Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 ggg gtc ttt aac cct ggt ggc gcc att tgg att gca gac gag gac cca 1056Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 cgt cct ttt tat cgc acc ctg tca gat cct gtt ttt gtc aga ggc gga 1104Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly

355 360 365 ttt ggg aat cct cat tat gtc ctg ggc ctt agg gga gtg gct ttc caa 1152Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 cag act ggc acc aac cac acc cgt acg ttt cgc aat agc ggg acc ata 1200Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 gat tct ctt gat gaa atc cca cct caa gat aac agc ggc gca cct tgg 1248Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 aac gat tat tcc cac gta tta aat cac gtt acg ttc gtc cgc tgg ccg 1296Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 ggt gag atc agc ggc agc gat tca tgg aga gca cca atg ttt tct tgg 1344Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 acg cac cgt tca gcc acc cct aca aat aca att gac ccg gag agg att 1392Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 act caa atc cca ttg gtc aaa gca cat aca ctt cag tct ggg acc acc 1440Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 gtg gtc aga ggg cct ggg ttc acg gga gga gac att ctt agg cgc aca 1488Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 tcc gga gga ccc ttc gct tat act atc gtt aat ata aat ggg cag ctc 1536Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 ccc cag cgc tat cgt gcc aga atc cgt tac gcc tct act aca aat ctc 1584Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 aga atc tac gtg acg gtt gcc ggt gag cgc att ttt gct ggt cag ttt 1632Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 aac aag acg atg gat act ggc gac cca ctg aca ttc caa tct ttc tca 1680Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 tac gca act att aat aca gct ttc aca ttc cca atg agc cag tca tct 1728Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 ttc acc gtc ggt gct gat acc ttc agc tct ggc aac gaa gtt tat ata 1776Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 gac aga ttt gag ttg att cca gtt act gca acg ttt gag tga 1818Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600 605 4605PRTArtificial SequenceSynthetic Construct 4Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600 605 51818DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry1Fa core toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 5atg gag aat aat atc cag aat caa tgc gtg cct tac aat tgt ctc aat 48Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 aat ccc gag gtg gag ata tta aac gag gag aga tcc act ggc aga ctg 96Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 cca ctc gac ata tcc ttg tcc ctt acc cgt ttc ctt ttg agc gaa ttt 144Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 gtt cct ggt gtg gga gtg gct ttc gga ctg ttc gat ctg ata tgg ggc 192Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 ttt atc act cct tct gat tgg agc ctc ttc ctt ctc cag att gag caa 240Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 ttg att gag cag aga ata gaa acc ttg gaa agg aac cgt gca atc acg 288Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 acc ttg cgc ggt ctc gcc gat agc tat gaa atc tac att gaa gca ctg 336Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 agg gag tgg gag gcc aac ccc aat aat gct caa tta agg gaa gat gtg 384Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 cgt att cgt ttt gct aat aca gac gac gct ctc atc aca gca atc aat 432Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 aat ttc aca ctt aca tcc ttt gaa atc ccg ctt ttg agc gtg tac gtt 480Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 caa gcc gcc aat ctc cac ctc tca ctt ctg agg gac gct gtc tcc ttt 528Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 ggg caa ggt tgg gga ctg gat atc gct act gtg aat aat cac tac aat 576Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180 185 190 aga tta atc aac ctg att cat aga tat acg aag cac tgc ttg gac aca 624Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 tac aat caa gga ctg gag aac ctt agg gga act aac act agg cag tgg 672Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 gca agg ttc aac cag ttc aga cgt gat ctc aca ctt act gtg ctg gat 720Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 atc gtt gct ctc ttt ccg aac tac gat gtt cgc acc tac cca atc cag 768Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 acg tca tcc caa tta aca agg gaa atc tac acc tcc tca gtg att gag 816Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 gac tct ccc gtt tct gct aac ata cct aac ggc ttc aac cgc gcc gag 864Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 ttc gga gtt aga ccg ccc cac ctt atg gac ttt atg aat agc ttg ttc 912Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 gtg act gct gag act gtt aga agc caa act gtg tgg ggc ggc cac ttg 960Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 gtc agc tca cgc aac acg gct ggc aac cgt atc aac ttc ccg tct tac 1008Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 ggg gtc ttt aac cct ggt ggc gcc att tgg att gca gac gag gac cca 1056Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 cgt cct ttt tac cgc acc ctg tca gat ccg gtt ttc gtc aga ggc gga 1104Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 ttt ggg aat cct cat tat gtc ctg ggc ctt agg gga gtg gct ttc caa 1152Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 cag act ggc acc aac cac acc cgt acg ttt cgc aat agc ggg acc ata 1200Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 gat tct ctt gat gaa atc cca cct caa gat aac agc ggc gca cct tgg 1248Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 aac gat tat tcc cac gta tta aat cac gtt acg ttc gtc cgc tgg ccg 1296Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 ggt gag atc agc ggc agc gat tca tgg aga gca cca atg ttc tct tgg 1344Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 acg cac cgt tca gcc acc cct aca aat aca att gac ccg gag agg att 1392Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 act caa atc cca ttg gtc aaa gca cat aca ctt cag tct ggg acc acc 1440Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 gtg gtc aga ggg cct ggg ttc acg gga gga gac att ctt agg cgc aca 1488Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 tcc gga gga ccc ttc gct tat act atc gtt aat ata aat ggg cag ctc 1536Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 ccc cag cgc tat cgt gcc aga atc cgt tac gcc tct act aca aat ctc 1584Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 aga atc tac gtg acg gtt gcc ggt gag cgc atc ttt gct ggt cag ttt 1632Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 aac aag acg atg gat act ggc gac cca ctg aca ttc caa tct ttc tca 1680Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 tac gca act att aat aca gct ttc aca ttc cca atg agc cag tca tct 1728Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 ttc acc gtc ggt gct gat acc ttc agc tct ggc aac gaa gtc tat atc 1776Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 gac aga ttt gag ttg att cca gtt act gca acg ttt gag tga 1818Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600 605 6605PRTArtificial SequenceSynthetic Construct 6Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5 10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu 20 25 30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40 45 Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55 60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70 75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85 90 95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105 110 Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120 125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135 140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150 155 160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165 170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180

185 190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195 200 205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210 215 220 Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230 235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245 250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260 265 270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275 280 285 Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295 300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310 315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325 330 335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340 345 350 Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355 360 365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375 380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390 395 400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405 410 415 Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420 425 430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440 445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450 455 460 Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470 475 480 Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485 490 495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500 505 510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520 525 Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535 540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550 555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser 565 570 575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580 585 590 Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600 605 7372DNABacillus thuringiensisCDS(1)..(372)Native DNA sequence encoding Bacillus thuringiensis Cry34Ab1 toxin 7atg tca gca cgt gaa gta cac att gat gta aat aat aag aca ggt cat 48Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 aca tta caa tta gaa gat aaa aca aaa ctt gat ggt ggt aga tgg cga 96Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 aca tca cct aca aat gtt gct aat gat caa att aaa aca ttt gta gca 144Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 gaa tca aat ggt ttt atg aca ggt aca gaa ggt act ata tat tat agt 192Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 ata aat gga gaa gca gaa att agt tta tat ttt gac aat cct ttt gca 240Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 ggt tct aat aaa tat gat gga cat tcc aat aaa tct caa tat gaa att 288Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 att acc caa gga gga tca gga aat caa tct cat gtt acg tat act att 336Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 caa acc aca tcc tca cga tat ggg cat aaa tca taa 372Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 8123PRTBacillus thuringiensis 8Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 9372DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus thuringiensis Cry34Ab1 toxin using codons optimized for maize and Table 1 sequences are maintained 9atg tca gca cgg gag gtc cac atc gat gta aat aat aag acg ggt cat 48Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 aca tta cag ttg gag gat aaa aca aag cta gac ggt ggc aga tgg aga 96Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 acc agt ccg acc aac gtt gct aac gat caa att aaa aca ttt gta gcc 144Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 gaa tca aac ggt ttt atg act ggc acg gag ggg act ata tat tat tcc 192Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 atc aac gga gaa gcc gag att tcg tta tat ttt gac aat cca ttc gcg 240Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 ggg tct aat aaa tac gac gga cac tcc aat aaa tct caa tat gaa atc 288Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 att aca caa ggc ggc agc gga aat caa agc cac gtc acg tat act atc 336Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 cag acc act tca tcg cgc tac ggg cat aaa tca tag 372Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 10123PRTArtificial SequenceSynthetic Construct 10Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 11372DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry34Ab1 toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 11atg tca gca cgg gag gtc cac atc gat gta aat aat aag acg ggt cat 48Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 aca tta cag ttg gag gat aaa aca aag cta gac ggt ggc aga tgg aga 96Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 acc agt ccg acc aac gtt gct aac gat caa att aaa aca ttt gta gcc 144Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 gaa tca aac ggt ttc atg act ggc acg gag ggg act atc tac tac tcc 192Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 atc aac gga gaa gcc gag att tcg ctg tac ttc gac aat cca ttc gcg 240Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 ggg tct aat aaa tac gac gga cac tcc aat aaa tct caa tat gaa atc 288Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 att aca caa ggc ggc agc gga aat caa agc cac gtc acg tat act atc 336Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 cag acc act tca tcg cgc tac ggg cat aaa tca tag 372Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 12123PRTArtificial SequenceSynthetic Construct 12Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120 131152DNABacillus thuringiensisCDS(1)..(1152)Native DNA sequence encoding Bacillus thuringiensis Cry35Ab1 toxin 13atg tta gat act aat aaa gtt tat gaa ata agc aat cat gct aat gga 48Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 cta tat gca gca act tat tta agt tta gat gat tca ggt gtt agt tta 96Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 atg aat aaa aat gat gat gat att gat gat tat aac tta aaa tgg ttt 144Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 tta ttt cct att gat gat gat caa tat att att aca agc tat gca gca 192Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 aat aat tgt aaa gtt tgg aat gtt aat aat gat aaa ata aat gtt tcg 240Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 act tat tct tca aca aat tca ata caa aaa tgg caa ata aaa gct aat 288Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 ggt tct tca tat gta ata caa agt gat aat gga aaa gtc tta aca gca 336Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 gga acc ggt caa gct ctt gga ttg ata cgt tta act gat gaa tcc tca 384Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 aat aat ccc aat caa caa tgg aat tta act tct gta caa aca att caa 432Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 ctt cca caa aaa cct ata ata gat aca aaa tta aaa gat tat ccc aaa 480Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 tat tca cca act gga aat ata gat aat gga aca tct cct caa tta atg 528Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 gga tgg aca tta gta cct tgt att atg gta aat gat cca aat ata gat 576Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 aaa aat act caa att aaa act act cca tat tat att tta aaa aaa tat 624Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 caa tat tgg caa cga gca gta gga agt aat gta gct tta cgt cca cat 672Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 gaa aaa aaa tca tat act tat gaa tgg ggc aca gaa ata gat caa aaa 720Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 aca aca att ata aat aca tta gga ttt caa atc aat ata gat tca gga 768Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 atg aaa ttt gat ata cca gaa gta ggt gga ggt aca gat gaa ata aaa 816Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 aca caa cta aat gaa gaa tta aaa ata gaa tat agt cat gaa act aaa 864Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 ata atg gaa aaa tat caa gaa caa tct gaa ata gat aat cca act gat 912Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 caa tca atg aat tct ata gga ttt ctt act att act tcc tta gaa tta 960Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 tat aga tat aat ggc tca gaa att cgt ata atg caa att caa acc tca 1008Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 gat aat gat act tat aat gtt act tct tat cca aat cat caa caa gct 1056Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 tta tta ctt ctt aca aat cat tca tat gaa gaa gta gaa gaa ata aca 1104Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 aat att cct aaa agt aca cta aaa aaa tta aaa aaa tat tat ttt taa 1152Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 14383PRTBacillus thuringiensis 14Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser

Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 151152DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus thuringiensis Cry35Ab1 toxin using codons optimized for maize and Table 1 sequences are maintained 15atg ctc gat act aat aaa gtg tat gaa ata agc aac cat gcc aac ggg 48Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 cta tat gcc gca act tat ttg agt ctg gac gac agc ggt gtg agc tta 96Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 atg aat aaa aac gac gac gac att gac gac tac aac ctc aag tgg ttt 144Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 tta ttt cct att gac gac gat caa tat att att aca agc tac gca gca 192Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 aat aat tgc aaa gtc tgg aac gtt aat aat gat aaa ata aat gtt tcg 240Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 acc tac agc tcc acc aac tca ata caa aag tgg caa ata aaa gct aat 288Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 gga tcg tcg tat gta ata cag agt gac aat ggg aag gtc ttg aca gcg 336Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 ggc act ggt caa gct ctt gga ctc ata agg ctc act gac gag tcc tca 384Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 aat aat ccc aat caa cag tgg aac ttg act tcc gtg cag acg atc caa 432Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 ctt cca cag aaa cct atc atc gat aca aaa tta aaa gat tac ccc aag 480Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 tac tcg cca acc ggc aac atc gat aat gga acg tct cct caa tta atg 528Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 ggc tgg acc ctc gta ccc tgt att atg gtg aac gac ccg aat atc gat 576Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 aaa aat act caa att aaa acc acg ccg tat tat ata ttg aaa aaa tac 624Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 caa tac tgg cag cgc gcg gtt ggc tca aac gtc gct ctg cgg cca cat 672Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 gaa aag aag tcc tac act tac gaa tgg ggc aca gag atc gat cag aaa 720Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 acg acc att ata aat aca tta gga ttc caa atc aat atc gac agc gga 768Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 atg aaa ttt gac atc ccg gaa gtg ggg ggc ggg acc gat gaa ata aaa 816Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 acg cag ctc aac gaa gaa tta aaa ata gag tac agt cat gaa act aaa 864Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 ata atg gaa aaa tat caa gag caa tct gaa atc gat aac ccg acc gac 912Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 caa tca atg aac tct atc ggt ttc ctt act att acc tcc ctg gag tta 960Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 tat aga tat aac ggc tct gag atc cgt ata atg cag att caa acc tca 1008Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 gac aat gac act tat aac gtc acc tct tac ccg aat cat caa caa gct 1056Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 tta ttg ctt ctt aca aac cac agt tat gaa gag gtg gaa gaa ata acg 1104Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 aac att cct aaa tcc aca cta aag aaa tta aaa aaa tat tat ttt tga 1152Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 16383PRTArtificial SequenceSynthetic Construct 16Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 171152DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry35Ab1 toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 17atg ctc gat act aat aaa gtg tat gaa ata tcg aac cat gcc aac ggg 48Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 cta tat gcc gca act tac ctg agt ctg gac gat agt ggt gtg agc tta 96Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 atg aat aaa aac gac gac gac att gac gac tac aac ctc aag tgg ttc 144Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 ctg ttt cct att gac gac gat cag tat atc att aca agc tac gca gcg 192Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 aat aat tgc aaa gtc tgg aac gtc aat aat gat aaa atc aat gtt tcg 240Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 acc tac agc tcc acc aac tca ata caa aag tgg caa atc aaa gct aat 288Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 ggc agc tcg tac gta ata cag agt gac aat ggg aag gtc ttg aca gcg 336Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 ggc act ggt caa gct ctt gga ctc ata agg ctc act gac gag tcc tcg 384Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 aat aat ccc aat caa cag tgg aac ttg act tcc gtg cag acg atc caa 432Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 ctt cca cag aaa cct atc atc gat aca aaa tta aaa gat tac ccc aag 480Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 tac tcg cca acc ggc aac atc gat aat gga acg tct cct caa tta atg 528Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 ggc tgg acc ctc gta ccc tgt ata atg gtg aac gac ccg aat atc gat 576Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 aaa aat act caa att aaa acc acg ccg tac tac ata ctc aaa aaa tac 624Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 caa tac tgg cag cgc gcg gtt ggc tca aac gtc gct ctg cgg cca cat 672Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 gaa aag aag tcc tac act tac gaa tgg ggc aca gag atc gat cag aaa 720Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 acg acc att ata aat aca tta gga ttc caa atc aat atc gac agc gga 768Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 atg aaa ttt gac atc ccg gaa gtg ggg ggc ggg acc gat gaa ata aaa 816Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 acg cag ctc aac gaa gaa tta aaa ata gag tac agt cat gaa act aaa 864Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 ata atg gaa aaa tat caa gag caa tct gaa atc gat aac ccg acc gac 912Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 caa tca atg aac tct atc ggt ttc ctt act att acc tcc ctg gag ttg 960Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 tac aga tat aac ggc tct gag atc cgt ata atg cag att caa acc tca 1008Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 gac aat gac act tat aac gtc acc tct tac ccg aat cat cag caa gcc 1056Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 ctg ctg ctt ctt aca aac cac agt tat gaa gag gtg gaa gag ata acg 1104Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 aac att cct aaa tcc aca cta aag aaa tta aaa aaa tat tat ttc tga 1152Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 18383PRTArtificial SequenceSynthetic Construct 18Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245

250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 191830DNABacillus thuringiensisCDS(1)..(1830)Native DNA sequence encoding Bacillus thuringiensis Cry1Ab1 core toxin 19atg gat aac aat ccg aac atc aat gaa tgc att cct tat aat tgt tta 48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gta gaa gta tta ggt gga gaa aga ata gaa act ggt 96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 tac acc cca atc gat att tcc ttg tcg cta acg caa ttt ctt ttg agt 144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 gaa ttt gtt ccc ggt gct gga ttt gtg tta gga cta gtt gat ata ata 192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 tgg gga att ttt ggt ccc tct caa tgg gac gca ttt ctt gta caa att 240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gaa cag tta att aac caa aga ata gaa gaa ttc gct agg aac caa gcc 288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 att tct aga tta gaa gga cta agc aat ctt tat caa att tac gca gaa 336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 tct ttt aga gag tgg gaa gca gat cct act aat cca gca tta aga gaa 384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 gag atg cgt att caa ttc aat gac atg aac agt gcc ctt aca acc gct 432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 att cct ctt ttt gca gtt caa aat tat caa gtt cct ctt tta tca gta 480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 tat gtt caa gct gca aat tta cat tta tca gtt ttg aga gat gtt tca 528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 gtg ttt gga caa agg tgg gga ttt gat gcc gcg act atc aat agt cgt 576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 tat aat gat tta act agg ctt att ggc aac tat aca gat tat gct gta 624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 cgc tgg tac aat acg gga tta gaa cgt gta tgg gga ccg gat tct aga 672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 gat tgg gta agg tat aat caa ttt aga aga gaa tta aca cta act gta 720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 tta gat atc gtt gct ctg ttc ccg aat tat gat agt aga aga tat cca 768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 att cga aca gtt tcc caa tta aca aga gaa att tat aca aac cca gta 816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 tta gaa aat ttt gat ggt agt ttt cga ggc tcg gct cag ggc ata gaa 864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 aga agt att agg agt cca cat ttg atg gat ata ctt aac agt ata acc 912Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 atc tat acg gat gct cat agg ggt tat tat tat tgg tca ggg cat caa 960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ata atg gct tct cct gta ggg ttt tcg ggg cca gaa ttc act ttt ccg 1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 cta tat gga act atg gga aat gca gct cca caa caa cgt att gtt gct 1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 caa cta ggt cag ggc gtg tat aga aca tta tcg tcc act tta tat aga 1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 aga cct ttt aat ata ggg ata aat aat caa caa cta tct gtt ctt gac 1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 ggg aca gaa ttt gct tat gga acc tcc tca aat ttg cca tcc gct gta 1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 tac aga aaa agc gga acg gta gat tcg ctg gat gaa ata ccg cca cag 1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 aat aac aac gtg cca cct agg caa gga ttt agt cat cga tta agc cat 1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 gtt tca atg ttt cgt tca ggc ttt agt aat agt agt gta agt ata ata 1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 aga gct cct atg ttc tct tgg ata cat cgt agt gct gaa ttt aat aat 1392Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 ata att cct tca tca caa att aca caa ata cct tta aca aaa tct act 1440Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470 475 480 aat ctt ggc tct gga act tct gtc gtt aaa gga cca gga ttt aca gga 1488Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495 gga gat att ctt cga aga act tca cct ggc cag att tca acc tta aga 1536Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505 510 gta aat att act gca cca tta tca caa aga tat cgg gta aga att cgc 1584Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520 525 tac gct tct acc aca aat tta caa ttc cat aca tca att gac gga aga 1632Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530 535 540 cct att aat cag ggg aat ttt tca gca act atg agt agt ggg agt aat 1680Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560 tta cag tcc gga agc ttt agg act gta ggt ttt act act ccg ttt aac 1728Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575 ttt tca aat gga tca agt gta ttt acg tta agt gct cat gtc ttc aat 1776Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590 tca ggc aat gaa gtt tat ata gat cga att gaa ttt gtt ccg gca gaa 1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595 600 605 gta acc 1830Val Thr 610 20610PRTBacillus thuringiensis 20Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470 475 480 Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505 510 Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530 535 540 Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575 Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595 600 605 Val Thr 610 211830DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus thuringiensis Cry1Ab1 core toxin using codons optimized for maize and Table 1 sequences are maintained 21atg gat aac aac ccg aac atc aat gag tgc atc ccg tat aac tgt ctc 48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gtg gag gtc tta ggt ggc gaa cgc atc gaa act ggt 96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 tac acc cca atc gac att agc ttg tcg ttg acg cag ttc ctt ttg tcc 144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 gag ttc gtg ccc ggt gcg ggt ttc gtg ctg ggg cta gtt gat ata atc 192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 tgg gga atc ttt ggt ccc tct cag tgg gac gcc ttt ctt gtg caa att 240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gag cag cta att aac caa aga ata gaa gag ttc gcg agg aac caa gcc 288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 att tcc aga ctg gag gga cta agc aac ctt tat caa atc tac gcg gag 336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 tct ttt agg gag tgg gag gca gat cct acg aac ccg gca ctg cgc gaa 384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 gag atg cgt att cag ttc aac gac atg aac agt gcc ctt aca acc gct 432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 att ccc ctt ttc gca gtt caa aat tac caa gtt ccc ctt ctc tca gtg 480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 tac gtt caa gcc gca aat tta cac cta agc gtt ctc cgc gat gtg tca 528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 gtg ttt ggc cag agg tgg gga ttt gat gcc gcc act atc aat agt cgt 576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 tat aat gat ctg acg agg ctt atc ggc aac tat acc gac tat gct gtc 624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 cgc tgg tac aat acg gga tta gag cgg gtc tgg ggt ccg gat tcc cga 672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 gac tgg gtg cgc tac aat caa ttc cgc cgc gaa tta acc ctc act gtc 720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 ctc gac atc gtg gcg ctg ttc ccg aac tac gac agt agg aga tac cca 768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 atc cgc aca gtt tcc caa tta acg cgg gaa att tac acc aac cca gtc

816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 ctg gag aat ttt gac ggg agc ttc cga ggc tcg gct caa ggc ata gaa 864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 cgc agc att agg tcg cca cac ttg atg gat atc ctt aac agc atc acc 912Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 atc tac acg gat gcc cat agg ggt tac tac tac tgg tcg ggg cat caa 960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ata atg gct tct cct gtc ggg ttt tcg ggg cca gag ttc acc ttc ccg 1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 ctc tac ggc act atg gga aat gcc gcg cca caa caa cgt atc gtc gct 1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 caa cta ggt caa ggc gtg tac cgg aca ctg tcg tcc act ctc tat cgg 1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 cgg cct ttc aat ata ggg ata aat aat caa cag ttg tct gtg ctg gac 1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 ggg aca gag ttt gct tac gga acc tca agc aac ttg cca tcc gct gta 1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 tac aga aaa agc ggc acg gtg gac tcg ctg gat gaa atc ccg ccc cag 1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 aat aac aac gtg ccc cct cgg caa ggc ttc agt cat cga ctg agc cac 1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 gtt agc atg ttc cgt tcg ggc ttc agc aac tcc tcc gta agt ata ata 1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 aga gca cct atg ttc agc tgg ata cat cgt tcc gcc gag ttt aat aat 1392Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 ata att ccc tcc tct caa atc aca cag atc cct ctg aca aag tct act 1440Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470 475 480 aat ctt ggc tct ggg act tct gtc gtt aag ggg cct ggc ttt acg ggc 1488Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495 ggc gat att ctg cgg aga act tca cct ggc cag att tcc acc ctg cgc 1536Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505 510 gtg aat atc acc gcg cca ttg tca caa cgt tac cgc gtg cgg att cgc 1584Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520 525 tac gct tct acc aca aac ctc cag ttc cat aca tct att gac ggc aga 1632Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530 535 540 ccc att aat caa ggg aat ttc tcc gcc acg atg tcg tcc ggc tcc aat 1680Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560 ctc cag tcc gga agt ttc cgc acc gta ggt ttt act acc ccg ttc aac 1728Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575 ttt tca aac ggc tca agt gtg ttt acg ctg tcc gct cat gtg ttc aac 1776Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590 tct ggc aat gag gtt tat atc gac cgg att gag ttc gtc ccg gca gaa 1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595 600 605 gtc acc 1830Val Thr 610 22610PRTArtificial SequenceSynthetic Construct 22Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470 475 480 Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505 510 Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530 535 540 Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575 Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595 600 605 Val Thr 610 231830DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry1Ab1 core toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 23atg gat aac aac ccg aac atc aat gag tgc atc ccg tat aac tgt ctc 48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agt aac cct gaa gtg gag gtc tta ggt ggc gaa cgc atc gaa act ggt 96Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 tac acc cca atc gac att agc ttg tcg ttg acg cag ttc ctc ttg tcc 144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 gag ttc gtg ccc ggt gcg ggt ttc gtg ctg ggg cta gtt gat ata atc 192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 tgg gga atc ttt ggt ccc tct cag tgg gac gcc ttt ctt gtg caa att 240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gag cag cta att aac caa aga ata gaa gag ttc gcg agg aac caa gcc 288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 att tcc aga ctg gag gga cta agc aac ctt tat caa atc tac gcg gag 336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 tct ttt agg gag tgg gag gca gat cct acg aac ccg gca ctg cgc gaa 384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 gag atg cgt att cag ttc aac gac atg aac agt gcc ctt aca acc gct 432Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 att ccc ctt ttc gca gtt caa aat tac caa gtt ccc ctt ctc tca gtg 480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 tac gtt caa gcc gca aat tta cac cta agc gtt ctc cgc gat gtg tca 528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 gtg ttc ggc cag agg tgg gga ttt gat gcc gcc act atc aat agt cgt 576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 tat aat gat ctg acg agg ctt atc ggc aac tat acc gac tat gct gtc 624Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 cgc tgg tac aat acg gga tta gag cgg gtc tgg ggt ccg gat tcc cga 672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 gac tgg gtg cgc tac aat caa ttc cgc cgc gaa tta acc ctc act gtc 720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 ctc gac atc gtg gcg ctg ttc ccg aac tac gac agt agg aga tac cca 768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 atc cgc aca gtt tcc caa tta acg cgg gaa att tac acc aac cca gtc 816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 ctg gag aat ttt gac ggg agc ttc cga ggc tcg gct caa ggc ata gaa 864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 cgc agc att agg tcg cca cac ttg atg gat atc ctt aac agc atc acc 912Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 atc tac acg gat gcc cat agg ggt tac tac tac tgg tcg ggg cat caa 960Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 ata atg gct tct cct gtc ggg ttt tcg ggg cca gag ttc acc ttc ccg 1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 ctc tac ggc act atg gga aat gcc gcg cca caa caa cgt atc gtc gct 1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 caa cta ggt caa ggc gtg tac cgg aca ctg tcg tcc act ctc tat cgg 1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 cgg cct ttc aat ata ggg atc aat aat caa cag ttg tct gtg ctg gac 1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 ggg aca gag ttt gct tac gga acc tca agc aac ttg cca tcc gct gta 1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 tac aga aaa agc ggc acg gtg gac tcg ctg gat gaa atc ccg ccc cag 1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 aat aac aac gtg ccc cct cgg caa ggc ttc agt cat cga ctg agc cac 1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 gtt agc atg ttc cgt tcg ggc ttc agc aac tcc tcc gta agt atc ata 1344Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 aga gca cct atg ttc agc tgg ata cat cgt tcc gcc gag ttc aat aat 1392Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 ata att ccc tcc tct caa atc aca cag atc cct ctg aca aag tct act 1440Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470 475 480 aat ctt ggc tct ggg act tct gtc gtt aag ggg cct ggc ttt acg ggc 1488Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495 ggc gat att ctg cgg aga act tca cct ggc cag att tcc acc ctg cgc 1536Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505 510 gtg aat atc acc gcg cca ttg tca caa cgt tac cgc gtg cgg att cgc 1584Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520 525 tac gct tct acc aca aac ctc cag ttc cat aca tct att gac ggc aga 1632Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530 535 540 ccc att aat caa ggg aat ttc tcc gcc acg atg tcg tcc ggc tcc aat 1680Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560 ctc cag tcc gga agt ttc cgc acc gta ggt ttt act acc ccg ttc aac 1728Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575 ttt tca aac ggc tca agt gtg ttt acg ctg tcc gct cat gtg ttc aac 1776Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590 tct ggc aat gag gtt tac atc gac cgg att gag ttc gtc ccg gca gaa 1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu

595 600 605 gtc acc 1830Val Thr 610 24610PRTArtificial SequenceSynthetic Construct 24Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450 455 460 Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470 475 480 Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505 510 Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530 535 540 Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550 555 560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565 570 575 Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580 585 590 Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595 600 605 Val Thr 610 251866DNABacillus thuringiensisCDS(1)..(1866)Native DNA sequence encoding Bacillus thuringiensis Cry1Ca core toxin 25atg gat aac aat ccg aac atc aat gaa tgc atc ccg tac aac tgc ctg 48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agc aac ccg gaa gaa gtg ctg ttg gat gga gaa cgg ata tca act ggt 96Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 aat tca tca att gat att tct ctg tca ctt gtt cag ttt ctg gta tct 144Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 aac ttc gtc cca ggc gga gga ttc ctg gtt gga tta ata gat ttt gta 192Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 tgg gga ata gtt ggc cct tct caa tgg gat gca ttt cta gta caa att 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gaa caa tta att aat gaa aga ata gct gaa ttt gct agg aat gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 att gct aat tta gaa gga tta gga aac aat ttc aat ata tat gtg gaa 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 gca ttt aaa gaa tgg gaa gaa gat cct aag aat cca gca acc agg acc 384Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 aga gta att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac 432Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 att cct tcg ttt cga att tct gga ttt gaa gta ccc ctt tta tcc gtt 480Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 tat gct caa gcg gcc aat ctg cat cta gct ata tta aga gat tct gta 528Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 att ttt gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac 576Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 tat aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca 624Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 aat acg tat aat cgg gga tta aat aat tta ccg aaa tct acg tat caa 672Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 gat tgg ata aca tat aat cga ttg cgg aga gac tta aca ttg act gta 720Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 tta gat atc gcc gct ttc ttt cca aac tat gac aat agg aga tat cca 768Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 att cag cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta 816Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 att aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt 864Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 aac gtt atg gag aac agc gca att aga aat cct cat tta ttt gat ata 912Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 ttg aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat 960Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 ttt tat tgg gga gga cat cga gta ata tct agc ctt ata gga ggt ggt 1008Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 aac ata aca tct cct ata tat gga aga gag gcg aac cag gag cct cca 1056Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 aga tcc ttt act ttt aat gga ccg gta ttt agg act tta tca aat cct 1104Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 act tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta 1152Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 cgt ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg 1200Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 tat cga gga aga ggt acg gtt gat tct tta act gaa ttg ccg cct gag 1248Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 gat aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta tgt cat 1296Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 gca act ttt gtt caa aga tct gga aca cct ttt tta aca act ggt gta 1344Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 gta ttt tct tgg acg cat cgt agt gca act ctt aca aat aca att gat 1392Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 cca gag aga att aat caa ata cct tta gtg aaa gga ttt aga gtt tgg 1440Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 ggg ggc acc tct gtc att aca gga cca gga ttt aca gga ggg gat atc 1488Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 ctt cga aga aat acc ttt ggt gat ttt gta tct cta caa gtc aat att 1536Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 aat tca cca att acc caa aga tac cgt tta aga ttt cgt tac gct tcc 1584Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 agt agg gat gca cga gtt ata gta tta aca gga gcg gca tcc aca gga 1632Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 gtg gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa 1680Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 ata ggg gag aac tta aca tct aga aca ttt aga tat acc gat ttt agt 1728Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 aat cct ttt tca ttt aga gct aat cca gat ata att ggg ata agt gaa 1776Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 caa cct cta ttt ggt gca ggt tct att agt agc ggt gaa ctt tat ata 1824Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 gat aaa att gaa att att cta gca gat gca aca ttt gaa taa 1866Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 26621PRTBacillus thuringiensis 26Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg

Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 271866DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus thuringiensis Cry1Ca core toxin using codons optimized for maize and Table 1 sequences are maintained 27atg gat aac aat ccg aac atc aat gag tgc atc ccg tac aac tgc ctg 48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agc aac ccg gaa gaa gtg ctg ttg gat gga gaa cgg ata tca act ggc 96Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 aat tca tcc att gat att tct ctg tca ctt gtt cag ttt ctg gtg tct 144Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 aac ttc gtc ccc ggc gga gga ttc ctg gtt gga tta ata gat ttt gta 192Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 tgg gga ata gtt ggc cct tct caa tgg gac gca ttt cta gta caa att 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gaa caa tta att aat gaa aga ata gct gaa ttt gct agg aac gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 att gct aat tta gaa gga tta gga aac aat ttc aat ata tat gtg gaa 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 gca ttt aag gaa tgg gaa gaa gat cct aag aat cca gca acg agg acc 384Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 aga gta att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac 432Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 att cct tcg ttt cga att tct gga ttt gaa gta ccc ctt tta tcc gtt 480Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 tat gct caa gcg gcc aat ctg cat cta gct ata tta aga gat tct gta 528Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 att ttt gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac 576Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 tat aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca 624Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 aat acg tat aat cgg gga tta aat aat tta ccg aaa tct acg tat caa 672Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 gat tgg ata aca tat aat cga ttg cgg aga gac tta aca ttg act gta 720Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 tta gat atc gcc gct ttc ttt cca aac tat gac aat agg aga tat cca 768Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 att cag cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta 816Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 att aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt 864Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 aac gtt atg gag aac agc gca att aga aat cct cat tta ttt gat ata 912Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 ttg aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aat 960Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 ttt tat tgg gga gga cat cga gta ata tct agc ctt ata gga ggt ggg 1008Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 aac atc aca tcg cct ata tat gga aga gag gcg aac caa gag cct cca 1056Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 aga tcc ttt act ttt aat gga ccc gtg ttt agg act tta tca aat cct 1104Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 act tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta 1152Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 cgt ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg 1200Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 tat cga gga aga ggg acg gtt gat tct tta act gaa ttg ccg cct gag 1248Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 gat aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta tgt cat 1296Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 gca act ttt gtt caa aga tcg gga aca cct ttt tta aca act ggt gta 1344Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 gta ttt tct tgg acg cat cgt agt gca act ctt aca aat aca atc gac 1392Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 cca gag aga att aat caa ata cct tta gtg aag gga ttt aga gtt tgg 1440Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 ggg ggc acc tct gtc att acc gga ccc gga ttt acc gga ggg gat atc 1488Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 ctt cga aga aat acc ttt ggt gat ttt gta tct cta caa gtc aac att 1536Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 aat tca cca att acc caa aga tac cgt tta aga ttt cgt tac gct tcc 1584Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 agt agg gat gca cga gtt ata gta tta acg gga gcg gca tcc acc gga 1632Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 gtg gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa 1680Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 ata ggg gag aac tta aca tcc aga aca ttt aga tat acc gat ttt agt 1728Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 aat cct ttt tca ttt aga gct aat cca gat ata att ggg ata agt gaa 1776Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 caa cct cta ttt ggg gcg ggt tct att agt agc ggt gaa ctt tat ata 1824Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 gat aaa att gaa att att cta gca gat gca aca ttt gaa tga 1866Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 28621PRTArtificial SequenceSynthetic Construct 28Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 291866DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry1Ca core toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 29atg gat aac aat ccg aac atc aat gag tgc atc ccg tac aac tgc ctg 48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 agc aac ccg gaa gaa gtg ctg ttg gat gga gaa cgg ata tca act ggc 96Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 aat tca tcc att gac att tct ctg tca ctt gtt cag ttt ctg gtg tct 144Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 aac ttc gtc ccc ggc gga gga ttc ctg gtt gga tta ata gat ttc gta 192Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 tgg gga ata gtt ggc cct tct caa tgg gac gca ttt cta gta caa att 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 gaa caa tta att aat gaa aga ata gct gaa ttt gct agg aac gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 att gct aat tta gaa gga tta gga aac aat ttc aac atc tat gtg gaa 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 gca ttt aag gaa tgg gaa gaa gat cct aag aat cca gca acg agg acc 384Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 aga gta att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac 432Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 att cct tcg ttt cga att tct gga ttt gaa gta ccc ctt ctc tcc gtt 480Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 tat gct caa gcg gcc aat ctg cat cta gct atc tta aga gat tct gtc 528Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 atc ttt gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac 576Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 tat aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca 624Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 aat acg tat aat cgg gga tta aat aat tta ccg aaa tct acg tat caa 672Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 gat tgg ata aca tat aat cga ttg cgg aga gac tta aca ttg act gta 720Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 tta gat atc gcc gct ttc ttt cca aac tat gac aat agg aga tat cca 768Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 att cag cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta 816Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 att aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt 864Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285

aac gtt atg gag aac agc gca att aga aat cct cat ttg ttc gac ata 912Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 ttg aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc aac 960Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 ttc tat tgg gga gga cat cga gta ata tct agc ctt ata gga ggt ggg 1008Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 aac atc aca tcg cct atc tat gga aga gag gcg aac caa gag cct cca 1056Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 aga tcc ttt act ttt aat gga ccc gtg ttt agg act tta tca aat cct 1104Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 act tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta 1152Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 cgt ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg 1200Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 tat cga gga aga ggg acg gtt gat tct tta act gaa ttg ccg cct gag 1248Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 gat aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta tgt cat 1296Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 gca acc ttt gtt caa aga tcg gga aca cct ttc tta aca act ggt gta 1344Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 gta ttc tct tgg acg cat cgt agt gca act ctt aca aat aca atc gac 1392Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 cca gag aga att aat caa ata cct tta gtg aag gga ttt aga gtt tgg 1440Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 ggg ggc acc tct gtc att acc gga ccc gga ttt acc gga ggg gat atc 1488Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 ctt cga aga aat acc ttt ggt gat ttc gta tct cta caa gtc aac att 1536Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 aat tca cca att acc caa aga tac cgt tta aga ttt cgt tac gct tcc 1584Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 agt agg gat gca cga gtt ata gta tta acg gga gcg gca tcc acc gga 1632Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 gtg gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa 1680Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 ata ggg gag aac tta aca tcc aga aca ttt aga tat acc gat ttt agt 1728Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 aat cct ttt tca ttt aga gct aat cca gat ata att ggg ata agt gaa 1776Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 caa cct cta ttt ggg gcg ggt tct att agt agc ggt gaa ctt tac ata 1824Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 gat aaa att gaa att att cta gca gat gca aca ttt gaa tga 1866Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 30621PRTArtificial SequenceSynthetic Construct 30Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25 30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35 40 45 Asn Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50 55 60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70 75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85 90 95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100 105 110 Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115 120 125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135 140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145 150 155 160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165 170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185 190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala 195 200 205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210 215 220 Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245 250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260 265 270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275 280 285 Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295 300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310 315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly 325 330 335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340 345 350 Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360 365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375 380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385 390 395 400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405 410 415 Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420 425 430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440 445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450 455 460 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470 475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485 490 495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 500 505 510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520 525 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535 540 Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550 555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565 570 575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 580 585 590 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595 600 605 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615 620 311428DNABacillus thuringiensisCDS(1)..(1428)Native DNA sequence encoding Bacillus thuringiensis Cry6Aa toxin 31atg att att gat agt aaa acg act tta cct aga cat tca ctt att cat 48Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15 aca att aaa tta aat tct aat aag aaa tat ggt cct ggt gat atg act 96Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30 aat gga aat caa ttt att att tca aaa caa gaa tgg gct acg att gga 144Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45 gca tat att cag act gga tta ggt tta cca gta aat gaa caa caa tta 192Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 aga aca cat gtt aat tta agt cag gat ata tca ata cct agt gat ttt 240Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 tct caa tta tat gat gtt tat tgt tct gat aaa act tca gca gaa tgg 288Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 tgg aat aaa aat tta tat cct tta att att aaa tct gct aat gat att 336Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105 110 gct tca tat ggt ttt aaa gtt gct ggt gat cct tct att aag aaa gat 384Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120 125 gga tat ttt aaa aaa ttg caa gat gaa tta gat aat att gtt gat aat 432Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135 140 aat tcc gat gat gat gca ata gct aaa gct att aaa gat ttt aaa gcg 480Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150 155 160 cga tgt ggt att tta att aaa gaa gct aaa caa tat gaa gaa gct gca 528Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 aaa aat att gta aca tct tta gat caa ttt tta cat ggt gat cag aaa 576Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 aaa tta gaa ggt gtt atc aat att caa aaa cgt tta aaa gaa gtt caa 624Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 aca gct ctt aat caa gcc cat ggg gaa agt agt cca gct cat aaa gag 672Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 tta tta gaa aaa gta aaa aat tta aaa aca aca tta gaa agg act att 720Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 aaa gct gaa caa gat tta gag aaa aaa gta gaa tat agt ttt cta tta 768Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 gga cca ttg tta gga ttt gtt gtt tat gaa att ctt gaa aat act gct 816Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 gtt cag cat ata aaa aat caa att gat gag ata aag aaa caa tta gat 864Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 tct gct cag cat gat ttg gat aga gat gtt aaa att ata gga atg tta 912Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 aat agt att aat aca gat att gat aat tta tat agt caa gga caa gaa 960Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 gca att aaa gtt ttc caa aag tta caa ggt att tgg gct act att gga 1008Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 gct caa ata gaa aat ctt aga aca acg tcg tta caa gaa gtt caa gat 1056Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350 tct gat gat gct gat gag ata caa att gaa ctt gag gac gct tct gat 1104Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360 365 gct tgg tta gtt gtg gct caa gaa gct cgt gat ttt aca cta aat gct 1152Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 tat tca act aat agt aga caa aat tta ccg att aat gtt ata tca gat 1200Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390 395 400 tca tgt aat tgt tca aca aca aat atg aca tca aat caa tac agt aat 1248Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 cca aca aca aat atg aca tca aat caa tat atg att tca cat gaa tat 1296Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 aca agt tta cca aat aat ttt atg tta tca aga aat agt aat tta gaa 1344Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 tat aaa tgt cct gaa aat aat ttt atg ata tat tgg tat aat aat tcg 1392Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 gat tgg tat aat aat tcg gat tgg tat aat aat tga 1428Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 32475PRTBacillus thuringiensis 32Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30 Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45 Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105 110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn

Thr Ala 260 265 270 Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350 Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360 365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 331428DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus thuringiensis Cry6Aa toxin using codons optimized for maize and Table 1 sequences are maintained 33atg atc atc gac tcc aag acg acc ctg cca cgg cac tcc ctt atc cac 48Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15 aca att aaa tta aat agc aat aag aag tac ggt ccc ggt gat atg act 96Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30 aac gga aat caa ttc att att tca aag caa gag tgg gct acc atc gga 144Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45 gcg tac atc cag act ggg ctg ggc cta cca gta aat gaa caa caa tta 192Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 agg acc cat gtc aac ctc agc caa gat atc agc atc cct agc gac ttt 240Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 tct cag ctc tac gac gtc tat tgc agc gat aaa act tcc gca gaa tgg 288Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 tgg aat aaa aac ctg tac ccc ctc atc att aaa tct gcc aac gat att 336Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105 110 gcc agc tac ggc ttc aag gtc gcg ggt gat cct tct att aag aag gac 384Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120 125 ggc tac ttc aag aag ctg caa gat gag ctg gac aac att gtt gac aat 432Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135 140 aat tcc gat gat gat gca ata gcg aaa gcc att aaa gac ttc aag gcg 480Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150 155 160 cga tgc ggc atc cta att aaa gaa gca aag cag tat gaa gag gca gcg 528Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 aaa aat atc gta aca tcc ctc gac caa ttt ctg cat ggc gat cag aag 576Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 aaa ttg gag ggt gtg atc aac atc caa aaa cgt ctg aag gag gtg cag 624Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 acg gct ctt aat caa gcc cac ggg gaa agt tca cca gct cat aaa gag 672Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 ctg tta gag aaa gtc aag aat ctc aag acc aca ctt gag agg acc att 720Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 aaa gct gag caa gac ctg gag aag aaa gtg gag tac agt ttc ctt ctc 768Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 ggc ccc ttg ctg ggc ttc gtc gtt tat gaa atc ctt gaa aat act gcc 816Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 gtc cag cat ata aaa aac caa att gac gag ata aag aag caa ctg gac 864Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 tct gcc cag cac gac ttg gac aga gac gtt aag atc ata ggg atg ctg 912Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 aac agt att aat aca gac att gat aac ttg tat agc caa gga caa gag 960Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 gca att aaa gtg ttc caa aag ctc caa ggc atc tgg gca act atc gga 1008Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 gcg cag ata gag aac ctt agg aca acg tcg ctc caa gaa gtg caa gac 1056Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350 tct gac gac gcc gat gag atc caa att gaa ctt gag gac gcg tct gat 1104Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360 365 gct tgg tta gtg gtg gcc caa gaa gct cgc gac ttc aca cta aat gcc 1152Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 tac tca act aac tcg cgt cag aat cta ccg att aat gtt ata tcc gat 1200Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390 395 400 tcc tgc aac tgt tcc aca acg aac atg acc tca aat caa tac agt aat 1248Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 cca acc aca aat atg acc tca aat caa tat atg atc tca cac gag tat 1296Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 acc tcg ttg ccg aat aat ttc atg ctc tca aga aat agc aat ctg gaa 1344Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 tat aag tgt cct gaa aat aat ttc atg ata tac tgg tac aat aat tcg 1392Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 gac tgg tac aat aat tcg gat tgg tac aat aat tga 1428Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 34475PRTArtificial SequenceSynthetic Construct 34Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30 Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45 Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105 110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350 Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360 365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 351428DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Bacillus thuringiensis Cry6Aa toxin using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 35atg atc atc gac tcc aag acg acc ctg cca cgg cac tcc ctt atc cac 48Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15 aca att aaa tta aat agc aat aag aag tac ggt ccc ggt gat atg act 96Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30 aac gga aat caa ttc att atc tca aag caa gag tgg gct acc atc gga 144Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45 gcg tac atc cag act ggg ctg ggc cta cca gta aat gaa caa caa tta 192Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 agg acc cat gtc aac ctc agc caa gat atc agc atc cct agc gac ttt 240Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 tct cag ctc tac gac gtc tat tgc agc gat aaa act tcc gca gaa tgg 288Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 tgg aat aaa aac ctg tac ccc ctc atc att aaa tct gcc aac gat att 336Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105 110 gcc agc tac ggc ttc aag gtc gcg ggt gat cct tct att aag aag gac 384Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120 125 ggc tac ttc aag aag ctg caa gat gag ctg gac aac att gtt gac aat 432Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135 140 aat tcc gat gat gat gca ata gcg aaa gcc att aaa gac ttc aag gcg 480Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150 155 160 cga tgc ggc atc cta att aaa gaa gca aag cag tat gaa gag gca gcg 528Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 aaa aat atc gta aca tcc ctc gac caa ttt ctg cat ggc gat cag aag 576Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 aaa ttg gag ggt gtg atc aac atc caa aaa cgt ctg aag gag gtg cag 624Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 acg gct ctt aat caa gcc cac ggg gaa agt tca cca gct cat aaa gag 672Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 ctg tta gag aaa gtc aag aat ctc aag acc aca ctt gag agg acc att 720Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 aaa gct gag caa gac ctg gag aag aaa gtg gag tac agt ttc ctt ctc 768Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 ggc ccc ttg ctg ggc ttc gtc gtt tat gaa atc ctt gaa aat act gcc 816Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 gtc cag cat ata aaa aac caa att gac gag ata aag aag caa ctg gac 864Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 tct gcc cag cac gac ttg gac aga gac gtt aag atc ata ggg atg ctg 912Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 aac agt att aat aca gac att gat aac ttg tat agc caa gga caa gag 960Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 gca att aaa gtg ttc caa aag ctc caa ggc atc tgg gca act atc gga 1008Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 gcg cag ata gag aac ctt agg aca acg tcg ctc caa gaa gtg caa gac 1056Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350 tct gac gac gcc gat gag atc caa att gaa ctt gag gac gcg tct gat 1104Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360 365 gct tgg tta gtg gtg gcc caa gaa gct cgc gac ttc aca cta aat gcc 1152Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 tac tca act aac tcg cgt cag aat cta ccg att aat gtt atc tcc gat 1200Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390 395 400 tcc tgc aac tgt tcc aca acg aac atg acc tca aat caa tac agt aat 1248Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 cca acc aca aat atg acc tca aat caa tac atg atc tca cac gag tat 1296Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430

acc tcg ttg ccg aat aat ttc atg ctc tca aga aat agc aat ctg gaa 1344Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 tat aag tgt cct gaa aat aat ttc atg ata tac tgg tac aat aat tcg 1392Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 gac tgg tac aat aat tcg gat tgg tac aat aat tga 1428Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 36475PRTArtificial SequenceSynthetic Construct 36Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 10 15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25 30 Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 40 45 Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55 60 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70 75 80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85 90 95 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100 105 110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210 215 220 Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315 320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325 330 335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345 350 Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360 365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470 475 37888DNASphingobiurn herbicidovoransCDS(1)..(888)Native DNA sequence encoding Sphingobiurn herbicidovorans AAD1 protein 37atg cat gct gca ctg tcc ccc ctc tcc cag cgc ttt gag cgc atc gcg 48Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5 10 15 gtc cag ccg ctg acc ggc gtc ctg ggc gcc gag atc acc ggc gtc gac 96Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30 ctg cgc gag ccg ctc gac gac agc acc tgg aac gaa atc ctc gac gcg 144Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45 ttc cac act tac cag gtc atc tat ttt ccc ggc cag gcg atc acc aac 192Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60 gaa cag cac atc gcc ttc agc cgg cgc ttc ggc ccc gtc gat ccc gtg 240Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70 75 80 ccc ctg ctc aag agc atc gaa ggg tat cca gag gtg cag atg atc cgc 288Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90 95 cgc gaa gcc aac gaa agc ggg cgt gtg atc ggt gat gac tgg cac acc 336Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100 105 110 gac agc acc ttc ctg gac gca ccg ccg gcc gcc gtg gtg atg cgc gcg 384Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120 125 atc gac gtg ccc gag cat ggc ggc gac acc ggt ttt ctg agc atg tac 432Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135 140 acc gcg tgg gag acg ctg tcg ccc acc atg cag gcc acc atc gaa ggg 480Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150 155 160 ttg aac gta gtg cac agc gcc acg cgt gtg ttc ggc tcg ctc tac cag 528Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175 gcc cag aac cgg cgc ttc agc aac acc agc gtc aag gtg atg gac gtc 576Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190 gac gcg ggc gac cgt gaa acc gtg cac ccc ctg gtg gtg acc cat ccg 624Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205 ggc agc ggc cgc aag ggc ctg tac gtg aac cag gtc tat tgc cag cgc 672Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215 220 atc gag ggc atg acc gat gcc gaa agc aaa ccg ctg ctg cag ttc ctg 720Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230 235 240 tac gag cat gcg aca cgg ttc gat ttc acc tgc cgc gtg cgc tgg aag 768Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245 250 255 aag gac cag gtc ctg gtc tgg gac aac ctg tgc acg atg cac cgg gcc 816Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260 265 270 gta ccc gac tac gcg ggc aag ttc cgc tac ctg acg cgc acc acg gtc 864Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285 ggt ggc gtg cgc ccg gcg cgc tag 888Gly Gly Val Arg Pro Ala Arg 290 295 38295PRTSphingobiurn herbicidovorans 38Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5 10 15 Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30 Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45 Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60 Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70 75 80 Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90 95 Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100 105 110 Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120 125 Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135 140 Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150 155 160 Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175 Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190 Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205 Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215 220 Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230 235 240 Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245 250 255 Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260 265 270 Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285 Gly Gly Val Arg Pro Ala Arg 290 295 39888DNAArtificial SequenceSynthetic DNA sequence encoding the AAD1 protein using codons optimized for maize and Table 1 and Table 2 sequences are maintained 39atg cac gct gca ctg tca cca ctc tca cag cgc ttt gag aga att gcg 48Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5 10 15 gtc cag ccg ctg act ggc gtc ttg ggc gct gag atc acc ggc gtc gat 96Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30 ctg agg gag cct ctc gac gat tca acg tgg aac gaa att ctc gac gcg 144Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45 ttc cat act tac caa gtc atc tat ttt ccc ggg caa gct att acc aac 192Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60 gaa caa cac atc gct ttc tct cgg cga ttc ggc ccc gtc gat cca gtg 240Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70 75 80 ccc tta ctc aag tct atc gaa ggc tac cca gag gtg cag atg ata aga 288Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90 95 agg gag gcc aac gaa agc ggg cgt gtg ata ggt gat gac tgg cac act 336Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100 105 110 gac agc aca ttc ctg gat gca ccg ccg gcc gct gtg gtg atg agg gca 384Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120 125 atc gac gtg ccc gag cac gga ggt gac act ggt ttc ttg agt atg tac 432Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135 140 act gct tgg gag acg ctt tcg cct act atg caa gcc aca atc gag ggg 480Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150 155 160 ttg aat gta gtt cac agc gcc acg cgt gtg ttc gga tct ctc tat caa 528Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175 gcc caa aac cgg cgc ttt tca aat acc tcc gtc aag gtg atg gac gtt 576Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190 gac gcg ggc gac cgt gaa acc gtg cac cct ctt gtt gta acc cat ccg 624Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205 ggc agt ggt cgc aag ggc cta tac gtt aac caa gtc tat tgc cag cgc 672Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215 220 atc gag gga atg aca gac gca gag agt aag ccg ctc ctg caa ttc ctg 720Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230 235 240 tac gag cac gcg aca cgg ttc gat ttc acc tgc cgc gtg cgc tgg aaa 768Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245 250 255 aag gat caa gtc ctt gta tgg gac aac ctt tgt acg atg cac cgg gcc 816Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260 265 270 gtt cct gac tac gcg ggc aag ttc aga tac ctg acg agg acc acg gtc 864Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285 ggt gga gtt agg cca gcg aga tga 888Gly Gly Val Arg Pro Ala Arg 290 295 40295PRTArtificial SequenceSynthetic Construct 40Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5 10 15 Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30 Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45 Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60 Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70 75 80 Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90 95 Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100 105 110 Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120 125 Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135 140 Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150 155 160 Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175 Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190 Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205 Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215 220 Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230 235 240 Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245 250 255 Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260 265 270 Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285 Gly Gly Val Arg Pro Ala Arg 290 295 41888DNAArtificial SequenceSynthetic DNA sequence encoding the AAD1 protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 41atg cac gct gca ctg tca cca ctc tca cag cgc ttt gag aga att gcg 48Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5 10 15 gtc cag

ccg ctg act ggc gtc ttg ggc gct gag atc acc ggc gtc gat 96Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30 ctg agg gag cct ctc gac gat tca acg tgg aac gaa att ctc gac gcg 144Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45 ttc cat act tac caa gtc atc tac ttt ccc ggg caa gct att acc aac 192Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60 gaa caa cac atc gct ttc tct cgg cga ttc ggc ccc gtc gat cca gtg 240Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70 75 80 ccc tta ctc aag tct atc gaa ggc tac cca gag gtg cag atg ata aga 288Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90 95 agg gag gcc aac gaa agc ggg cgt gtg ata ggt gat gac tgg cac act 336Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100 105 110 gac agc aca ttc ctg gat gca ccg ccg gcc gct gtg gtg atg agg gca 384Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120 125 atc gac gtg ccc gag cac gga ggt gac act ggt ttc ttg agt atg tac 432Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135 140 act gct tgg gag acg ctt tcg cct act atg caa gcc aca atc gag ggg 480Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150 155 160 ttg aat gta gtt cac agc gcc acg cgt gtg ttc gga tct ctc tat caa 528Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175 gcc caa aac cgg cgc ttt tca aat acc tcc gtc aag gtg atg gac gtt 576Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190 gac gcg ggc gac cgt gaa acc gtg cac cct ctt gtt gta acc cat ccg 624Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205 ggc agt ggt cgc aag ggc cta tac gtt aac caa gtc tat tgc cag cgc 672Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215 220 atc gag gga atg aca gac gca gag agt aag ccg ctc ctg caa ttc ctg 720Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230 235 240 tac gag cac gcg aca cgg ttc gat ttc acc tgc cgc gtg cgc tgg aaa 768Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245 250 255 aag gat caa gtc ctt gta tgg gac aac ctt tgt acg atg cac cgg gcc 816Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260 265 270 gtt cct gac tac gcg ggc aag ttc aga tac ctg acg agg acc acg gtc 864Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285 ggt gga gtt agg cca gcg aga tga 888Gly Gly Val Arg Pro Ala Arg 290 295 42295PRTArtificial SequenceSynthetic Construct 42Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5 10 15 Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20 25 30 Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40 45 Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55 60 Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70 75 80 Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85 90 95 Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100 105 110 Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120 125 Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135 140 Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150 155 160 Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165 170 175 Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180 185 190 Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro 195 200 205 Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210 215 220 Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230 235 240 Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245 250 255 Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260 265 270 Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275 280 285 Gly Gly Val Arg Pro Ala Arg 290 295 431368DNAAspergillus nidulansCDS(1)..(1368)Native DNA sequence encoding Aspergillus nidulans delta-9 fatty acid desaturase protein 43atg tct gca cca acg gcg gac atc agg gct cgc gcc ccg gag gcc aaa 48Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5 10 15 aag gtt cac atc gct gac act gct atc aac cgc cat aac tgg tac aag 96Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25 30 cat gtg aac tgg ctg aac gtt ttc ctg atc atc ggt atc ccg ctt tat 144His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40 45 ggg tgc att cag gcg ttc tgg gtg cca ctg cag ctg aag act gcc atc 192Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50 55 60 tgg gcc gtc atc tac tac ttt ttc acc ggt ctc ggt atc aca gca ggt 240Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70 75 80 tac cat cgt cta tgg gct cac tgc tcg tac tcc gcc acc ctt cct ttg 288Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85 90 95 cgt atc tgg ctc gct gcc gtt ggt ggt ggt gcc gtc gaa ggt tct atc 336Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100 105 110 cgc tgg tgg gct cgt gac cac cgc gct cac cac cgc tac acc gat acc 384Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115 120 125 gac aaa gac ccg tac tcc gtt cgc aag ggt ctg ctc tac tct cac ctt 432Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135 140 ggc tgg atg gtg atg aag cag aac cct aag cgt att ggc cgt acc gat 480Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145 150 155 160 att tcc gac ctg aac gag gac ccc gtc gtt gtc tgg cag cac cgc aac 528Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165 170 175 tac ctc aag gtc gtt ttc acg atg gga ttg gct gtg cct atg ctt gtt 576Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180 185 190 gct ggt ctt gga tgg ggt gac tgg ttg ggc ggc ttc gtg tat gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195 200 205 att ctg cgt atc ttc ttc gtc cag cag gcg act ttc tgc gtc aac tct 672Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 ttg gcc cac tgg ctc ggt gac cag ccc ttc gat gac cgc aac tca cct 720Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225 230 235 240 cgt gac cac gtt atc acc gct ctc gtc acc ctt gga gag ggc tac cac 768Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245 250 255 aac ttc cac cac gag ttc ccc tcg gac tac cgt aac gcc atc gaa tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260 265 270 cac cag tat gat ccc acc aag tgg tcc atc tgg gcc tgg aag cag ctt 864His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu 275 280 285 ggt ctt gcc tac gac ctg aag aag ttc cgt gcc aac gag att gag aag 912Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295 300 ggt cgt gtc cag cag ctc cag aag aag ctt gac cgt aag cgt gcc act 960Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310 315 320 ctc gat tgg ggt act cct ctt gac cag ctc ccc gtc atg gag tgg gac 1008Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp 325 330 335 gac tac gtc gag cag gct aag aac ggc cgc ggt ctc gtg gct att gcc 1056Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345 350 ggt gtt gtc cac gat gtc acg gac ttc atc aaa gac cac ccc ggt ggc 1104Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360 365 aag gcc atg atc agc tcc ggt att ggg aag gac gcc acc gcc atg ttc 1152Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375 380 aac ggt ggt gtc tac tac cac tcc aac gcc gca cac aac ctc ctc tct 1200Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385 390 395 400 acc atg cgt gtt ggt gtt atc cgc ggc ggc tgt gaa gtc gaa atc tgg 1248Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405 410 415 aag cgt gcc cag aag gag aac gtg gag tac gtg cgt gat ggc tct ggc 1296Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420 425 430 cag cgc gtc atc cgt gcc ggc gag cag cca acc aag atc cca gaa ccc 1344Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440 445 att ccc aca gcg gat gcg gcg tga 1368Ile Pro Thr Ala Asp Ala Ala 450 455 44455PRTAspergillus nidulans 44Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5 10 15 Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25 30 His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40 45 Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50 55 60 Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70 75 80 Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85 90 95 Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100 105 110 Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115 120 125 Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135 140 Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145 150 155 160 Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165 170 175 Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180 185 190 Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195 200 205 Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225 230 235 240 Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245 250 255 Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260 265 270 His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu 275 280 285 Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295 300 Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310 315 320 Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp 325 330 335 Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345 350 Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360 365 Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375 380 Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385 390 395 400 Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405 410 415 Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420 425 430 Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440 445 Ile Pro Thr Ala Asp Ala Ala 450 455 451368DNAArtificial SequenceSynthetic DNA sequence encoding Aspergillus nidulans delta-9 fatty acid desaturase protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained 45atg agt gca cca acg gcg gac ata agg gcg cgc gcc ccg gag gca aaa 48Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5 10 15 aag gtt cac att gct gac act gct atc aat cgc cat aac tgg tat aag 96Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25 30 cat gtg aat tgg ctg aac gtt ttt ctg atc atc ggc atc ccg ctt tat 144His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40 45 ggg tgt att caa gcg ttc tgg gtg cca ctc cag ctc aag act gcc atc 192Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50 55 60 tgg gcc gta atc tac tac ttc ttt acc ggt ttg gga atc aca gcg ggt 240Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70 75 80 tat cac aga ttg tgg gca cac tgc tcg tac tcc gcc acc ctt cct tta 288Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85 90 95 cgt ata tgg ctc gct gcc gta gga gga ggc gcc gtc gaa ggt tca atc 336Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100 105 110 cgt tgg tgg gct aga gac cat cgt gct cat cat aga tat acc gat aca 384Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115 120 125 gac aaa gac ccg tac tcc gtt

cgc aag ggg ctg cta tac tct cac ctt 432Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135 140 ggc tgg atg gtg atg aag cag aac cct aag cgt att ggc aga acc gat 480Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145 150 155 160 att tcc gac ctg aac gag gac ccc gtc gtt gtc tgg cag cac cgg aac 528Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165 170 175 tac ctc aag gtc gtt ttc acg atg gga ttg gct gtg cct atg ctt gtt 576Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180 185 190 gct ggg ctt ggc tgg gga gac tgg ttg ggc ggc ttc gtg tat gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195 200 205 ata ctg aga atc ttt ttc gtc cag caa gcg act ttt tgc gtc aac tct 672Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 ttg gcc cac tgg ctc gga gat cag ccg ttc gat gac cgg aac agt cct 720Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225 230 235 240 agg gac cac gtt atc act gct ctc gtc acc cta gga gag ggc tac cac 768Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245 250 255 aac ttc cat cac gag ttc ccc tcg gac tac cgg aac gcc atc gaa tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260 265 270 cac cag tat gat cca acg aag tgg agc atc tgg gcc tgg aag cag ctt 864His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu 275 280 285 ggt tta gcc tac gac ctg aag aaa ttc aga gcc aac gag att gag aaa 912Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295 300 ggg cgt gtc caa cag ctg caa aag aaa ctg gac cgt aag cgg gcg act 960Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310 315 320 ctc gat tgg gga aca cct ctg gat cag ctc ccc gtc atg gag tgg gac 1008Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp 325 330 335 gac tac gtg gag caa gca aag aac ggt cgc ggt ctc gtg gca ata gcg 1056Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345 350 ggc gtg gtg cac gat gtc acg gat ttc atc aaa gat cac ccg ggg ggc 1104Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360 365 aag gcc atg atc agc tcc ggg att ggc aag gac gca acc gcc atg ttc 1152Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375 380 aat ggg gga gtc tac tac cac agc aac gca gca cac aat ctc ttg tca 1200Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385 390 395 400 aca atg agg gtg ggt gtt att agg ggc ggc tgt gaa gtc gaa atc tgg 1248Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405 410 415 aag agg gcg caa aag gag aat gtg gag tac gtg cga gat ggc tct ggt 1296Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420 425 430 caa cgc gtg atc aga gcg ggc gag cag cca acc aag ata cca gaa ccg 1344Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440 445 att ccc aca gcg gat gcg gcg tag 1368Ile Pro Thr Ala Asp Ala Ala 450 455 46455PRTArtificial SequenceSynthetic Construct 46Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5 10 15 Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25 30 His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40 45 Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50 55 60 Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70 75 80 Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85 90 95 Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100 105 110 Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115 120 125 Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135 140 Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145 150 155 160 Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165 170 175 Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180 185 190 Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195 200 205 Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225 230 235 240 Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245 250 255 Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260 265 270 His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu 275 280 285 Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295 300 Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310 315 320 Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp 325 330 335 Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345 350 Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360 365 Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375 380 Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385 390 395 400 Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405 410 415 Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420 425 430 Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440 445 Ile Pro Thr Ala Asp Ala Ala 450 455 471368DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Aspergillus nidulans delta-9 fatty acid desaturase protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 47atg agt gca cca acg gcg gac ata agg gcg cgc gcc ccg gag gca aaa 48Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5 10 15 aag gtt cac att gct gac act gct atc aat cgc cat aac tgg tat aag 96Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25 30 cat gtg aat tgg ctg aac gtt ttt ctg atc atc ggc atc ccg ctt tat 144His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40 45 ggg tgt att caa gcg ttc tgg gtg cca ctc cag ctc aag act gcc atc 192Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50 55 60 tgg gcc gta atc tac tac ttc ttt acc ggt ttg gga atc aca gcg ggt 240Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70 75 80 tat cac aga ttg tgg gca cac tgc tcg tac tcc gcc acc ctt cct tta 288Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85 90 95 cgt ata tgg ctc gct gcc gta gga gga ggc gcc gtc gaa ggt tca atc 336Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100 105 110 cgt tgg tgg gct aga gac cat cgt gct cat cat aga tat acc gat aca 384Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115 120 125 gac aaa gac ccg tac tcc gtt cgc aag ggg ctg cta tac tct cac ctt 432Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135 140 ggc tgg atg gtg atg aag cag aac cct aag cgt att ggc aga acc gat 480Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145 150 155 160 att agc gac ctg aac gag gac ccc gtc gtt gtc tgg cag cac cgg aac 528Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165 170 175 tac ctc aag gtc gtt ttc acg atg gga ttg gct gtg cct atg ctt gtt 576Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180 185 190 gct ggg ctt ggc tgg gga gac tgg ttg ggc ggc ttc gtg tat gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195 200 205 ata ctg aga atc ttt ttc gtc cag caa gcg act ttt tgc gtc aac tct 672Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 ttg gcc cac tgg ctc gga gat cag ccg ttc gat gac cgg aac agt cct 720Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225 230 235 240 agg gac cac gtt atc act gct ctc gtc acc cta gga gag ggc tac cac 768Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245 250 255 aac ttc cat cac gag ttc ccc tcg gac tac cgg aac gcc atc gaa tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260 265 270 cac cag tat gat cca acg aag tgg agc atc tgg gcc tgg aag cag ctt 864His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu 275 280 285 ggt tta gcc tac gac ctg aag aaa ttc aga gcc aac gag att gag aaa 912Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295 300 ggg cgt gtc caa cag ctg caa aag aaa ctg gac cgt aag cgg gcg act 960Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310 315 320 ctc gat tgg gga aca cct ctg gat cag ctc ccc gtc atg gag tgg gac 1008Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp 325 330 335 gac tac gtg gag caa gca aag aac ggt cgc ggt ctc gtg gca ata gcg 1056Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345 350 ggc gtg gtg cac gat gtc acg gat ttc atc aaa gat cac ccg ggg ggc 1104Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360 365 aag gcc atg atc agc tcc ggg att ggc aag gac gca acc gcc atg ttc 1152Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375 380 aat ggg gga gtc tac tac cac agc aac gca gca cac aat ctc ttg tca 1200Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385 390 395 400 aca atg agg gtg ggt gtt att agg ggc ggc tgt gaa gtc gaa atc tgg 1248Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405 410 415 aag agg gcg caa aag gag aat gtg gag tac gtg cga gat ggc tct ggt 1296Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420 425 430 caa cgc gtg atc aga gcg ggc gag cag cca acc aag ata cca gaa ccg 1344Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440 445 att ccc aca gcg gat gcg gcg tag 1368Ile Pro Thr Ala Asp Ala Ala 450 455 48455PRTArtificial SequenceSynthetic Construct 48Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5 10 15 Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25 30 His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40 45 Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50 55 60 Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70 75 80 Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85 90 95 Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100 105 110 Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115 120 125 Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135 140 Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145 150 155 160 Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165 170 175 Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180 185 190 Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195 200 205 Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225 230 235 240 Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245 250 255 Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260 265 270 His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu 275 280 285 Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295 300 Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310 315 320 Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp 325 330 335 Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345 350 Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360 365 Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375 380 Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385 390 395 400 Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405 410 415 Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420 425 430 Gln Arg Val Ile Arg Ala Gly

Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440 445 Ile Pro Thr Ala Asp Ala Ala 450 455 49798DNAXerophyta viscosaCDS(1)..(798)Native DNA sequence encoding Xerophyta viscosa SAP1 protein 49atg agg aac gag ggt ttt ctg aaa atg aag acc gac gtt gga gtc gcc 48Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5 10 15 gac gag gtg atc tcc gga gat ctc aag cag ctt ggt gac gct gca aag 96Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30 cgg cta gct aaa cat gcg atc aag ctc ggc gcc agc ttc ggg gtt ggc 144Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45 tct acc ata gtc cag gct att gct tcg atc gct gct atc tat ttg ttg 192Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60 ata ttg gac cgg aca aac tgg cgt aca aat atc ttg aca tca ctt cta 240Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70 75 80 att cca tat gtt tac ttg agt ctt cct tca gtg ata ttc aac cta ttc 288Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95 agg ggc gac ctg ggc aga tgg ctt tca ttc att ggc gta gta atg aag 336Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110 ctc ttc ttc cac cga cac ttc cca gtt acc ttg gaa ctg ctt gtg tct 384Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125 ctc att ctc ctg att gtg gtt tcc ccc act ttc att gcc cac aca atc 432Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140 aga ggc agt ctc att gga gtc ttc atc ttc ctt gtc atc gcc tgc tac 480Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150 155 160 ctc ctc caa gag cac att aga tca gct ggt ggc ttc aaa aac gcg ttc 528Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175 aca aag agc aat ggg att tca aac agc gtc ggg atc atc att cta ctg 576Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190 atc cac ccg atc tgg agc ttg gtg gtg tat ttc ctc tac acg tct ttg 624Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205 ctg caa ctt ctt gca tac tct cct tcc cct tgt tgt tgc ata tta tac 672Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220 aat aag tgg ttt aat ttc atg cat gtt tgt aaa tgt gta agc ctt cat 720Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230 235 240 atg tat tct cag tca att ggg tca tgc gtg tcc ata ttt ttc gtg cag 768Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255 ttt gta ttc atc tat gaa gct gaa ttt taa 798Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 265 50265PRTXerophyta viscosa 50Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5 10 15 Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30 Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45 Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60 Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70 75 80 Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95 Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110 Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125 Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140 Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150 155 160 Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175 Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190 Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205 Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220 Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230 235 240 Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255 Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 265 51798DNAArtificial SequenceSynthetic DNA sequence encoding Xerophyta viscosa SAP1 protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained 51atg aga aac gaa ggt ttt ctg aag atg aaa acg gac gtt ggg gtt gct 48Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5 10 15 gac gaa gtc atc agc ggt gat ttg aag cag ttg ggt gat gct gcc aaa 96Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30 cgc ctt gct aag cac gct atc aaa ctg gga gcc agc ttt ggt gtt ggt 144Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45 tca act atc gtt caa gcc atc gca tca ata gca gcc atc tat ctt ctg 192Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60 att ctc gat agg acc aac tgg agg acc aac atc ttg acg tcc ctc ctc 240Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70 75 80 att ccc tac gtg tat ctg tcc ctc ccg agc gtc atc ttc aat ctc ttt 288Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95 cgt ggg gac ctc ggg aga tgg ctg tca ttc ata ggc gtt gtg atg aag 336Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110 ctg ttc ttt cat agg cac ttt cct gtt act ttg gag ctg ctt gtg agc 384Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125 ctc att ctt ttg att gtc gtg tca cct acc ttc ata gct cat aca att 432Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140 cgt gga tct ttg att ggg gtg ttc atc ttc ttg gtg ata gca tgt tat 480Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150 155 160 ctg ctt caa gag cac att aga tca gct ggt ggc ttc aag aac gcc ttt 528Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175 aca aag tct aat gga atc tcc aac agc gtg ggc atc atc atc ctt ctg 576Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190 atc cac ccg att tgg tct ctc gtc gtc tac ttc ctc tac act tca ctt 624Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205 ctc cag ctt ttg gcc tac tca cca tcc ccg tgc tgc tgc ata tta tac 672Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220 aac aag tgg ttc aac ttc atg cat gtt tgc aag tgc gtc tct ttg cac 720Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230 235 240 atg tac tct cag tcc ata ggc tca tgt gtt tca ata ttt ttc gtc cag 768Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255 ttc gtg ttc atc tat gag gct gag ttt taa 798Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 265 52265PRTArtificial SequenceSynthetic Construct 52Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5 10 15 Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30 Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45 Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60 Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70 75 80 Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95 Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110 Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125 Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140 Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150 155 160 Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175 Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190 Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205 Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220 Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230 235 240 Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255 Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 265 53798DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Xerophyta viscosa SAP1 protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 53atg aga aac gaa ggt ttt ctg aag atg aaa acg gac gtt ggg gtt gct 48Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5 10 15 gac gaa gtc atc agc ggt gat ttg aag cag ttg ggt gat gct gcc aaa 96Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30 cgc ctt gct aag cac gct atc aaa ctg gga gcc agc ttt ggt gtt ggt 144Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45 tca act atc gtt caa gcc atc gca tca ata gca gcc atc tat ctt ctg 192Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60 att ctc gat agg acc aac tgg agg acc aac atc ttg acg tcc ctc ctc 240Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70 75 80 att ccc tac gtg tat ctg tcc ctc ccg agc gtc atc ttc aat ctc ttt 288Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95 cgt ggg gac ctc ggg aga tgg ctg tca ttc ata ggc gtt gtg atg aag 336Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110 ctg ttc ttt cat agg cac ttt cct gtt act ttg gag ctg ctt gtg agc 384Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125 ctc att ctt ttg att gtc gtg tct cct acc ttc ata gct cat aca att 432Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140 cgt gga tct ttg att ggg gtg ttc atc ttc ttg gtg ata gca tgt tat 480Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150 155 160 ctg ctt caa gag cac att aga tca gct ggt ggc ttc aag aac gcc ttt 528Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175 aca aag tct aat gga atc tcc aac agc gtg ggc atc atc atc ctt ctg 576Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190 atc cac ccg att tgg tct ctc gtc gtc tac ttc ctc tac act tca ctt 624Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205 ctc cag ctt ttg gcc tac tca cca tcc cca tgc tgc tgt att ctt tac 672Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220 aac aaa tgg ttc aac ttc atg cac gtg tgc aag tgc gtc tct ttg cac 720Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230 235 240 atg tac tct cag tcc att ggc tca tgt gtt tca atc ttc ttt gtc cag 768Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255 ttc gtg ttc atc tat gag gct gag ttt taa 798Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 265 54265PRTArtificial SequenceSynthetic Construct 54Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5 10 15 Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25 30 Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35 40 45 Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55 60 Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70 75 80 Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85 90 95 Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100 105 110 Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115 120 125 Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130 135 140 Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150 155 160 Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165 170 175 Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185 190 Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu 195 200 205 Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210 215 220 Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230 235 240 Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245 250 255 Phe Val Phe Ile Tyr Glu Ala Glu Phe 260 265 55717DNAAequorea victoriaCDS(1)..(717)Native DNA sequence encoding Aequorea victoria

GFP1 protein 55atg agt aaa gga gaa gaa ctt ttc act gga gtg gtc cca gtt ctt gtt 48Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5 10 15 gaa tta gat ggc gat gtt aat ggg caa aaa ttc tct gtc agt gga gag 96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aat ttt att tgc 144Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35 40 45 act act ggg aag cta cct gtt cca tgg cca aca ctt gtc act act ttc 192Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtt caa tgc ttc tca aga tac cca gat cat atg aaa cag 240Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga 288His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt tac aaa gat gac ggg aac tac aag aca cgt gct gaa gtc 336Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggt att 384Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa atg gaa tac aac 432Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 tat aac tca cat aat gta tac atc atg gga gac aaa cca aag aat ggc 480Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttc aaa att aga cac aac att aaa gat gga agc gtt 528Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 caa tta gca gac cat tat caa caa aat act cca att ggc gat ggc cct 576Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gac aac cat tac ctg tcc aca caa tct gcc ctt tcc 624Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaa aag aga gat cac atg atc ctt ctt gag ttt gta 672Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 aca gct gct agg att aca cat ggc atg gat gaa cta tac aaa taa 717Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 56238PRTAequorea victoria 56Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 57717DNAArtificial SequenceSynthetic DNA sequence encoding Aequorea victoria GFP1 protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained 57atg agt aaa ggg gaa gaa ctt ttc acc ggc gtg gtc cca gtc ctc gtt 48Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5 10 15 gag ttg gat ggc gat gtg aat ggg caa aaa ttc tct gtc tcc ggg gag 96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gag ggt gat gca acc tac gga aag ctg acc cta aat ttt att tgc 144Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35 40 45 acg act ggg aag ttg cct gtg cct tgg ccg aca ctg gtg acg acg ttc 192Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtg cag tgt ttc tca cgc tac ccg gat cat atg aaa cag 240Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag tcg gcc atg cca gaa ggc tat gta caa gag aga 288His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt tac aag gac gac ggg aac tac aag aca cgt gct gag gtg 336Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttc gag ggt gat acc ctt gtt aat cgg atc gag cta aag ggc att 384Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gac ttt aag gag gac gga aac att ctg gga cac aaa atg gaa tac aac 432Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 tat aac tcg cac aac gta tac atc atg gga gac aaa cca aag aat ggc 480Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145 150 155 160 ata aag gtt aac ttc aag att cga cac aac att aaa gac ggc agc gtt 528Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 cag ttg gcc gac cac tat caa caa aat act cca att ggc gat ggc cct 576Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctc tta ccc gac aac cat tac ctg tcc acg caa tca gcg ctc agc 624Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aag gac ccc aac gag aag agg gat cac atg atc ctc ctt gag ttt gtc 672Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 acc gca gct agg ata acc cac ggc atg gat gaa ctg tac aag taa 717Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 58238PRTArtificial SequenceSynthetic Construct 58Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 59717DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Aequorea victoria GFP1 protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 59atg agt aaa ggg gaa gaa ctt ttc acc ggc gtg gtc cca gtc ctc gtt 48Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5 10 15 gag ttg gat ggc gat gtg aat ggg caa aaa ttc tct gtc tcc ggg gag 96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gag ggt gat gca acc tac gga aag ctg acc cta aat ttc atc tgc 144Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35 40 45 acg act ggg aag ttg cct gtg cct tgg ccg aca ctg gtg acg acg ttc 192Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 tct tat ggt gtg cag tgt ttc tca cgc tac ccg gat cat atg aaa cag 240Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aag tcg gcc atg cca gaa ggc tat gta caa gag aga 288His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act atc ttt tac aag gac gac ggg aac tac aag aca cgt gct gag gtg 336Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttc gag ggt gat acc ctt gtt aat cgg atc gag cta aag ggc att 384Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gac ttt aag gag gac gga aac att ctg gga cac aaa atg gaa tac aac 432Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 tat aac tcg cac aac gta tac atc atg gga gac aaa cca aag aat ggc 480Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145 150 155 160 ata aag gtt aac ttc aag att cga cac aac att aaa gac ggc agc gtt 528Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 cag ttg gcc gac cac tat caa caa aat act cca att ggc gat ggc cct 576Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctc tta ccc gac aac cat tac ctg tcc acg caa tca gcg ctc agc 624Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aag gac ccc aac gag aag agg gat cac atg atc ctc ctt gag ttt gtc 672Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 acc gca gct agg ata acc cac ggc atg gat gaa ctg tac aag taa 717Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 60238PRTArtificial SequenceSynthetic Construct 60Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230 235 611350DNALeptosphaeria nodorumCDS(1)..(1350)Native DNA sequence encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein 61atg gcg gcc ttg gac agc att cca gag gat aag gct acc tcg tcg aaa 48Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5 10 15 tcg act cat att caa tat caa gaa gta act ttt cgg aac tgg tat aag 96Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20 25 30 aag ata aat tgg ctc aac acg acg ctg gtg gtg ctc ata ccc gct ctt 144Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35 40 45 gga ctc tac cta aca cgc acc acg cca ctt aca cga cct acg ctc atc 192Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55 60 tgg tcc gtc ctg tac tac ttc tgc aca gct ttc ggc atc aca ggc gga 240Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70 75 80 tat cat cga cta tgg agt cat cgc agc tac tcc gct cgt cta ccg cta 288Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90 95 cgc tta ttc cta gcc ttc aca ggc gcc gga gcc atc caa ggt agt gct 336Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105 110 cga tgg tgg agc gca aat cac cgc gcc cac cac cga tgg acc gac aca 384Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115 120 125 atg aag gac ccc tac tcc gtt atg cgc ggc cta tta ttc tcg cac atc 432Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130 135 140 gga tgg atg gta ttg aac agc gac ccc aaa gtc aaa ggc cga aca gac 480Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150 155

160 gtc agt gat ctc gac agc gac ccc gtc gta gtc tgg cag cac aag cac 528Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His 165 170 175 tac ggc aag tgc ctg ctg ttc gcc gcg tgg ata ttc ccc atg atc gta 576Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180 185 190 gcc ggc ctc gga tgg gga gat tgg tgg gga ggc ctt gtc tac gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200 205 atc att cga gcg tgt ttc gtc cag cag gcg aca ttt tgc gtg aac tct 672Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 ctc gcg cat tgg atc ggc gag cag ccg ttc gac gac aga cgc acg cct 720Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225 230 235 240 cga gac cac gtt ttg aca gcg ttg gta acg atg gga gaa gga tat cat 768Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245 250 255 aac ttc cac cac gaa ttc cca agc gat tat cgc aac gcg atc atc tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260 265 270 tac caa tac gac cct acc aaa tgg ctc att tac ctc ttc tcc ctc ggc 864Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275 280 285 ccc ttc ccc ctc gca tac tcg ctc aaa acc ttc cgg tcc aat gag att 912Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295 300 gaa aaa ggg cgg ttg caa caa caa caa aaa gcc ctg gac aag aag cgc 960Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310 315 320 tca gga ctt gat tgg ggc cta ccc ctc ttc caa ctc cct gtc ata tcg 1008Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325 330 335 tgg gac gac ttc caa gcg cgt tgc aaa gag tcc ggc gag atg ctg gtt 1056Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340 345 350 gct gtc gca ggt gtg att cac gac gtc agc cag ttt att gaa gat cac 1104Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355 360 365 cct gga ggc agg agt ttg att cgg agt gcg gtg ggc aaa gat ggg aca 1152Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375 380 ggg atg ttt aat gga ggc gta tat gag cac agt aat gcg gcg cat aat 1200Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390 395 400 ctg ttg tcg aca atg agg gtg gga gtg ctt aga ggt ggg cag gag gtg 1248Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405 410 415 gag gtg tgg aag aag cag aga gtg gat gtt tta ggg aag agc gac att 1296Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420 425 430 ttg aga cag gtt acg cgg gtg gag agg ttg gtt gag ggg gct gtg gct 1344Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440 445 gcg tag 1350Ala 62449PRTLeptosphaeria nodorum 62Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5 10 15 Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20 25 30 Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35 40 45 Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55 60 Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70 75 80 Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90 95 Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105 110 Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115 120 125 Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130 135 140 Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150 155 160 Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His 165 170 175 Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180 185 190 Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200 205 Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225 230 235 240 Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245 250 255 Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260 265 270 Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275 280 285 Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295 300 Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310 315 320 Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325 330 335 Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340 345 350 Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355 360 365 Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375 380 Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390 395 400 Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405 410 415 Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420 425 430 Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440 445 Ala 631350DNAArtificial SequenceSynthetic DNA sequence encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained 63atg gca gcc ctt gac agc atc cca gag gat aag gct acc tcg tct aaa 48Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5 10 15 tcg act cat att cag tac caa gaa gtg act ttt cgg aac tgg tac aaa 96Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20 25 30 aag ata aac tgg ctc aac acg acg ctg gtg gtg ctc ata cca gct ctt 144Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35 40 45 ggt ctt tac cta aca agg acc acg cca ctt act agg cca acg ctc atc 192Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55 60 tgg tcc gtc ctg tac tac ttt tgc acc gct ttc ggc att acc ggc gga 240Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70 75 80 tat cat aga cta tgg agt cat cgc agc tac tcc gct cgt cta ccg ctt 288Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90 95 cgc ttg ttc ctg gcc ttc act ggc gcc ggg gcc atc caa ggt tca gct 336Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105 110 agg tgg tgg agc gca aat cac cgc gcc cat cat agg tgg acc gac aca 384Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115 120 125 atg aag gac ccc tac tcc gtt atg cgc ggt cta tta ttc tcg cac atc 432Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130 135 140 ggt tgg atg gtt cta aac agc gac ccc aaa gtc aaa ggc cgc act gac 480Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150 155 160 gtc tca gac cta gat agc gac ccc gtc gtt gtc tgg cag cac aag cac 528Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His 165 170 175 tac ggc aag tgc ctg cta ttt gcc gca tgg ata ttc ccg atg atc gta 576Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180 185 190 gcc ggc ctc gga tgg gga gat tgg tgg gga ggc ctt gtc tac gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200 205 atc att agg gcg tgt ttc gtc cag caa gca acc ttt tgc gtg aac tct 672Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 ctc gcg cac tgg atc ggc gag cag ccg ttc gac gac aga cgc acc cct 720Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225 230 235 240 aga gac cac gtt ttg acc gcg ttg gtc act atg gga gaa ggt tat cac 768Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245 250 255 aac ttc cac cac gag ttc ccg tct gat tat agg aac gcg atc atc tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260 265 270 tat cag tac gac cct acc aaa tgg ctc ata tac ctc ttc tcc ctc ggc 864Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275 280 285 ccg ttc cca ctg gca tac tcg ctc aaa acc ttc cgg tct aac gag atc 912Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295 300 gaa aag ggg cgg ttg caa caa caa caa aag gcc ctg gat aag aag cgc 960Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310 315 320 tct ggc ctt gat tgg ggc ctg ccc ctc ttc cag ctc cct gtg ata tct 1008Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325 330 335 tgg gac gac ttc caa gcg cgt tgt aag gag tcc ggc gag atg ctg gtt 1056Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340 345 350 gct gtc gcc ggt gtg att cac gac gtc tca cag ttt att gaa gat cac 1104Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355 360 365 cct gga ggg agg agt ctg att cgg tct gcg gtg ggc aag gat ggg act 1152Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375 380 ggg atg ttt aat gga ggc gtt tat gag cac agt aat gcg gcg cac aat 1200Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390 395 400 ctg ttg tca aca atg agg gtg ggt gtg ctt aga ggt ggg caa gag gtg 1248Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405 410 415 gag gtg tgg aag aag cag cgt gtg gat gtt tta ggg aag agc gat atc 1296Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420 425 430 ttg cgt caa gtt acg cgg gtg gag agg ctg gtt gag ggg gct gtg gct 1344Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440 445 gcc tag 1350Ala 64449PRTArtificial SequenceSynthetic Construct 64Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5 10 15 Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20 25 30 Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35 40 45 Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55 60 Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70 75 80 Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90 95 Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105 110 Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115 120 125 Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130 135 140 Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150 155 160 Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His 165 170 175 Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180 185 190 Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200 205 Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225 230 235 240 Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245 250 255 Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260 265 270 Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275 280 285 Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295 300 Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310 315 320 Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325 330 335 Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340 345 350 Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355 360 365 Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375 380 Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390 395 400 Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405 410 415 Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420 425 430 Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440 445 Ala 651350DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 65atg gca gcc ctt gac agc atc cca gag gat aag gct acc tcg tct aaa 48Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5 10 15 tcg act cat att cag tac caa gaa gtg act ttt

cgg aac tgg tac aaa 96Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20 25 30 aag ata aac tgg ctc aac acg acg ctg gtg gtg ctc ata cca gct ctt 144Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35 40 45 ggt ctt tac cta aca agg acc acg cca ctt act agg cca acg ctc atc 192Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55 60 tgg tcc gtc ctg tac tac ttt tgc acc gct ttc ggc att acc ggc gga 240Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70 75 80 tat cat aga cta tgg agt cat cgc agc tac tcc gct cgt cta ccg ctt 288Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90 95 cgc ttg ttc ctg gcc ttc act ggc gcc ggg gcc atc caa ggt tca gct 336Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105 110 agg tgg tgg agc gca aat cac cgc gcc cat cat agg tgg acc gac aca 384Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115 120 125 atg aag gac ccc tac tcc gtt atg cgc ggt ctg tta ttc tcg cac atc 432Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130 135 140 ggt tgg atg gtt cta aac agc gac ccc aaa gtc aaa ggc cgc act gac 480Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150 155 160 gtc tca gac cta gat agc gac ccc gtc gtt gtc tgg cag cac aag cac 528Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His 165 170 175 tac ggc aag tgc ctg cta ttt gcc gca tgg ata ttc ccg atg atc gta 576Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180 185 190 gcc ggc ctc gga tgg gga gat tgg tgg gga ggc ctt gtc tac gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200 205 atc att agg gcg tgt ttc gtc cag caa gca acc ttt tgc gtg aac tct 672Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 ctc gcg cac tgg atc ggc gag cag ccg ttc gac gac aga cgc acc cct 720Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225 230 235 240 aga gac cac gtt ttg acc gcg ttg gtc act atg gga gaa ggt tat cac 768Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245 250 255 aac ttc cac cac gag ttc ccg tct gat tat agg aac gcg atc atc tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260 265 270 tat cag tac gac cct acc aaa tgg ctc ata tac ctc ttc tcc ctc ggc 864Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275 280 285 ccg ttc cca ctg gca tac tcg ctc aaa acc ttc cgg tct aac gag atc 912Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295 300 gaa aag ggg cgg ttg caa caa caa caa aag gcc ctg gat aag aag cgc 960Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310 315 320 tct ggc ctt gat tgg ggc ctg ccc ctc ttc cag ctc cct gtg ata tct 1008Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325 330 335 tgg gac gac ttc caa gcg cgt tgt aag gag tcc ggc gag atg ctg gtt 1056Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340 345 350 gct gtc gcc ggt gtg att cac gac gtc tca cag ttc att gaa gat cac 1104Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355 360 365 cct gga ggg agg agt ctg att cgg tct gcg gtg ggc aag gat ggg act 1152Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375 380 ggg atg ttt aat gga ggc gtt tat gag cac agt aat gcg gcg cac aat 1200Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390 395 400 ctg ttg tca aca atg agg gtg ggt gtg ctt aga ggt ggg caa gag gtg 1248Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405 410 415 gag gtg tgg aag aag cag cgt gtg gat gta tta ggg aag agc gat atc 1296Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420 425 430 ttg cgt caa gtt acg cgg gtg gag agg ctg gtt gag ggg gct gtg gct 1344Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440 445 gcc tag 1350Ala 66449PRTArtificial SequenceSynthetic Construct 66Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5 10 15 Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20 25 30 Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35 40 45 Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55 60 Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70 75 80 Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90 95 Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105 110 Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115 120 125 Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130 135 140 Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150 155 160 Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His 165 170 175 Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180 185 190 Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200 205 Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215 220 Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225 230 235 240 Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245 250 255 Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260 265 270 Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275 280 285 Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295 300 Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310 315 320 Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325 330 335 Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340 345 350 Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355 360 365 Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375 380 Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390 395 400 Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405 410 415 Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420 425 430 Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440 445 Ala 67660DNAXerophyta viscosaCDS(1)..(660)Native DNA sequence encoding Xerophyta viscosa PER1 protein 67atg ccg ggg ctc acc att ggc gac acg atc ccc aac ctg gag ctt gac 48Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1 5 10 15 acc acc cag ggt agg atc aaa atc cac gat tac gtc ggc aac ggc tac 96Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30 gtc atc ttg ttc tca cac cct gga gac ttc act cct gtc tgc acc acc 144Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 gaa ctt gga aag atg gct gct tac gcc gac gag ttc agc aag cgc ggg 192Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55 60 gtt aag ctt ctt ggt ctt tcc tgc gac gat gta cag agc cac aag gag 240Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70 75 80 tgg atc aag gat atc gaa gcc tat acg ccg gga tgt cac gta aaa tat 288Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85 90 95 cct atc gcg gcg gac cca acc cgc gag att atc cag cag cta aac atg 336Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100 105 110 gta gac cca gac gag aca gag tcc agc aaa tgc gcc gtg cct tcg cga 384Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115 120 125 gct ctg cac atc att ggg ccc gac aag agg atc aag ctg agt ttc ctg 432Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135 140 tac ccc gcg tcg acg ggg cga aac atg gat gag gtg ctg agg gca gtg 480Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150 155 160 gag tcg ctc cag cag gcg gca aag cac aag gtg gca acg ccg gcg aac 528Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165 170 175 tgg aag cct ggt gaa cct gtt gtg atc aag cct gat gtg tcc agc gag 576Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu 180 185 190 gag gcc aag aag ctt ttc ccg cag ggt tat aaa agt gtt gat ctt cca 624Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200 205 tcc aag aag gat tac ctt cgt ttt acg aac gtc tga 660Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215 68219PRTXerophyta viscosa 68Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1 5 10 15 Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30 Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55 60 Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70 75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85 90 95 Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100 105 110 Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115 120 125 Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135 140 Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150 155 160 Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165 170 175 Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu 180 185 190 Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200 205 Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215 69660DNAArtificial SequenceSynthetic DNA sequence encoding Xerophyta viscosa PER1 protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained 69atg cct gga ttg act att ggt gac aca att ccc aac ttg gag ctg gat 48Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1 5 10 15 acg aca caa ggt cgc atc aag atc cac gac tat gtc ggg aat gga tac 96Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30 gtg att ctc ttc tca cat cct ggt gat ttc act ccg gtg tgt acc acc 144Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 gaa ttg ggc aag atg gct gct tat gcc gac gag ttc tct aag cgt ggt 192Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55 60 gtg aag ctg ctt ggg ttg tcc tgt gat gat gtc caa tca cat aag gag 240Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70 75 80 tgg atc aaa gac ata gag gct tac aca cct ggc tgt cac gta aaa tat 288Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85 90 95 ccg att gct gct gat cca acc aga gaa atc ata cag cag ctg aac atg 336Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100 105 110 gtg gac cct gat gag acg gaa agc tct aag tgc gct gtg cct tct agg 384Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115 120 125 gca ctt cac atc ata gga cca gat aag agg atc aag ctg tcc ttc ctc 432Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135 140 tac cct gcc tct act ggt cgc aac atg gac gaa gtt ctt aga gcc gtt 480Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150 155 160 gag tct ctt cag caa gca gct aaa cac aaa gtt gca act cct gct aac 528Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165 170 175 tgg aaa cct ggc gaa cca gtc gtc atc aaa cca gac gtc agc tcc gag 576Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu 180 185 190 gag gcc aag aag ctc ttt cct caa ggt tat aaa agc gtt gat ttg cct 624Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200 205 tca aag aag gac tac ttg agg ttc acc aat gtt tga 660Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215 70219PRTArtificial SequenceSynthetic Construct 70Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1 5 10 15 Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30 Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55 60 Val Lys Leu Leu Gly Leu

Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70 75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85 90 95 Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100 105 110 Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115 120 125 Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135 140 Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150 155 160 Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165 170 175 Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu 180 185 190 Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200 205 Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215 71660DNAArtificial SequenceSynthetic DNA sequence in accordance with the invention encoding Xerophyta viscosa PER1 protein using codons optimized for maize and with sequences identified in Table 2 removed and Table 1 sequences are maintained 71atg cct gga ttg act att ggt gac aca att ccc aac ttg gag ctg gat 48Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1 5 10 15 acg aca caa ggt cgc atc aag atc cac gac tat gtc ggg aat gga tac 96Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30 gtg att ctc ttc tca cat cct ggt gat ttc act ccg gtg tgt acc acc 144Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 gaa ttg ggc aag atg gct gct tat gcc gac gag ttc tct aag cgt ggt 192Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55 60 gtg aag ctg ctt ggg ttg tcc tgt gat gat gtc caa tca cat aag gag 240Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70 75 80 tgg atc aaa gac ata gag gct tac aca cct ggc tgt cac gta aaa tat 288Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85 90 95 ccg att gct gct gat cca acc aga gaa atc ata cag cag ctg aac atg 336Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100 105 110 gtg gac cct gat gag acg gaa agc tct aag tgc gct gtg cct tct agg 384Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115 120 125 gca ctt cac atc ata gga cca gat aag agg atc aag ctg tcc ttc ctc 432Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135 140 tac cct gcc tct act ggt cgc aac atg gac gaa gtt ctt aga gcc gtt 480Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150 155 160 gag tct ctt cag caa gca gct aaa cac aaa gtt gca act cct gct aac 528Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165 170 175 tgg aaa cct ggc gaa cca gtc gtc atc aaa cca gac gtc agc tcc gag 576Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu 180 185 190 gag gcc aag aag ctc ttt cct caa ggt tat aaa agc gtt gat ttg cct 624Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200 205 tca aag aag gac tac ttg agg ttc acc aat gtt tga 660Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215 72219PRTArtificial SequenceSynthetic Construct 72Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1 5 10 15 Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30 Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55 60 Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70 75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85 90 95 Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100 105 110 Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115 120 125 Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135 140 Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150 155 160 Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165 170 175 Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu 180 185 190 Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200 205 Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215

Claims

1-6. (canceled)

7. A synthetic DNA sequence for expressing a protein of interest in soybean cells which comprises: a) a codon-optimized DNA sequence encoding the protein of interest, b) at least one polyadenylation signal sequence chosen from the group consisting of Class I and Class III wherein; Class I is chosen from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATITAAA, AATITAA, AATACA, and CATAAA; and Class III is chosen from the group consisting of ATTTTT, TATTTT, TTATTT, TTTATT, TTTTTTT, TTTAT, AATTIT, TTTTTA, ATATAT, TAATTT, TTAATT, AAATTT, AAATAA, ATATTT, TTTGTT TTTGTTT, ATTTAT, ATTTTA, TTTAAT, and TTTTAA, and wherein said codon-optimized DNA sequence contains at least one polyadenylation signal sequence from Class III and wherein said synthetic DNA sequence contains fewer Class III polyadenylation signal sequences than the protein's native DNA sequence and contains the same number of Class I polyadenylation signal sequences compared to said native DNA sequence.

8. The synthetic DNA sequence of claim 7 that is substantially devoid of class III polyadenylation signal sequences.

9. The synthetic DNA sequence of claim 7 that is devoid of class III polyadenylation signal sequences.

10. The synthetic DNA sequence of claim 7 wherein said synthetic DNA sequence encodes a native protein selected from the group consisting of insecticidal proteins, herbicide tolerance proteins, stress tolerance-related proteins, and oil profile modification proteins.

11. The synthetic DNA sequence of claim 10 wherein said synthetic DNA sequence encodes an insecticidal protein.

12. The synthetic DNA sequence of claim 10 wherein said synthetic DNA sequence encodes aryloxyalkanoate dioxygenase 1 protein.

13. A synthetic DNA sequence chosen from the group consisting of SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59, SEQ ID NO:65, and SEQ ID NO:71.

14. (canceled)

15. A DNA construct for expression of a protein of interest comprising a 5' non-translated sequence, a coding sequence for a protein of interest, and a 3' non-translated region, wherein said 5' nontranslated sequence contains a promoter functional in a plant cell, said coding sequence is the synthetic DNA coding sequence of claim 7, and wherein said 3' nontranslated sequence comprises transcription termination sequence and a polyadenylation signal.

16. (canceled)

17. A transgenic plant containing the synthetic DNA sequence of claim 7.

18. (canceled)

19. A method of controlling pests in grain or seed which comprises obtaining said grain or seed from plants containing the synthetic DNA of claim 11.

20-22. (canceled)

23. A composition derived from the transgenic plant of claim 17 wherein said composition is a commodity product selected from the group consisting of meal, flour, protein concentrate, or oil.