Patent application title: SYNTHETIC GENES
Inventors:
IPC8 Class: AC12N1582FI
USPC Class:
1 1
Class name:
Publication date: 2021-07-15
Patent application number: 20210214745
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.Claims:
1. A synthetic DNA sequence for expressing a protein of interest in maize
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 II wherein; 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 Class II is chosen from the group
consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTT, TrTTT,
ATTTr, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TrTTAT, ATATTT, TATTAT,
TGTTTG, TTATAT, TGTAAT, and AAATAA; and 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 said native DNA sequence.
2. The synthetic DNA sequence of claim 1 that is substantially devoid of class II polyadenylation signal sequences.
3. The synthetic DNA sequence of claim 1 that is devoid of class II polyadenylation signal sequences.
4. The synthetic DNA sequence of claim 1 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.
5. The synthetic DNA sequence of claim 4 wherein said synthetic DNA sequence encodes an insecticidal protein.
6. The synthetic DNA sequence of claim 4 wherein said synthetic DNA sequence encodes aryloxyalkanoate dioxygenase 1 protein.
7-12. (canceled)
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. 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 1, and wherein said 3' nontranslated sequence comprises a transcription termination sequence and a polyadenylation signal.
15. (canceled)
16. A transgenic plant containing the synthetic DNA sequence of claim 1.
17. (canceled)
18. A method of controlling pests in grain or seed which comprises obtaining said grain or seed from plants containing the synthetic DNA of claim 5.
19. (canceled)
20. A method of controlling pests in grain or seed which comprises obtaining said grain or seed from plants containing the synthetic DNA of claim 5.
21. A method of controlling pests in meal or flour which comprises obtaining said meal or flour from the grain containing the synthetic DNA of claim 5.
22. A composition derived from the transgenic plant of claim 16 wherein said composition is a commodity product selected from the group consisting of meal, flour, protein concentrate, or oil.
23. (canceled)
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. Nos. 5,380,301, 5,625,136, 6,218,188, 6,340,593, 6,673,990, 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 U.S. Pat. No. 7,7411,18, Table 11. 1 AATAAA 2 AATAAT 3 AACCAA 4 ATATAA 5 AATCAA 6 ATACTA 7 ATAAAA 8 ATGAAA 9 AAGCAT 10 ATTAAT 11 ATACAT 12 AAAATA 13 ATTAAA 14 AATTAA 15 AATACA 16 CATAAA
[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 2 TTGTTT 3 TTTTGT 4 TGTTTT 5 TATATA 6 TATTTT 7 TTTTTT 8 ATTTTT 9 TTATTT 10 TTTATT 11 TAATAA 12 ATTTAT 13 TATATT 14 TTTTAT 15 ATATTT !6 TATTAT 17 TGTTTG 18 TTATAT 19 TGTAAT 20 AAATAA
[0007] Table 3 below identifies 20 potential polyadenylation signal sequences that occur frequently in soybean genes.
TABLE-US-00003 TABLE 3 Potential poiyadenyiation signal sequences found in soybean genes. 1 ATTTTT 2 TATTTT 3 TTATTT 4 TTTATT 5 TTTTTT 6 TTTTAT 7 AATTTT 8 TTTTTA 9 TAATTT 10 TTAATT 11 AAATTT 12 AAATAA 13 ATATTT 14 TTTGTT 15 TTGTTT 16 ATATAT 17 ATTATT 18 ATTTTA 19 TTTAAT 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 Class I and Class II wherein;
[0011] Class 1 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
[0012] 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
[0013] 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.
[0014] The present invention also provides a synthetic DNA sequence for expressing a protein of interest in soybean cells which comprises:
[0015] a) a codon-optimized DNA sequence encoding the protein of interest,
[0016] b) at least one polyadenylation signal sequence chosen from the group consisting of Class I and Class III wherein:
[0017] 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
[0018] Class III is chosen from the group consisting of ATTTTT, TATTTT, TTATTT, TTTATT, TTTTT, TTTTAT, AATTTT, TTTTTA, ATATAT, TAATTT, TTAATT, AAATTT, AAATAA, ATATTT, TTTGTT TTGTTT, ATTATT, ATTTTA, TTTAAT, and TITTAA, and
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] The invention also provides a transgenic plant containing the synthetic DNA sequences of the invention.
[0026] 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.
[0027] 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 ryloxykanoate gioxygenase (AADI) see WO/2005/107437, or phosphinothricin acetytransferase, or 5-enolpyruvylshikimate-3-phosphate synthase enzymes.
[0028] 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.
[0029] 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 (SAPI); US Patent Publication No: 2010/0275327, and 1-Cys peroxiredoxin (Perl) proteins (Mowla, et al., 2002, Planta 215:716-726).
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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
[0034] SEQ ID NO:1 is the native DNA sequence encoding Bacillus thuringiensis Cry1Fa core toxin.
[0035] SEQ ID NO:2 is Bacillus thuringiensis Cry1Fa core toxin sequence.
[0036] 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.
[0037] SEQ ID NO:4 is Bacillus thuringiensis Cry1Fa core toxin sequence.
[0038] 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.
[0039] SEQ ID NO 6 is Bacillus thuringiensis Cry1Fa core toxin sequence.
[0040] SEQ ID NO:7 is the native DNA sequence encoding Bacillus thuringiensis Cry34Ab1 toxin.
[0041] SEQ ID NO:8 is Bacillus thuringiensis Cry34Ab1 toxin sequence.
[0042] 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.
[0043] SEQ ID NO:10 is Bacillus thuringiensis Cry34Ab1 toxin sequence.
[0044] 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.
[0045] SEQ ID NO:12 is Bacillus thuringiensis Cry34Ab1 toxin sequence.
[0046] SEQ ID NO: 13 is the native DNA sequence encoding Bacillus thuringiensis Cry35Ab1 toxin.
[0047] SEQ ID NO:14 is Bacillus thuringiensis Cry35Ab1 toxin sequence.
[0048] 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
[0049] SEQ ID NO:16 is Bacillus thuringiensis Cry35Ab1 toxin sequence.
[0050] 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.
[0051] SEQ ID NO:18 is Bacillus thuringiensis Cry35Ab1 toxin sequence.
[0052] SEQ ID NO:19 is the native DNA sequence encoding Bacillus thuringiensis Cry1Ab1 core toxin.
[0053] SEQ ID NO:20 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.
[0054] 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.
[0055] SEQ ID NO:22 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.
[0056] 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.
[0057] SEQ ID NO:24 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.
[0058] SEQ ID NO:25 is the native DNA sequence encoding Bacillus thuringiensis Cry1Ca core toxin.
[0059] SEQ ID NO:26 is encoding Bacillus thuringiensis Cry1Ca core toxin sequence.
[0060] SEQ ID NO:27 is a synthetic DNA sequence encoding Bacillus thuringiensis Cry1Ca core toxin using codons optimized for maize and Table 1 sequences are maintained.
[0061] SEQ ID NO:28 is encoding Bacillus thuringiensis Cry1Ca core toxin sequence.
[0062] SEQ ID NO:29 is a synthetic 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.
[0063] SEQ ID NO:30 is encoding Bacillus thuringiensis Cry1Ca core toxin sequence.
[0064] SEQ ID NO:31 is the native DNA sequence encoding Bacillus thuringiensis Cry6Aa toxin.
[0065] SEQ ID NO:32 is Bacillus thuringiensis Cry6Aa toxin sequence.
[0066] 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.
[0067] SEQ ID NO:34 is Bacillus thuringiensis Cry6Aa toxin sequence.
[0068] 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.
[0069] SEQ ID NO:36 is Bacillus thuringiensis Cry6Aa toxin sequence.
[0070] SEQ ID NO:37 is the native DNA sequence encoding Sphingobiurn herbicidovorans AAD1 protein.
[0071] SEQ ID NO:38 is Sphingobiurn herbicidovorans AADI protein sequence.
[0072] 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.
[0073] SEQ ID NO:40 is Sphingobiurn herbicidovorans AADI protein sequence.
[0074] 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.
[0075] SEQ ID NO:42 is Sphingobiurn herbicidovorans AADI protein sequence.
[0076] SEQ ID NO:43 is the native DNA sequence encoding Aspergillus nidulans delta-9 fatty acid desaturase protein.
[0077] SEQ ID NO:44 is Aspergillus nidulans delta-9 fatty acid desaturase protein sequence.
[0078] 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.
[0079] SEQ ID NO:46 is Aspergillus nidulans delta-9 fatty acid desaturase protein sequence.
[0080] 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.
[0081] SEQ ID NO:48 is Aspergillus nidulans delta-9 fatty acid desaturase protein.
[0082] SEQ ID NO:49 is the native DNA sequence encoding Xerophyta viscosa SAPI protein.
[0083] SEQ ID NO:50 is Xerophyta viscosa SAPI protein sequence.
[0084] 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
[0085] SEQ ID NO:52 is Xerophyta viscosa SAPI protein sequence.
[0086] 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 1 sequences are maintained
[0087] SEQ ID NO:54 is Xerophyta viscosa SAPI protein sequence.
[0088] SEQ ID NO:55 is the native DNA sequence encoding Aequorea victoria GFPI protein.
[0089] SEQ ID NO:56 is Aequorea victoria GFPI protein sequence.
[0090] SEQ ID NO:57 is a synthetic DNA sequence encoding Aequorea victoria GFPI protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained.
[0091] SEQ ID NO:58 is Aequorea victoria GFPI protein sequence.
[0092] 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 1 sequences are maintained.
[0093] SEQ ID NO:60 is Aequorea victoria GFPI protein sequence.
[0094] SEQ ID NO:61 is the native DNA sequence encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein.
[0095] SEQ ID NO:62 is Leptosphaeria nodorum delta-9 fatty acid desaturase protein sequence.
[0096] 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 1 & Table 2 sequences are maintained
[0097] SEQ ID NO:64 is Leptosphaeria nodorum delta-9 fatty acid desaturase protein sequence.
[0098] 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
[0099] SEQ ID NO:66 is Leptosphaeria nodorum delta-9 fatty acid desaturase protein sequence.
[0100] SEQ ID NO:67 is the native DNA sequence encoding Xerophyta viscosa PERI protein.
[0101] SEQ ID NO:68 is Xerophyta viscosa PERI protein sequence.
[0102] SEQ ID NO:69 is a synthetic DNA sequence encoding Xerophyta viscosa PERI protein using codons optimized for maize and Table 1 & Table 2 sequences are maintained.
[0103] SEQ ID NO:70 is Xerophyta viscosa PERI protein sequence.
[0104] SEQ ID NO:71 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 1 sequences are maintained.
[0105] SEQ ID NO:72 is Xerophyta viscosa PERI protein sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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 1 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 vey important to remove the polyadenylation signal sequences listed in Tables 2 and 3, particularly where they occur in nested multimeric form.
[0111] 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.
[0112] 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. Soy- Soy- Amino Maize bean Amino Maize bean Acid Codon % % 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 12.3 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 220 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 ASM (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 CYS (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 !9.0 319 ACG 22 0 0.0 GGC 12.0 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 12.4 32.0 ATT 28.0 46.7 GTT 22.8 38.7
Transgenic Plants
[0113] 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. Nos. 4,945,050 and 5,141,131). Plants may be transformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010, European Patent No. EP131624B1, European Patent No. EP159418B1, European Patent No. EP17611281, U.S. Pat. No. 5,149,645, EP120516B1, U.S. Pat. Nos. 5,464,763, 4,693,976, European Patent No. EP116718B1, European Patent No. EP290799B1, European Patent No. EP320500B1, European Patent No. EP604662B1, U.S. Pat. Nos. 7,060,876, 6,037,526, 6,376,234, European Patent No. EP292435B1, U.S. Pat. Nos. 5,231,019, 5,463,174, 4,762,785, 5,608,142, and 5,159,135. Other transformation technology includes WHISKERS.TM. technology, see U.S. Pat. Nos. 5,302,523 and 5,464,765. Electroporation technology has also been used to transform plants, see WO1987006614, U.S. Pat. Nos. 5,472,869, 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.
[0114] 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.
[0115] 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.
[0116] 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 1297-341).
[0117] 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, corn silk specific, cotton fiber specific, seed endosperm specific, phloem specific, and the like.
[0118] 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.
[0119] Transgenic crops containing insect resistance (IR) traits are prevalent in corn 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.I. 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.
[0120] 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.
[0121] 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.
[0122] 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
[0123] 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.
[0124] 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.
[0125] The synthetic coding region of SEQ ID NO:5 was optimized for expression in maize.
[0126] 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.
[0127] 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 pDAB111440 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. Nat. Acad. Sci. U.S.A. 107:20240-5) under the transcriptional control of a sugarcane bacilliform badnavirus (ScBV) promoter (Schenk e 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. nt No. nt Sites Location Sites Location in in in rede- in rede- Native Native signed signed Cry1Fa Cry1Fa Cry1Fa Cry1Fa core core core core sequence sequence sequence sequence Table 1 (SEQ ID (SEQ ID (SEQ ID (SEQ ID Sequence NO: 1) NO: 1) NO: 5) NO: 5) 1 AATAAA 2 426; 582 0 NA* 2 AATAAT 5 7; 46; 5 7; 46; 358; 430; 358; 662 430; 562 3 AACCAA 0 NA 0 NA 4 ATATAA 1 1520 1 1520 5 AACCAA 2 19; 628 4 19; 426; 582; 628 6 ATACTA 1 1508 1 1508 7 ACAAAA 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; 3 400; 1369; 1693 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: 5) No. nt No. nt Sites Location Sites Location in in in in rede- rede- Native Native signed signed Cry1Fa Cry1Fa Cry1Fa Cry1Fa core core core core Se- Se- Se- Se- Table 2 quence quence quence quence Se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 1) NO: 1) NO: 5) NO: 5) 1 ATATAT 1 104 0 NA* 2 TTGTTT 3 39; 0 NA 612; 907 3 TTTTGT 1 1089 0 NA 4 TGTTTT 2 1086; 0 NA 1334 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; 0 NA 217 10 TTTATT 0 NA 0 NA 11 TAATAA 1 357; 0 NA 416; 561; 581 12 ATTTAT 3 319; 0 NA 497; 793 13 TATATT 1 322 0 NA 14 TTTTAT 3 192; 0 NA 464; 1063 15 ATATTT 0 NA 0 NA 16 TATTAT 0 NA 0 NA 17 TGTTTG 2 613; 0 NA 908 18 TTATAT 2 321; 0 NA 1770 19 TGTAAT 0 NA 0 NA 20 AAATAA 2 45; 0 NA 429 Total 28 0 NA *NA = Not Applicable
Example 2
Synthetic Coding Region Encoding Bacillus thuringiensis Cry34A Toxin
[0128] 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.
[0129] 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.
[0130] 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.
[0131] The synthetic coding region of SEQ ID NO:5 was optimized for expression in maize.
[0132] 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 in the redesigned version (SEQ ID NO: 11) No. nt No. nt Sites Location Sites Location in in in in rede- rede- native native signed signed Cry34Ab1 Cry34Ab1 Cry34Ab1 Cry34Ab1 Table 1 sequence sequence sequence sequence Se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 7) NO: 7) NO: 11) NO: 11) 1 AATAAA 2 247; 2 247; 268 268 2 AATAAT 1 31 1 31 3 AACCAA 0 NA* 8 NA 4 ATATAA 0 NA 0 NA 5 AATCAA 2 146; 2 146; 310 310 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 13 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) No. nt nt Sites Location Location in in No. Sites in rede- rede- in native native signed signed Cry34Ab1 Cry34Ab1 Cry34Ab1 Cry34Abl Table 2 sequence sequence sequence sequence Se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 7) NO: 7) NO: 11) 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 C NA 11 TAATAA 2 33; 2 33; 246 246 12 ATTTAT 0 NA 0 NA 13 TATATT 2 182; C NA 218 14 TTTTAT 1 156 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 1 *NA= Not Applicable
Example 3
Synthetic Coding Region Encoding Bacillus thuringiensis Cry35Ab1 Toxin
[0133] 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.
[0134] 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 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.
[0135] 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.
[0136] The synthetic coding region of SEQ ID NO:17 was optimized for expression in maize.
[0137] 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 region (SEQ ID NO: 13) and in the redesigned version (SEQ ID NO : 17) No. nt No. nt Sites Location Sites in Location in in rede- in native native signed rede- Cry35Ab1 Cry35Ab1 Cry35Ab1 signed se- se- se- Cry35Ab1 Table 1 quence quence quence sequence se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 13) NO: 13) NO: 17) NO: 17) 1 AATAAA 5 13; 3 13; 100; 100; 810 228; 276; 810 2 AATAAT 4 193; 4 193; 217; 217; 385; 864 385; 864 3 AACCAA 0 NA* 0 NA 4 ATATAA 1 966 1 966 5 AATCAA 3 394; 5 228; 276; 750; 394; 750; 914 914 6 ATACTA 1 8 1 8 7 ATAAAA 3 101; 5 101; 224; 224; 277; 573; 277; 811 575; 811 8 ATGAAA 5 23; 5 23; 671; 671; 769; 806; 769; 354 806; 854 9 AAGCAT 0 NA 0 NA 10 ATTAAT 1 522 1 522 11 ATACAT 1 734 1 734 12 AAAATA 7 226; n 226; 578; 578; 618; 838; 618; 862; 873; 838; 1137 862; 873; 1137 13 ATTAAA 4 462; 4 462; 589; 589; 834; 1131 834; 1131 14 AATTAA 5 461; 5 461; 521; 521; 588; 833; 588; 1130 833; 1130 15 AATACA 3 261; 3 261 ;303; 303; 733 733 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. No. nt Sites nt Sites Location in Location in in native in rede- rede- Cry35Ab1 native signed signed sequence Cry35Ab1 Cry35Ab1 Cry35Ab1 Table 2 (SEQ sequence sequence sequence Se- ID NO: (SEQ ID (SEQ ID (SEQ ID quence 13) NO: 13) NO: 17) 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; 0 NA 1144 7 TTTTTT 0 NA 0 NA 8 ATTTTT 1 1145 0 NA 9 TTATTT 3 63; 1 1143 145; 1143 10 TTTATT 2 144; 0 NA 1056 11 TAATAA 2 12; 1 12 216 12 ATTTAT 0 NA 0 NA 13 TATATT 2 169; 0 NA 607 14 TTTTAT 1 143 0 NA 15 ATATTT 1 608 0 NA 16 TATTAT 4 171; 1 1141 549; 604; 1141 17 TGTTTG 0 NA 0 NA 18 TTATAT 7 606; 0 NA 958 19 TGTAAT 1 300 0 NA 20 AAATAA 8 26; 2 809; 192; 863 227; 275; 384; 809; 863; 1097 Total 31 5 *NA = Not Applicable
Example 4
Synthetic Coding Region Encoding Bacillus thuringiensis Cry1Ab Core Toxin
[0138] 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.
[0139] 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.
[0140] The synthetic coding region of SEQ ID NO:23 was optimized for expression in maize.
[0141] 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.
[0142] 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, pDAB11449, containing the aforementioned gene expression cassette, was constructed and utilized in the production of transgenic maize plants. Plasmid pDABI 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 Cry1Ab core toxin coding region (SEQ ID NO: 19) and in the redesigned version (SEQ ID NO: 23) No. nt No. Sites Location Sites nt in in in Location rede- rede- Native in signed signed Cry1Ab Native Cry1Ab Cry1Ab core Cry1Ab core core se- core se- se- Table 1 quence sequence quence quence Se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 19) NO: 19) NO: 23) NO: 23) 1 AATAAA 0 NA* 0 NA 2 AATAAT 3 960, 3 960, 1126, 1126, 1387 1387 3 AACCAA 2 253, 2 253, 280 280 4 ATATAA 2 185, 2 185, 1391 1391 5 AATCAA 2 688, 3 688, 1129 1129, 1639 6 ATACTA 0 NA 0 NA 7 ATAAAA 0 NA 0 NA 8 ATGAAA 1 1232 1 1232 9 AAGCAT 0 NA 0 NA 10 ATTAAT 1 1636 1 1636 11 ATACAT 2 1366, 2 1366, 1613 1613 12 AAAATA 0 NA 0 NA 13 ATTAAA 3 249, 3 249, 704, 704, 785 785 13 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. nt No. nt Sites Location Sites Location in in in in rede- rede- Native Native signed signed Cry1Ab Cry1Ab Cry1Ab Cry1Ab core core core core Table 2 sequence sequence sequence sequence Se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 19) NO: 19) NO: 23) 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, 0 NA 1792 6 TATTTT 0 NA 0 NA 7 TTTTTT 0 NA 0 NA 8 ATTTTT 2 199, 0 NA 1649 9 TTATTT 0 NA 0 NA 10 TTTATT 1 470 0 NA 11 TAATAA 2 1340, 0 NA 1386 12 ATTTAT 2 503, 0 NA 799 13 TATATT 0 NA 0 NA 14 TTTTAT 0 NA 0 NA 15 ATATTT 1 110 0 NA 16 TATTAT 2 937, 0 NA 940 17 TGTTTG 1 530 0 NA 18 TTATAT 2 1096, 0 NA 1791 19 TGTAAT 0 NA 0 NA 20 AAATAA 2 959, 1 959 1125 Total 18 1 *NA = Not Applicable
Examples
Synthetic Coding Region Encoding Bacillus thuringiensis CryICa Core Toxin
[0143] 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.
[0144] 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.
[0145] 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.
[0146] The synthetic gene of SEQ ID NO:29 was optimized for expression in maize.
[0147] 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. nt No. nt Sites Location Sites Location in in in in rede- rede- Native Native signed signed Cry1Ca Cry1Ca Cry1Ca Cry1Ca core core core core se- se- se- se- Table 1 quence quence quence quence Se- (SEQ ID (SEQ ID (SEQ ID (SEQ ID quence NO: 25) NO: 25) NO: 29) NO: 29) 1 AATAAA 0 NA* 0 NA 2 AATAAT 2 646, 2 646, 916 916 3 AACCAA 0 NA 1 1042 4 ATATAA 2 684, 2 684, 1757 1757 5 AATCAA 1 1405 1 1405 6 ATACTA 0 NA 0 NA 7 ATAAAA 1 1826 1 1826 8 ATGAAA 2 254, 254, 569 569 9 AAGCAT 1 335 1 335 10 ATTAAT 7 177, 7 177, 246, 246, 250, 250, 813, 813, 817, 817, 1402, 1402, 1534 1534 11 ATACAT 0 NA 0 NA 12 AAAATA 0 NA 0 NA 13 ATTAAA 4 245, 4 245, 249, 249, 816, 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 CryICa core CryICa core CryICa core CryICa 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
[0148] 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.
[0149] 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.
[0150] 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.
[0151] The synthetic coding region of SEQ ID NO:35 was optimized for expression in maize.
[0152] 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, 493, 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
[0153] 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.
[0154] 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 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.
[0155] 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.
[0156] The synthetic coding region of SEQ ID NO:41 was optimized for expression in maize.
[0157] 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 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 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
[0158] 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.
[0159] 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.
[0160] 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.
[0161] The synthetic coding region of SEQ ID NO:47 was optimized for expression in maize.
[0162] 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 SAPI
[0163] 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.
[0164] 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.
[0165] 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.
[0166] The synthetic coding region of SEQ ID NO:53 was optimized for expression in maize.
[0167] 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 ATTTTT 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 GFP1
[0168] 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 codon 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.
[0169] 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.
[0170] 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.
[0171] The synthetic coding region of SEQ ID NO:59 was optimized for expression in maize.
[0172] 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 GFP 1 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 AAAATAA 0 NA 0 NA Total 6 0 *NA = Not Applicable
Example 11
Synthetic Coding Region Encoding Leptosphaeria nodurum FAD9
[0173] 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.
[0174] 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.
[0175] 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.
[0176] The synthetic coding region of SEQ ID NO:65 was optimized for expression in maize.
[0177] 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 ATT6TT 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
[0178] 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.
[0179] 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.
[0180] 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.
[0181] The synthetic coding region of SEQ ID NO:71 was optimized for expression in maize.
[0182] 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 at 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 AACCAAA 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 at 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
[0183] 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. A pat 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.
[0184] 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).
[0185] 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.
[0186] 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-.sup.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
[0187] 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.
[0188] 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.
[0189] 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!) 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.
[0190] Electro-competent cells of Agrobacterium tumeficiens 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 280 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 28.degree.. Well separated single colonies are selected and streaked onto fresh YEP+agar plates with spectinomycin and streptomycin as before, and incubated at 280 for 1-3 days.
[0191] 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) 94.degree. for 3 minutes; Step 2) 94.degree. for 45 seconds; Step 3) 550 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 for 10 minutes. The reaction is maintained at 4 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.
[0192] 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.
[0193] 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
[0194] Arabidopsis Transformation. 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 280 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 28.degree. 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.
[0195] Arabidopsis Growth and Selection. Freshly harvested Ti 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/ 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.
[0196] 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.
[0197] 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.
[0198] Insect Bioassays of transgenic Arabidopsis. 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
[0199] The Agrobacterium 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 pSB1, 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
[0200] Agrobacterium-Mediated Transformation of Maize. 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.
[0201] Infection and co-cultivation. 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 28 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.
[0202] 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.
[0203] 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.
[0204] Regeneration and seed production. 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) 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.
[0205] 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.
[0206] Ear sterilization and embryo isolation. 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.
[0207] Agrobacterium Culture Initiation. 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.
[0208] Agrobacterium culture and Co-cultivation. 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.
[0209] Callus Selection and Regeneration of Putative Events. 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.2sec-.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.
[0210] 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 1 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.
[0211] 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.
[0212] 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.
[0213] Proteins extracted from measured areas of leaf tissue were analyzed for expression of Cry1Fa protein and AAD-1 protein by standard ELISA (Enzyme-Linked Immunosorbant Assay) or protein immunoblots (western blots). For Cry1Fa ELISA detection, reagents from an ENVIROLOGIX ELISA kit (Cat. No. AP 016 NW VlO; 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.
[0214] The ELISA results obtained from extracts of pDAB111440-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 ofCry1Fa and AAD-1 proteins extracted from maize plants transformed with plasmid pDAB1 11440, as detectedby ELISA methods, Sample ID CryIFa 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
[0215] 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).
[0216] 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 jluorescens 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.
[0217] Methods of controlling insect pests. 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 Asn1 5
10 15aat 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 30ccg
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 45gtt 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
60ttt 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 Gln65
70 75 80ttg 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 95aca 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
110aga 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 125cgt 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
140aat 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 Val145
150 155 160caa 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 175ggg 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
190aga 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 205tac 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
220gca 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 Asp225
230 235 240atc 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 255acg 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
270gat 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 285ttt 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
300gta 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 Leu305
310 315 320gtt 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 335ggg 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
350cgt 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 365ttt 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
380caa 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 Ile385
390 395 400gat 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 415aat 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
430ggt 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 445acg 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
460act 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 Thr465
470 475 480gtt 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 495agt 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
510ccc 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 525aga 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
540aac 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 Ser545
550 555 560tac 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 575ttc 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
590gac 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 6052605PRTBacillus thuringiensis 2Met Glu
Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn1 5
10 15Asn Pro Glu Val Glu Ile Leu Asn
Glu Glu Arg Ser Thr Gly Arg Leu 20 25
30Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu
Phe 35 40 45Val Pro Gly Val Gly
Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55
60Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile
Glu Gln65 70 75 80Leu
Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95Thr Leu Arg Gly Leu Ala Asp
Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100 105
110Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu
Asp Val 115 120 125Arg Ile Arg Phe
Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130
135 140Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu
Ser Val Tyr Val145 150 155
160Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175Gly Gln Gly Trp Gly
Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180
185 190Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His
Cys Leu Asp Thr 195 200 205Tyr Asn
Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210
215 220Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr
Leu Thr Val Leu Asp225 230 235
240Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255Thr Ser Ser Gln
Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260
265 270Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly
Phe Asn Arg Ala Glu 275 280 285Phe
Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290
295 300Val Thr Ala Glu Thr Val Arg Ser Gln Thr
Val Trp Gly Gly His Leu305 310 315
320Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser
Tyr 325 330 335Gly Val Phe
Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340
345 350Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro
Val Phe Val Arg Gly Gly 355 360
365Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370
375 380Gln Thr Gly Thr Asn His Thr Arg
Thr Phe Arg Asn Ser Gly Thr Ile385 390
395 400Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser
Gly Ala Pro Trp 405 410
415Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro
420 425 430Gly Glu Ile Ser Gly Ser
Asp Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440
445Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu
Arg Ile 450 455 460Thr Gln Ile Pro Leu
Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr465 470
475 480Val Val Arg Gly Pro Gly Phe Thr Gly Gly
Asp Ile Leu Arg Arg Thr 485 490
495Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu
500 505 510Pro Gln Arg Tyr Arg
Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515
520 525Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe
Ala Gly Gln Phe 530 535 540Asn Lys Thr
Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser545
550 555 560Tyr Ala Thr Ile Asn Thr Ala
Phe Thr Phe Pro Met Ser Gln Ser Ser 565
570 575Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn
Glu Val Tyr Ile 580 585 590Asp
Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595
600 60531818DNAArtificial SequenceSynthetic DNA sequence
encoding Bacillus thuringiensis Cry1Fa core toxin using codons
optimized for maize and Table 1 sequences are
maintainedCDS(1)..(1818) 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 Asn1 5 10
15aat 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 30cca 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
45gtt 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 60ttt 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 Gln65 70
75 80ttg 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
95acc 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 110agg 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
125cgt 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 140aat 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 Val145 150
155 160caa 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
175ggg 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 190aga 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
205tac 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 220gca 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 Asp225 230
235 240atc 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
255acg 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 270gac 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
285ttc 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 300gtg 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 Leu305 310
315 320gtc 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
335ggg 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 350cgt 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
365ttt 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 380cag 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 Ile385 390
395 400gat 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
415aac 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 430ggt 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
445acg 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 460act 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 Thr465 470
475 480gtg 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
495tcc 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 510ccc 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
525aga 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 540aac 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 Ser545 550
555 560tac 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
575ttc 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 590gac 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
6054605PRTArtificial SequenceSynthetic Construct 4Met Glu Asn Asn Ile
Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn1 5
10 15Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg
Ser Thr Gly Arg Leu 20 25
30Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe
35 40 45Val Pro Gly Val Gly Val Ala Phe
Gly Leu Phe Asp Leu Ile Trp Gly 50 55
60Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln65
70 75 80Leu Ile Glu Gln Arg
Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr 85
90 95Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile
Tyr Ile Glu Ala Leu 100 105
110Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125Arg Ile Arg Phe Ala Asn Thr
Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135
140Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr
Val145 150 155 160Gln Ala
Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175Gly Gln Gly Trp Gly Leu Asp
Ile Ala Thr Val Asn Asn His Tyr Asn 180 185
190Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu
Asp Thr 195 200 205Tyr Asn Gln Gly
Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210
215 220Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu
Thr Val Leu Asp225 230 235
240Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255Thr Ser Ser Gln Leu
Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu 260
265 270Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe
Asn Arg Ala Glu 275 280 285Phe Gly
Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290
295 300Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val
Trp Gly Gly His Leu305 310 315
320Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335Gly Val Phe Asn
Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340
345 350Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val
Phe Val Arg Gly Gly 355 360 365Phe
Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370
375 380Gln Thr Gly Thr Asn His Thr Arg Thr Phe
Arg Asn Ser Gly Thr Ile385 390 395
400Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro
Trp 405 410 415Asn Asp Tyr
Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420
425 430Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg
Ala Pro Met Phe Ser Trp 435 440
445Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450
455 460Thr Gln Ile Pro Leu Val Lys Ala
His Thr Leu Gln Ser Gly Thr Thr465 470
475 480Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Thr 485 490
495Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu
500 505 510Pro Gln Arg Tyr Arg Ala
Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520
525Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly
Gln Phe 530 535 540Asn Lys Thr Met Asp
Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser545 550
555 560Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe
Pro Met Ser Gln Ser Ser 565 570
575Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590Asp Arg Phe Glu Leu
Ile Pro Val Thr Ala Thr Phe Glu 595 600
60551818DNAArtificial 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 maintainedCDS(1)..(1818)
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 Asn1
5 10 15aat 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
30cca 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 45gtt 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 60ttt 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 Gln65 70 75
80ttg 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 95acc 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 110agg 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 125cgt 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 140aat 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 Val145 150 155
160caa 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 175ggg 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 190aga 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 205tac 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 220gca 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 Asp225 230 235
240atc 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 255acg 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 270gac 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 285ttc 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 300gtg 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 Leu305 310 315
320gtc 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 335ggg 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 350cgt 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 365ttt 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 380cag 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 Ile385 390 395
400gat 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 415aac 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 430ggt 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 445acg 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 460act 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 Thr465 470 475
480gtg 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 495tcc 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 510ccc 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 525aga 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 540aac 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 Ser545 550 555
560tac 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 575ttc 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 590gac 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 6056605PRTArtificial
SequenceSynthetic Construct 6Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro
Tyr Asn Cys Leu Asn1 5 10
15Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30Pro Leu Asp Ile Ser Leu Ser
Leu Thr Arg Phe Leu Leu Ser Glu Phe 35 40
45Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp
Gly 50 55 60Phe Ile Thr Pro Ser Asp
Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln65 70
75 80Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg
Asn Arg Ala Ile Thr 85 90
95Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110Arg Glu Trp Glu Ala Asn
Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120
125Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala
Ile Asn 130 135 140Asn Phe Thr Leu Thr
Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val145 150
155 160Gln Ala Ala Asn Leu His Leu Ser Leu Leu
Arg Asp Ala Val Ser Phe 165 170
175Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn
180 185 190Arg Leu Ile Asn Leu
Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr 195
200 205Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn
Thr Arg Gln Trp 210 215 220Ala Arg Phe
Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp225
230 235 240Ile Val Ala Leu Phe Pro Asn
Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245
250 255Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser
Ser Val Ile Glu 260 265 270Asp
Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275
280 285Phe Gly Val Arg Pro Pro His Leu Met
Asp Phe Met Asn Ser Leu Phe 290 295
300Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu305
310 315 320Val Ser Ser Arg
Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr 325
330 335Gly Val Phe Asn Pro Gly Gly Ala Ile Trp
Ile Ala Asp Glu Asp Pro 340 345
350Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly
355 360 365Phe Gly Asn Pro His Tyr Val
Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375
380Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr
Ile385 390 395 400Asp Ser
Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415Asn Asp Tyr Ser His Val Leu
Asn His Val Thr Phe Val Arg Trp Pro 420 425
430Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe
Ser Trp 435 440 445Thr His Arg Ser
Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450
455 460Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln
Ser Gly Thr Thr465 470 475
480Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495Ser Gly Gly Pro Phe
Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500
505 510Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser
Thr Thr Asn Leu 515 520 525Arg Ile
Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530
535 540Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr
Phe Gln Ser Phe Ser545 550 555
560Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575Phe Thr Val Gly
Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580
585 590Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr
Phe Glu 595 600 6057372DNABacillus
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 His1 5 10
15aca 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 30aca 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
45gaa 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 60ata 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 Ala65 70
75 80ggt 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
95att 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 110caa acc aca tcc tca cga
tat ggg cat aaa tca taa 372Gln Thr Thr Ser Ser Arg
Tyr Gly His Lys Ser 115 1208123PRTBacillus
thuringiensis 8Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr
Gly His1 5 10 15Thr Leu
Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20
25 30Thr Ser Pro Thr Asn Val Ala Asn Asp
Gln Ile Lys Thr Phe Val Ala 35 40
45Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50
55 60Ile Asn Gly Glu Ala Glu Ile Ser Leu
Tyr Phe Asp Asn Pro Phe Ala65 70 75
80Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr
Glu Ile 85 90 95Ile Thr
Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100
105 110Gln Thr Thr Ser Ser Arg Tyr Gly His
Lys Ser 115 1209372DNAArtificial SequenceSynthetic
DNA sequence encoding Bacillus thuringiensis Cry34Ab1 toxin using
codons optimized for maize and Table 1 sequences are
maintainedCDS(1)..(372) 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 His1 5 10
15aca 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 30acc 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
45gaa 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 60atc 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 Ala65 70
75 80ggg 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
95att 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 110cag acc act tca tcg cgc
tac ggg cat aaa tca tag 372Gln Thr Thr Ser Ser Arg
Tyr Gly His Lys Ser 115 12010123PRTArtificial
SequenceSynthetic Construct 10Met Ser Ala Arg Glu Val His Ile Asp Val Asn
Asn Lys Thr Gly His1 5 10
15Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg
20 25 30Thr Ser Pro Thr Asn Val Ala
Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40
45Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr
Ser 50 55 60Ile Asn Gly Glu Ala Glu
Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala65 70
75 80Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys
Ser Gln Tyr Glu Ile 85 90
95Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile
100 105 110Gln Thr Thr Ser Ser Arg
Tyr Gly His Lys Ser 115 12011372DNAArtificial
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 maintainedCDS(1)..(372) 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 His1 5
10 15aca 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
30acc 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 45gaa 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
60atc 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 Ala65
70 75 80ggg 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 95att 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
110cag acc act tca tcg cgc tac ggg cat aaa tca tag
372Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115
12012123PRTArtificial SequenceSynthetic Construct 12Met Ser Ala Arg Glu
Val His Ile Asp Val Asn Asn Lys Thr Gly His1 5
10 15Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp
Gly Gly Arg Trp Arg 20 25
30Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala
35 40 45Glu Ser Asn Gly Phe Met Thr Gly
Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55
60Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala65
70 75 80Gly Ser Asn Lys Tyr
Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile 85
90 95Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His
Val Thr Tyr Thr Ile 100 105
110Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser 115
120131152DNABacillus 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 Gly1 5
10 15cta 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
30atg 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 45tta 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
60aat 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 Ser65
70 75 80act 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 95ggt 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
110gga 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 125aat 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
140ctt 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 Lys145
150 155 160tat 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 175gga 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
190aaa 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 205caa 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
220gaa 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 Lys225
230 235 240aca 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 255atg 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
270aca 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 285ata 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
300caa 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 Leu305
310 315 320tat 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 335gat 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
350tta 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 365aat 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
38014383PRTBacillus thuringiensis 14Met Leu Asp Thr Asn Lys Val Tyr Glu
Ile Ser Asn His Ala Asn Gly1 5 10
15Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser
Leu 20 25 30Met Asn Lys Asn
Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35
40 45Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr
Ser Tyr Ala Ala 50 55 60Asn Asn Cys
Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser65 70
75 80Thr Tyr Ser Ser Thr Asn Ser Ile
Gln Lys Trp Gln Ile Lys Ala Asn 85 90
95Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu
Thr Ala 100 105 110Gly Thr Gly
Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115
120 125Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser
Val Gln Thr Ile Gln 130 135 140Leu Pro
Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys145
150 155 160Tyr Ser Pro Thr Gly Asn Ile
Asp Asn Gly Thr Ser Pro Gln Leu Met 165
170 175Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp
Pro Asn Ile Asp 180 185 190Lys
Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195
200 205Gln Tyr Trp Gln Arg Ala Val Gly Ser
Asn Val Ala Leu Arg Pro His 210 215
220Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys225
230 235 240Thr Thr Ile Ile
Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245
250 255Met Lys Phe Asp Ile Pro Glu Val Gly Gly
Gly Thr Asp Glu Ile Lys 260 265
270Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys
275 280 285Ile Met Glu Lys Tyr Gln Glu
Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295
300Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu
Leu305 310 315 320Tyr Arg
Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser
325 330 335Asp Asn Asp Thr Tyr Asn Val
Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345
350Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu
Ile Thr 355 360 365Asn Ile Pro Lys
Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375
380151152DNAArtificial SequenceSynthetic DNA sequence
encoding Bacillus thuringiensis Cry35Ab1 toxin using codons
optimized for maize and Table 1 sequences are
maintainedCDS(1)..(1152) 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 Gly1 5 10
15cta 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 30atg 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
45tta 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 60aat 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 Ser65 70
75 80acc 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
95gga 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 110ggc 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
125aat 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 140ctt 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 Lys145 150
155 160tac 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
175ggc 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 190aaa 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
205caa 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 220gaa 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 Lys225 230
235 240acg 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
255atg 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 270acg 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
285ata 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 300caa 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 Leu305 310
315 320tat 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
335gac 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 350tta 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
365aac 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 38016383PRTArtificial
SequenceSynthetic Construct 16Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser
Asn His Ala Asn Gly1 5 10
15Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu
20 25 30Met Asn Lys Asn Asp Asp Asp
Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40
45Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala
Ala 50 55 60Asn Asn Cys Lys Val Trp
Asn Val Asn Asn Asp Lys Ile Asn Val Ser65 70
75 80Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp
Gln Ile Lys Ala Asn 85 90
95Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala
100 105 110Gly Thr Gly Gln Ala Leu
Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120
125Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr
Ile Gln 130 135 140Leu Pro Gln Lys Pro
Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys145 150
155 160Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly
Thr Ser Pro Gln Leu Met 165 170
175Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp
180 185 190Lys Asn Thr Gln Ile
Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195
200 205Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala
Leu Arg Pro His 210 215 220Glu Lys Lys
Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys225
230 235 240Thr Thr Ile Ile Asn Thr Leu
Gly Phe Gln Ile Asn Ile Asp Ser Gly 245
250 255Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr
Asp Glu Ile Lys 260 265 270Thr
Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275
280 285Ile Met Glu Lys Tyr Gln Glu Gln Ser
Glu Ile Asp Asn Pro Thr Asp 290 295
300Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu305
310 315 320Tyr Arg Tyr Asn
Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325
330 335Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr
Pro Asn His Gln Gln Ala 340 345
350Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr
355 360 365Asn Ile Pro Lys Ser Thr Leu
Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375
380171152DNAArtificial 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 maintainedCDS(1)..(1152) 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 Gly1 5
10 15cta 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
30atg 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 45ctg 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
60aat 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 Ser65 70 75 80acc
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 95ggc 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
110ggc 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 125aat 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 140ctt 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 Lys145 150 155
160tac 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 175ggc 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 190aaa 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 205caa 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 220gaa 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 Lys225 230 235
240acg 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 255atg 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 270acg 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 285ata 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 300caa 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 Leu305 310 315
320tac 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 335gac 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 350ctg 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 365aac 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 38018383PRTArtificial SequenceSynthetic Construct
18Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly1
5 10 15Leu Tyr Ala Ala Thr Tyr
Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25
30Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu
Lys Trp Phe 35 40 45Leu Phe Pro
Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50
55 60Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys
Ile Asn Val Ser65 70 75
80Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn
85 90 95Gly Ser Ser Tyr Val Ile
Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100
105 110Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr
Asp Glu Ser Ser 115 120 125Asn Asn
Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130
135 140Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu
Lys Asp Tyr Pro Lys145 150 155
160Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met
165 170 175Gly Trp Thr Leu
Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180
185 190Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr
Ile Leu Lys Lys Tyr 195 200 205Gln
Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210
215 220Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly
Thr Glu Ile Asp Gln Lys225 230 235
240Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser
Gly 245 250 255Met Lys Phe
Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260
265 270Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu
Tyr Ser His Glu Thr Lys 275 280
285Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290
295 300Gln Ser Met Asn Ser Ile Gly Phe
Leu Thr Ile Thr Ser Leu Glu Leu305 310
315 320Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln
Ile Gln Thr Ser 325 330
335Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala
340 345 350Leu Leu Leu Leu Thr Asn
His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360
365Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr
Phe 370 375 380191830DNABacillus
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 Leu1 5 10
15agt 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 30tac 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 45gaa 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 60tgg 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 Ile65 70
75 80gaa 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
95att 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 110tct 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 125gag 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 140att 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 Val145
150 155 160tat 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 175gtg 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 190tat
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 205cgc 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
220gat 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 Val225
230 235 240tta 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 255att 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
270tta 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 285aga 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
300atc 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 Gln305
310 315 320ata 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 335cta 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
350caa 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 365aga 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
380ggg 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 Val385
390 395 400tac 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 415aat 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
430gtt 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 445aga 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
460ata 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 Thr465
470 475 480aat 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 495gga 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
510gta 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 525tac 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
540cct 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 Asn545
550 555 560tta 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 575ttt 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
590tca 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 605gta acc
1830Val Thr 61020610PRTBacillus
thuringiensis 20Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn
Cys Leu1 5 10 15Ser Asn
Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20
25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser
Leu Thr Gln Phe Leu Leu Ser 35 40
45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50
55 60Trp Gly Ile Phe Gly Pro Ser Gln Trp
Asp Ala Phe Leu Val Gln Ile65 70 75
80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn
Gln Ala 85 90 95Ile Ser
Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100
105 110Ser Phe Arg Glu Trp Glu Ala Asp Pro
Thr Asn Pro Ala Leu Arg Glu 115 120
125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140Ile Pro Leu Phe Ala Val Gln
Asn Tyr Gln Val Pro Leu Leu Ser Val145 150
155 160Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
Arg Asp Val Ser 165 170
175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190Tyr Asn Asp Leu Thr Arg
Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200
205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp
Ser Arg 210 215 220Asp Trp Val Arg Tyr
Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val225 230
235 240Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr
Asp Ser Arg Arg Tyr Pro 245 250
255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270Leu Glu Asn Phe Asp
Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275
280 285Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu
Asn Ser Ile Thr 290 295 300Ile Tyr Thr
Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln305
310 315 320Ile Met Ala Ser Pro Val Gly
Phe Ser Gly Pro Glu Phe Thr Phe Pro 325
330 335Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln
Arg Ile Val Ala 340 345 350Gln
Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355
360 365Arg Pro Phe Asn Ile Gly Ile Asn Asn
Gln Gln Leu Ser Val Leu Asp 370 375
380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val385
390 395 400Tyr Arg Lys Ser
Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405
410 415Asn Asn Asn Val Pro Pro Arg Gln Gly Phe
Ser His Arg Leu Ser His 420 425
430Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445Arg Ala Pro Met Phe Ser Trp
Ile His Arg Ser Ala Glu Phe Asn Asn 450 455
460Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser
Thr465 470 475 480Asn Leu
Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly
485 490 495Gly Asp Ile Leu Arg Arg Thr
Ser Pro Gly Gln Ile Ser Thr Leu Arg 500 505
510Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg
Ile Arg 515 520 525Tyr Ala Ser Thr
Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530
535 540Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser
Ser Gly Ser Asn545 550 555
560Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn
565 570 575Phe Ser Asn Gly Ser
Ser Val Phe Thr Leu Ser Ala His Val Phe Asn 580
585 590Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe
Val Pro Ala Glu 595 600 605Val Thr
610211830DNAArtificial SequenceSynthetic DNA sequence encoding
Bacillus thuringiensis Cry1Ab1 core toxin using codons optimized for
maize and Table 1 sequences are maintainedCDS(1)..(1830) 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 Leu1 5
10 15agt 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
30tac 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 45gag 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
60tgg 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 Ile65 70 75 80gag
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 95att 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
110tct 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 125gag 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 140att 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 Val145 150 155
160tac 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 175gtg 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 190tat 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 205cgc 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 220gac 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 Val225 230 235
240ctc 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 255atc 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 270ctg 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 285cgc 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 300atc 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 Gln305 310 315
320ata 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 335ctc 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 350caa 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 365cgg 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 380ggg 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 Val385 390 395
400tac 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 415aat 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 430gtt 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 445aga 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 460ata 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 Thr465 470 475
480aat 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 495ggc 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 510gtg 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 525tac 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 540ccc 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 Asn545 550 555
560ctc 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 575ttt 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 590tct 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 605gtc acc
1830Val Thr
61022610PRTArtificial SequenceSynthetic Construct 22Met Asp Asn Asn Pro
Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1 5
10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu
Arg Ile Glu Thr Gly 20 25
30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45Glu Phe Val Pro Gly Ala Gly Phe
Val Leu Gly Leu Val Asp Ile Ile 50 55
60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65
70 75 80Glu Gln Leu Ile Asn
Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85
90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr
Gln Ile Tyr Ala Glu 100 105
110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125Glu Met Arg Ile Gln Phe Asn
Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135
140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser
Val145 150 155 160Tyr Val
Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175Val Phe Gly Gln Arg Trp Gly
Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185
190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr
Ala Val 195 200 205Arg Trp Tyr Asn
Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210
215 220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu
Thr Leu Thr Val225 230 235
240Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro
245 250 255Ile Arg Thr Val Ser
Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260
265 270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala
Gln Gly Ile Glu 275 280 285Arg Ser
Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290
295 300Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr
Trp Ser Gly His Gln305 310 315
320Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340
345 350Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser
Ser Thr Leu Tyr Arg 355 360 365Arg
Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370
375 380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser
Asn Leu Pro Ser Ala Val385 390 395
400Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro
Gln 405 410 415Asn Asn Asn
Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420
425 430Val Ser Met Phe Arg Ser Gly Phe Ser Asn
Ser Ser Val Ser Ile Ile 435 440
445Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450
455 460Ile Ile Pro Ser Ser Gln Ile Thr
Gln Ile Pro Leu Thr Lys Ser Thr465 470
475 480Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro
Gly Phe Thr Gly 485 490
495Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg
500 505 510Val Asn Ile Thr Ala Pro
Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520
525Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp
Gly Arg 530 535 540Pro Ile Asn Gln Gly
Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn545 550
555 560Leu Gln Ser Gly Ser Phe Arg Thr Val Gly
Phe Thr Thr Pro Phe Asn 565 570
575Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
580 585 590Ser Gly Asn Glu Val
Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595
600 605Val Thr 610231830DNAArtificial
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 maintainedCDS(1)..(1830) 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 Leu1 5
10 15agt 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
30tac 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 45gag 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
60tgg 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 Ile65
70 75 80gag 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 95att 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
110tct 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 125gag 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
140att 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 Val145
150 155 160tac 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 175gtg 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
190tat 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 205cgc 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
220gac 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 Val225
230 235 240ctc 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 255atc 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
270ctg 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 285cgc 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
300atc 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 Gln305
310 315 320ata 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 335ctc 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
350caa 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 365cgg 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
380ggg 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 Val385
390 395 400tac 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 415aat 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
430gtt 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 445aga 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
460ata 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 Thr465
470 475 480aat 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 495ggc 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
510gtg 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 525tac 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
540ccc 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 Asn545
550 555 560ctc 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 575ttt 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
590tct 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 605gtc acc
1830Val Thr
61024610PRTArtificial SequenceSynthetic Construct 24Met Asp Asn Asn Pro
Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1 5
10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu
Arg Ile Glu Thr Gly 20 25
30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45Glu Phe Val Pro Gly Ala Gly Phe
Val Leu Gly Leu Val Asp Ile Ile 50 55
60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65
70 75 80Glu Gln Leu Ile Asn
Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85
90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr
Gln Ile Tyr Ala Glu 100 105
110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125Glu Met Arg Ile Gln Phe Asn
Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135
140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser
Val145 150 155 160Tyr Val
Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175Val Phe Gly Gln Arg Trp Gly
Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185
190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr
Ala Val 195 200 205Arg Trp Tyr Asn
Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210
215 220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu
Thr Leu Thr Val225 230 235
240Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro
245 250 255Ile Arg Thr Val Ser
Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260
265 270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala
Gln Gly Ile Glu 275 280 285Arg Ser
Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290
295 300Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr
Trp Ser Gly His Gln305 310 315
320Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340
345 350Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser
Ser Thr Leu Tyr Arg 355 360 365Arg
Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370
375 380Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser
Asn Leu Pro Ser Ala Val385 390 395
400Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro
Gln 405 410 415Asn Asn Asn
Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420
425 430Val Ser Met Phe Arg Ser Gly Phe Ser Asn
Ser Ser Val Ser Ile Ile 435 440
445Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450
455 460Ile Ile Pro Ser Ser Gln Ile Thr
Gln Ile Pro Leu Thr Lys Ser Thr465 470
475 480Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro
Gly Phe Thr Gly 485 490
495Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg
500 505 510Val Asn Ile Thr Ala Pro
Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515 520
525Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp
Gly Arg 530 535 540Pro Ile Asn Gln Gly
Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn545 550
555 560Leu Gln Ser Gly Ser Phe Arg Thr Val Gly
Phe Thr Thr Pro Phe Asn 565 570
575Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
580 585 590Ser Gly Asn Glu Val
Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595
600 605Val Thr 610251866DNABacillus
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 Leu1 5 10
15agc 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 30aat 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
45aac 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 60tgg 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 Ile65 70
75 80gaa 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
95att 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 110gca 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
125aga 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 140att 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 Val145 150
155 160tat 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
175att 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 190tat 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
205aat 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 220gat 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 Val225 230
235 240tta 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
255att 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 270att 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
285aac 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 300ttg 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 Asn305 310
315 320ttt 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
335aac 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 350aga 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
365act 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 380cgt 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 Thr385 390
395 400tat 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
415gat 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 430gca 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
445gta 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 460cca 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 Trp465 470
475 480ggg 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
495ctt 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 510aat 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
525agt 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 540gtg 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 Glu545 550
555 560ata 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
575aat 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 590caa 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
605gat 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 62026621PRTBacillus thuringiensis
26Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1
5 10 15Ser Asn Pro Glu Glu Val
Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20 25
30Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe
Leu Val Ser 35 40 45Asn Phe Val
Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50
55 60Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile65 70 75
80Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala
85 90 95Ile Ala Asn Leu Glu Gly
Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100
105 110Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro
Ala Thr Arg Thr 115 120 125Arg Val
Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130
135 140Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val
Pro Leu Leu Ser Val145 150 155
160Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
165 170 175Ile Phe Gly Glu
Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180
185 190Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr
Ala Asp His Cys Ala 195 200 205Asn
Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210
215 220Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg
Asp Leu Thr Leu Thr Val225 230 235
240Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr
Pro 245 250 255Ile Gln Pro
Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260
265 270Ile Asn Phe Asn Pro Gln Leu Gln Ser Val
Ala Gln Leu Pro Thr Phe 275 280
285Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290
295 300Leu Asn Asn Leu Thr Ile Phe Thr
Asp Trp Phe Ser Val Gly Arg Asn305 310
315 320Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu
Ile Gly Gly Gly 325 330
335Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro
340 345 350Arg Ser Phe Thr Phe Asn
Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360
365Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe
Asn Leu 370 375 380Arg Gly Val Glu Gly
Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr385 390
395 400Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu
Thr Glu Leu Pro Pro Glu 405 410
415Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His
420 425 430Ala Thr Phe Val Gln
Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val 435
440 445Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr
Asn Thr Ile Asp 450 455 460Pro Glu Arg
Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp465
470 475 480Gly Gly Thr Ser Val Ile Thr
Gly Pro Gly Phe Thr Gly Gly Asp Ile 485
490 495Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu
Gln Val Asn Ile 500 505 510Asn
Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515
520 525Ser Arg Asp Ala Arg Val Ile Val Leu
Thr Gly Ala Ala Ser Thr Gly 530 535
540Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu545
550 555 560Ile Gly Glu Asn
Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565
570 575Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp
Ile Ile Gly Ile Ser Glu 580 585
590Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile
595 600 605Asp Lys Ile Glu Ile Ile Leu
Ala Asp Ala Thr Phe Glu 610 615
620271866DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus
thuringiensis Cry1Ca core toxin using codons optimized for maize
and Table 1 sequences are maintainedCDS(1)..(1866) 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 Leu1 5
10 15agc 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
30aat 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 45aac 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
60tgg 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 Ile65
70 75 80gaa 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 95att 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
110gca 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 125aga 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
140att 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 Val145
150 155 160tat 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 175att 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
190tat 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 205aat 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
220gat 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 Val225
230 235 240tta 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 255att 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
270att 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 285aac 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
300ttg 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 Asn305
310 315 320ttt 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 335aac 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
350aga 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 365act 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
380cgt 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 Thr385
390 395 400tat 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 415gat 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
430gca 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 445gta 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
460cca 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 Trp465
470 475 480ggg 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 495ctt 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
510aat 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 525agt 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
540gtg 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 Glu545
550 555 560ata 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 575aat 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
590caa 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 605gat 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
62028621PRTArtificial SequenceSynthetic Construct 28Met Asp Asn Asn Pro
Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1 5
10 15Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu
Arg Ile Ser Thr Gly 20 25
30Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser
35 40 45Asn Phe Val Pro Gly Gly Gly Phe
Leu Val Gly Leu Ile Asp Phe Val 50 55
60Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65
70 75 80Glu Gln Leu Ile Asn
Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85
90 95Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe
Asn Ile Tyr Val Glu 100 105
110Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr
115 120 125Arg Val Ile Asp Arg Phe Arg
Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135
140Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser
Val145 150 155 160Tyr Ala
Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
165 170 175Ile Phe Gly Glu Arg Trp Gly
Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185
190Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His
Cys Ala 195 200 205Asn Thr Tyr Asn
Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210
215 220Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu
Thr Leu Thr Val225 230 235
240Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro
245 250 255Ile Gln Pro Val Gly
Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260
265 270Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln
Leu Pro Thr Phe 275 280 285Asn Val
Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290
295 300Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe
Ser Val Gly Arg Asn305 310 315
320Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
325 330 335Asn Ile Thr Ser
Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340
345 350Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg
Thr Leu Ser Asn Pro 355 360 365Thr
Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370
375 380Arg Gly Val Glu Gly Val Glu Phe Ser Thr
Pro Thr Asn Ser Phe Thr385 390 395
400Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro
Glu 405 410 415Asp Asn Ser
Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420
425 430Ala Thr Phe Val Gln Arg Ser Gly Thr Pro
Phe Leu Thr Thr Gly Val 435 440
445Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450
455 460Pro Glu Arg Ile Asn Gln Ile Pro
Leu Val Lys Gly Phe Arg Val Trp465 470
475 480Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr
Gly Gly Asp Ile 485 490
495Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile
500 505 510Asn Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520
525Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser
Thr Gly 530 535 540Val Gly Gly Gln Val
Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu545 550
555 560Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe
Arg Tyr Thr Asp Phe Ser 565 570
575Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu
580 585 590Gln Pro Leu Phe Gly
Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595
600 605Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe
Glu 610 615 620291866DNAArtificial
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 maintainedCDS(1)..(1866) 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 Leu1 5
10 15agc 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
30aat 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 45aac 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
60tgg 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 Ile65
70 75 80gaa 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 95att 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
110gca 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 125aga 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
140att 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 Val145
150 155 160tat 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 175atc 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
190tat 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 205aat 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
220gat 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 Val225
230 235 240tta 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 255att 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
270att 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 285aac 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
300ttg 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 Asn305
310 315 320ttc 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 335aac 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
350aga 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 365act 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
380cgt 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 Thr385
390 395 400tat 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 415gat 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
430gca 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 445gta 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
460cca 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 Trp465
470 475 480ggg 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 495ctt 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
510aat 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 525agt 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
540gtg 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 Glu545
550 555 560ata 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 575aat 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
590caa 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 605gat 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
62030621PRTArtificial SequenceSynthetic Construct 30Met Asp Asn Asn Pro
Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1 5
10 15Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu
Arg Ile Ser Thr Gly 20 25
30Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser
35 40 45Asn Phe Val Pro Gly Gly Gly Phe
Leu Val Gly Leu Ile Asp Phe Val 50 55
60Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65
70 75 80Glu Gln Leu Ile Asn
Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85
90 95Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe
Asn Ile Tyr Val Glu 100 105
110Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr
115 120 125Arg Val Ile Asp Arg Phe Arg
Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135
140Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser
Val145 150 155 160Tyr Ala
Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
165 170 175Ile Phe Gly Glu Arg Trp Gly
Leu Thr Thr Ile Asn Val Asn Glu Asn 180 185
190Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His
Cys Ala 195 200 205Asn Thr Tyr Asn
Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210
215 220Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu
Thr Leu Thr Val225 230 235
240Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro
245 250 255Ile Gln Pro Val Gly
Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu 260
265 270Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln
Leu Pro Thr Phe 275 280 285Asn Val
Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290
295 300Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe
Ser Val Gly Arg Asn305 310 315
320Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
325 330 335Asn Ile Thr Ser
Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340
345 350Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg
Thr Leu Ser Asn Pro 355 360 365Thr
Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370
375 380Arg Gly Val Glu Gly Val Glu Phe Ser Thr
Pro Thr Asn Ser Phe Thr385 390 395
400Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro
Glu 405 410 415Asp Asn Ser
Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420
425 430Ala Thr Phe Val Gln Arg Ser Gly Thr Pro
Phe Leu Thr Thr Gly Val 435 440
445Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450
455 460Pro Glu Arg Ile Asn Gln Ile Pro
Leu Val Lys Gly Phe Arg Val Trp465 470
475 480Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr
Gly Gly Asp Ile 485 490
495Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile
500 505 510Asn Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515 520
525Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser
Thr Gly 530 535 540Val Gly Gly Gln Val
Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu545 550
555 560Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe
Arg Tyr Thr Asp Phe Ser 565 570
575Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu
580 585 590Gln Pro Leu Phe Gly
Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595
600 605Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe
Glu 610 615 620311428DNABacillus
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 His1 5 10
15aca 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 30aat 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
45gca 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 60aga 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 Phe65 70
75 80tct 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
95tgg 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 110gct 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
125gga 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 140aat 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 Ala145 150
155 160cga 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
175aaa 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 190aaa 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
205aca 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 220tta 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 Ile225 230
235 240aaa 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
255gga 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 270gtt 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
285tct 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 300aat 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 Glu305 310
315 320gca 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
335gct 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 350tct 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
365gct 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 380tat 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 Asp385 390
395 400tca 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
415cca 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 430aca 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
445tat 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 460gat tgg tat aat aat
tcg gat tgg tat aat aat tga 1428Asp Trp Tyr Asn Asn
Ser Asp Trp Tyr Asn Asn465 470
47532475PRTBacillus thuringiensis 32Met Ile Ile Asp Ser Lys Thr Thr Leu
Pro Arg His Ser Leu Ile His1 5 10
15Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met
Thr 20 25 30Asn Gly Asn Gln
Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35
40 45Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn
Glu Gln Gln Leu 50 55 60Arg Thr His
Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe65 70
75 80Ser Gln Leu Tyr Asp Val Tyr Cys
Ser Asp Lys Thr Ser Ala Glu Trp 85 90
95Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn
Asp Ile 100 105 110Ala Ser Tyr
Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115
120 125Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp
Asn Ile Val Asp Asn 130 135 140Asn Ser
Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala145
150 155 160Arg Cys Gly Ile Leu Ile Lys
Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165
170 175Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His
Gly Asp Gln Lys 180 185 190Lys
Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln 195
200 205Thr Ala Leu Asn Gln Ala His Gly Glu
Ser Ser Pro Ala His Lys Glu 210 215
220Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile225
230 235 240Lys Ala Glu Gln
Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245
250 255Gly Pro Leu Leu Gly Phe Val Val Tyr Glu
Ile Leu Glu Asn Thr Ala 260 265
270Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp
275 280 285Ser Ala Gln His Asp Leu Asp
Arg Asp Val Lys Ile Ile Gly Met Leu 290 295
300Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln
Glu305 310 315 320Ala Ile
Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly
325 330 335Ala Gln Ile Glu Asn Leu Arg
Thr Thr Ser Leu Gln Glu Val Gln Asp 340 345
350Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala
Ser Asp 355 360 365Ala Trp Leu Val
Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370
375 380Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn
Val Ile Ser Asp385 390 395
400Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn
405 410 415Pro Thr Thr Asn Met
Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420
425 430Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn
Ser Asn Leu Glu 435 440 445Tyr Lys
Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450
455 460Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn
Asn465 470 475331428DNAArtificial
SequenceSynthetic DNA sequence encoding Bacillus thuringiensis
Cry6Aa toxin using codons optimized for maize and Table 1 sequences
are maintainedCDS(1)..(1428) 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 His1 5 10
15aca 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 30aac 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
45gcg 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 60agg 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 Phe65 70
75 80tct 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
95tgg 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 110gcc 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
125ggc 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 140aat 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 Ala145 150
155 160cga 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
175aaa 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 190aaa 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
205acg 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 220ctg 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 Ile225 230
235 240aaa 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
255ggc 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 270gtc 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
285tct 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 300aac 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 Glu305 310
315 320gca 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
335gcg 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 350tct 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
365gct 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 380tac 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 Asp385 390
395 400tcc 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
415cca 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 430acc 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
445tat 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 460gac tgg tac aat aat
tcg gat tgg tac aat aat tga 1428Asp Trp Tyr Asn Asn
Ser Asp Trp Tyr Asn Asn465 470
47534475PRTArtificial SequenceSynthetic Construct 34Met Ile Ile Asp Ser
Lys Thr Thr Leu Pro Arg His Ser Leu Ile His1 5
10 15Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly
Pro Gly Asp Met Thr 20 25
30Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly
35 40 45Ala Tyr Ile Gln Thr Gly Leu Gly
Leu Pro Val Asn Glu Gln Gln Leu 50 55
60Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe65
70 75 80Ser Gln Leu Tyr Asp
Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 85
90 95Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys
Ser Ala Asn Asp Ile 100 105
110Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp
115 120 125Gly Tyr Phe Lys Lys Leu Gln
Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135
140Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys
Ala145 150 155 160Arg Cys
Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala
165 170 175Lys Asn Ile Val Thr Ser Leu
Asp Gln Phe Leu His Gly Asp Gln Lys 180 185
190Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu
Val Gln 195 200 205Thr Ala Leu Asn
Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210
215 220Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu
Glu Arg Thr Ile225 230 235
240Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu
245 250 255Gly Pro Leu Leu Gly
Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260
265 270Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys
Lys Gln Leu Asp 275 280 285Ser Ala
Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290
295 300Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr
Ser Gln Gly Gln Glu305 310 315
320Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly
325 330 335Ala Gln Ile Glu
Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340
345 350Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu
Glu Asp Ala Ser Asp 355 360 365Ala
Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370
375 380Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro
Ile Asn Val Ile Ser Asp385 390 395
400Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser
Asn 405 410 415Pro Thr Thr
Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420
425 430Thr Ser Leu Pro Asn Asn Phe Met Leu Ser
Arg Asn Ser Asn Leu Glu 435 440
445Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450
455 460Asp Trp Tyr Asn Asn Ser Asp Trp
Tyr Asn Asn465 470 475351428DNAArtificial
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 maintainedCDS(1)..(1428) 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 His1 5 10
15aca 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 30aac 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 45gcg 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 60agg
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 Phe65
70 75 80tct 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 95tgg 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 110gcc
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 125ggc 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
140aat 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 Ala145
150 155 160cga 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 175aaa 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
190aaa 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 205acg 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
220ctg 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 Ile225
230 235 240aaa 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 255ggc 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
270gtc 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 285tct 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
300aac 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 Glu305
310 315 320gca 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 335gcg 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
350tct 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 365gct 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
380tac 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 Asp385
390 395 400tcc 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 415cca 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
430acc 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 445tat 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
460gac tgg tac aat aat tcg gat tgg tac aat aat tga
1428Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn465 470
47536475PRTArtificial SequenceSynthetic Construct 36Met
Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His1
5 10 15Thr Ile Lys Leu Asn Ser Asn
Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 25
30Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr
Ile Gly 35 40 45Ala Tyr Ile Gln
Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55
60Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro
Ser Asp Phe65 70 75
80Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp
85 90 95Trp Asn Lys Asn Leu Tyr
Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100
105 110Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser
Ile Lys Lys Asp 115 120 125Gly Tyr
Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130
135 140Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile
Lys Asp Phe Lys Ala145 150 155
160Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala
165 170 175Lys Asn Ile Val
Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180
185 190Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg
Leu Lys Glu Val Gln 195 200 205Thr
Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210
215 220Leu Leu Glu Lys Val Lys Asn Leu Lys Thr
Thr Leu Glu Arg Thr Ile225 230 235
240Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu
Leu 245 250 255Gly Pro Leu
Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260
265 270Val Gln His Ile Lys Asn Gln Ile Asp Glu
Ile Lys Lys Gln Leu Asp 275 280
285Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290
295 300Asn Ser Ile Asn Thr Asp Ile Asp
Asn Leu Tyr Ser Gln Gly Gln Glu305 310
315 320Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp
Ala Thr Ile Gly 325 330
335Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp
340 345 350Ser Asp Asp Ala Asp Glu
Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360
365Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu
Asn Ala 370 375 380Tyr Ser Thr Asn Ser
Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp385 390
395 400Ser Cys Asn Cys Ser Thr Thr Asn Met Thr
Ser Asn Gln Tyr Ser Asn 405 410
415Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr
420 425 430Thr Ser Leu Pro Asn
Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu 435
440 445Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp
Tyr Asn Asn Ser 450 455 460Asp Trp Tyr
Asn Asn Ser Asp Trp Tyr Asn Asn465 470
47537888DNASphingobiurn 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 Ala1 5
10 15gtc 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
30ctg 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 45ttc 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
60gaa 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 Val65
70 75 80ccc 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 95cgc 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
110gac 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 125atc 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
140acc 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 Gly145
150 155 160ttg 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 175gcc 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
190gac 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 205ggc 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
220atc 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 Leu225
230 235 240tac 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 255aag 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
270gta 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 285ggt ggc gtg cgc ccg gcg cgc
tag 888Gly Gly Val Arg Pro Ala Arg
290 29538295PRTSphingobiurn herbicidovorans 38Met His
Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala1 5
10 15Val Gln Pro Leu Thr Gly Val Leu
Gly Ala Glu Ile Thr Gly Val Asp 20 25
30Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp
Ala 35 40 45Phe His Thr Tyr Gln
Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55
60Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp
Pro Val65 70 75 80Pro
Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg
85 90 95Arg Glu Ala Asn Glu Ser Gly
Arg Val Ile Gly Asp Asp Trp His Thr 100 105
110Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met
Arg Ala 115 120 125Ile Asp Val Pro
Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130
135 140Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala
Thr Ile Glu Gly145 150 155
160Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln
165 170 175Ala Gln Asn Arg Arg
Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180
185 190Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val
Val Thr His Pro 195 200 205Gly Ser
Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210
215 220Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro
Leu Leu Gln Phe Leu225 230 235
240Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys
245 250 255Lys Asp Gln Val
Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260
265 270Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu
Thr Arg Thr Thr Val 275 280 285Gly
Gly Val Arg Pro Ala Arg 290 29539888DNAArtificial
SequenceSynthetic DNA sequence encoding the AAD1 protein using
codons optimized for maize and Table 1 and Table 2 sequences are
maintainedCDS(1)..(888) 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 Ala1 5 10
15gtc 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 30ctg 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
45ttc 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 60gaa 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 Val65 70
75 80ccc 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
95agg 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 110gac 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
125atc 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 140act 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 Gly145 150
155 160ttg 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
175gcc 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 190gac 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
205ggc 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 220atc 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 Leu225 230
235 240tac 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
255aag 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 270gtt 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
285ggt gga gtt agg cca gcg aga tga
888Gly Gly Val Arg Pro Ala Arg 290
29540295PRTArtificial SequenceSynthetic Construct 40Met His Ala Ala Leu
Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala1 5
10 15Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu
Ile Thr Gly Val Asp 20 25
30Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala
35 40 45Phe His Thr Tyr Gln Val Ile Tyr
Phe Pro Gly Gln Ala Ile Thr Asn 50 55
60Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val65
70 75 80Pro Leu Leu Lys Ser
Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85
90 95Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly
Asp Asp Trp His Thr 100 105
110Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala
115 120 125Ile Asp Val Pro Glu His Gly
Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135
140Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu
Gly145 150 155 160Leu Asn
Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln
165 170 175Ala Gln Asn Arg Arg Phe Ser
Asn Thr Ser Val Lys Val Met Asp Val 180 185
190Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr
His Pro 195 200 205Gly Ser Gly Arg
Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210
215 220Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu
Leu Gln Phe Leu225 230 235
240Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys
245 250 255Lys Asp Gln Val Leu
Val Trp Asp Asn Leu Cys Thr Met His Arg Ala 260
265 270Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr
Arg Thr Thr Val 275 280 285Gly Gly
Val Arg Pro Ala Arg 290 29541888DNAArtificial
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 maintainedCDS(1)..(888) 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 Ala1 5
10 15gtc 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
30ctg 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 45ttc 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
60gaa 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 Val65 70 75 80ccc
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 95agg 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
110gac 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 125atc 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 140act 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 Gly145 150 155
160ttg 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 175gcc 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 190gac 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 205ggc 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 220atc 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 Leu225 230 235
240tac 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 255aag 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 270gtt 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 285ggt gga
gtt agg cca gcg aga tga 888Gly Gly
Val Arg Pro Ala Arg 290 29542295PRTArtificial
SequenceSynthetic Construct 42Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg
Phe Glu Arg Ile Ala1 5 10
15Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp
20 25 30Leu Arg Glu Pro Leu Asp Asp
Ser Thr Trp Asn Glu Ile Leu Asp Ala 35 40
45Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr
Asn 50 55 60Glu Gln His Ile Ala Phe
Ser Arg Arg Phe Gly Pro Val Asp Pro Val65 70
75 80Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu
Val Gln Met Ile Arg 85 90
95Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr
100 105 110Asp Ser Thr Phe Leu Asp
Ala Pro Pro Ala Ala Val Val Met Arg Ala 115 120
125Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser
Met Tyr 130 135 140Thr Ala Trp Glu Thr
Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly145 150
155 160Leu Asn Val Val His Ser Ala Thr Arg Val
Phe Gly Ser Leu Tyr Gln 165 170
175Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val
180 185 190Asp Ala Gly Asp Arg
Glu Thr Val His Pro Leu Val Val Thr His Pro 195
200 205Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val
Tyr Cys Gln Arg 210 215 220Ile Glu Gly
Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu225
230 235 240Tyr Glu His Ala Thr Arg Phe
Asp Phe Thr Cys Arg Val Arg Trp Lys 245
250 255Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr
Met His Arg Ala 260 265 270Val
Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275
280 285Gly Gly Val Arg Pro Ala Arg 290
295431368DNAAspergillus 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 Lys1
5 10 15aag 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 30cat 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 45ggg
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 60tgg 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 Gly65 70 75
80tac 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 95cgt 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 110cgc 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
125gac 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 140ggc 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 Asp145 150
155 160att 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
175tac 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 190gct 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
205att 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 220ttg 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 Pro225 230
235 240cgt 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
255aac 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 270cac 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
285ggt 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 300ggt 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 Thr305 310
315 320ctc 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
335gac 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 350ggt 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
365aag 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 380aac 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 Ser385 390
395 400acc 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
415aag 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 430cag 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
445att ccc aca gcg gat gcg gcg tga
1368Ile Pro Thr Ala Asp Ala Ala 450
45544455PRTAspergillus nidulans 44Met Ser Ala Pro Thr Ala Asp Ile Arg Ala
Arg Ala Pro Glu Ala Lys1 5 10
15Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys
20 25 30His Val Asn Trp Leu Asn
Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35 40
45Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr
Ala Ile 50 55 60Trp Ala Val Ile Tyr
Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly65 70
75 80Tyr His Arg Leu Trp Ala His Cys Ser Tyr
Ser Ala Thr Leu Pro Leu 85 90
95Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile
100 105 110Arg Trp Trp Ala Arg
Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115
120 125Asp Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu
Tyr Ser His Leu 130 135 140Gly Trp Met
Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp145
150 155 160Ile Ser Asp Leu Asn Glu Asp
Pro Val Val Val Trp Gln His Arg Asn 165
170 175Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val
Pro Met Leu Val 180 185 190Ala
Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly 195
200 205Ile Leu Arg Ile Phe Phe Val Gln Gln
Ala Thr Phe Cys Val Asn Ser 210 215
220Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro225
230 235 240Arg Asp His Val
Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245
250 255Asn Phe His His Glu Phe Pro Ser Asp Tyr
Arg Asn Ala Ile Glu Trp 260 265
270His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu
275 280 285Gly Leu Ala Tyr Asp Leu Lys
Lys Phe Arg Ala Asn Glu Ile Glu Lys 290 295
300Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala
Thr305 310 315 320Leu Asp
Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp
325 330 335Asp Tyr Val Glu Gln Ala Lys
Asn Gly Arg Gly Leu Val Ala Ile Ala 340 345
350Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro
Gly Gly 355 360 365Lys Ala Met Ile
Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370
375 380Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His
Asn Leu Leu Ser385 390 395
400Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp
405 410 415Lys Arg Ala Gln Lys
Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420
425 430Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys
Ile Pro Glu Pro 435 440 445Ile Pro
Thr Ala Asp Ala Ala 450 455451368DNAArtificial
SequenceSynthetic DNA sequence encoding Aspergillus nidulans delta-9
fatty acid desaturase protein using codons optimized for maize and
Table 1 & Table 2 sequences are maintainedCDS(1)..(1368) 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 Lys1 5
10 15aag 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
30cat 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 45ggg 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
60tgg 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 Gly65
70 75 80tat 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 95cgt 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
110cgt 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 125gac 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
140ggc 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 Asp145
150 155 160att 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 175tac 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
190gct 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 205ata 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
220ttg 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 Pro225
230 235 240agg 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 255aac 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
270cac 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 285ggt 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
300ggg 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 Thr305
310 315 320ctc 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 335gac 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
350ggc 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 365aag 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
380aat 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 Ser385
390 395 400aca 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 415aag 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
430caa 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 445att ccc aca gcg gat gcg gcg
tag 1368Ile Pro Thr Ala Asp Ala Ala
450 45546455PRTArtificial SequenceSynthetic Construct
46Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys1
5 10 15Lys Val His Ile Ala Asp
Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20 25
30His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile
Pro Leu Tyr 35 40 45Gly Cys Ile
Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50
55 60Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly
Ile Thr Ala Gly65 70 75
80Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu
85 90 95Arg Ile Trp Leu Ala Ala
Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100
105 110Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg
Tyr Thr Asp Thr 115 120 125Asp Lys
Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130
135 140Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg
Ile Gly Arg Thr Asp145 150 155
160Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn
165 170 175Tyr Leu Lys Val
Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180
185 190Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly
Phe Val Tyr Ala Gly 195 200 205Ile
Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210
215 220Leu Ala His Trp Leu Gly Asp Gln Pro Phe
Asp Asp Arg Asn Ser Pro225 230 235
240Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr
His 245 250 255Asn Phe His
His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260
265 270His Gln Tyr Asp Pro Thr Lys Trp Ser Ile
Trp Ala Trp Lys Gln Leu 275 280
285Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290
295 300Gly Arg Val Gln Gln Leu Gln Lys
Lys Leu Asp Arg Lys Arg Ala Thr305 310
315 320Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val
Met Glu Trp Asp 325 330
335Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala
340 345 350Gly Val Val His Asp Val
Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355 360
365Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala
Met Phe 370 375 380Asn Gly Gly Val Tyr
Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser385 390
395 400Thr Met Arg Val Gly Val Ile Arg Gly Gly
Cys Glu Val Glu Ile Trp 405 410
415Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly
420 425 430Gln Arg Val Ile Arg
Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro 435
440 445Ile Pro Thr Ala Asp Ala Ala 450
455471368DNAArtificial 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
maintainedCDS(1)..(1368) 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 Lys1 5 10
15aag 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 30cat 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
45ggg 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 60tgg 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 Gly65 70
75 80tat 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
95cgt 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 110cgt 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
125gac 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 140ggc 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 Asp145 150
155 160att 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
175tac 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 190gct 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
205ata 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 220ttg 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 Pro225 230
235 240agg 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
255aac 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 270cac 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
285ggt 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 300ggg 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 Thr305 310
315 320ctc 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
335gac 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 350ggc 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
365aag 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 380aat 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 Ser385 390
395 400aca 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
415aag 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 430caa 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
445att ccc aca gcg gat gcg gcg tag
1368Ile Pro Thr Ala Asp Ala Ala 450
45548455PRTArtificial SequenceSynthetic Construct 48Met Ser Ala Pro Thr
Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys1 5
10 15Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg
His Asn Trp Tyr Lys 20 25
30His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr
35 40 45Gly Cys Ile Gln Ala Phe Trp Val
Pro Leu Gln Leu Lys Thr Ala Ile 50 55
60Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly65
70 75 80Tyr His Arg Leu Trp
Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu 85
90 95Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala
Val Glu Gly Ser Ile 100 105
110Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr
115 120 125Asp Lys Asp Pro Tyr Ser Val
Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135
140Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr
Asp145 150 155 160Ile Ser
Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn
165 170 175Tyr Leu Lys Val Val Phe Thr
Met Gly Leu Ala Val Pro Met Leu Val 180 185
190Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr
Ala Gly 195 200 205Ile Leu Arg Ile
Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210
215 220Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp
Arg Asn Ser Pro225 230 235
240Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His
245 250 255Asn Phe His His Glu
Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260
265 270His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala
Trp Lys Gln Leu 275 280 285Gly Leu
Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290
295 300Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp
Arg Lys Arg Ala Thr305 310 315
320Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp Asp
325 330 335Asp Tyr Val Glu
Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala 340
345 350Gly Val Val His Asp Val Thr Asp Phe Ile Lys
Asp His Pro Gly Gly 355 360 365Lys
Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370
375 380Asn Gly Gly Val Tyr Tyr His Ser Asn Ala
Ala His Asn Leu Leu Ser385 390 395
400Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile
Trp 405 410 415Lys Arg Ala
Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420
425 430Gln Arg Val Ile Arg Ala Gly Glu Gln Pro
Thr Lys Ile Pro Glu Pro 435 440
445Ile Pro Thr Ala Asp Ala Ala 450
45549798DNAXerophyta 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 Ala1 5 10
15gac 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 30cgg 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 45tct 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 60ata 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 Leu65 70
75 80att 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
95agg 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 110ctc 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 125ctc 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 140aga 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 Tyr145
150 155 160ctc 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 175aca 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 190atc
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 205ctg 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
220aat 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 His225
230 235 240atg 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 255ttt gta ttc atc tat gaa gct gaa ttt taa
798Phe Val Phe Ile Tyr Glu Ala Glu Phe
260 26550265PRTXerophyta viscosa 50Met Arg Asn Glu Gly
Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala1 5
10 15Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu
Gly Asp Ala Ala Lys 20 25
30Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly
35 40 45Ser Thr Ile Val Gln Ala Ile Ala
Ser Ile Ala Ala Ile Tyr Leu Leu 50 55
60Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu65
70 75 80Ile Pro Tyr Val Tyr
Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85
90 95Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile
Gly Val Val Met Lys 100 105
110Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser
115 120 125Leu Ile Leu Leu Ile Val Val
Ser Pro Thr Phe Ile Ala His Thr Ile 130 135
140Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys
Tyr145 150 155 160Leu Leu
Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe
165 170 175Thr Lys Ser Asn Gly Ile Ser
Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185
190Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr
Ser Leu 195 200 205Leu Gln Leu Leu
Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210
215 220Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys
Val Ser Leu His225 230 235
240Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln
245 250 255Phe Val Phe Ile Tyr
Glu Ala Glu Phe 260 26551798DNAArtificial
SequenceSynthetic DNA sequence encoding Xerophyta viscosa SAP1
protein using codons optimized for maize and Table 1 & Table 2 sequences
are maintainedCDS(1)..(798) 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 Ala1 5 10
15gac 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 30cgc 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
45tca 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 60att 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 Leu65 70
75 80att 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
95cgt 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 110ctg 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
125ctc 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 140cgt 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 Tyr145 150
155 160ctg 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
175aca 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 190atc 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
205ctc 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 220aac 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 His225 230
235 240atg 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
255ttc gtg ttc atc tat gag gct gag ttt taa
798Phe Val Phe Ile Tyr Glu Ala Glu Phe 260
26552265PRTArtificial SequenceSynthetic Construct 52Met Arg Asn Glu Gly
Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala1 5
10 15Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu
Gly Asp Ala Ala Lys 20 25
30Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly
35 40 45Ser Thr Ile Val Gln Ala Ile Ala
Ser Ile Ala Ala Ile Tyr Leu Leu 50 55
60Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu65
70 75 80Ile Pro Tyr Val Tyr
Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85
90 95Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile
Gly Val Val Met Lys 100 105
110Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser
115 120 125Leu Ile Leu Leu Ile Val Val
Ser Pro Thr Phe Ile Ala His Thr Ile 130 135
140Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys
Tyr145 150 155 160Leu Leu
Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe
165 170 175Thr Lys Ser Asn Gly Ile Ser
Asn Ser Val Gly Ile Ile Ile Leu Leu 180 185
190Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr
Ser Leu 195 200 205Leu Gln Leu Leu
Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210
215 220Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys
Val Ser Leu His225 230 235
240Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln
245 250 255Phe Val Phe Ile Tyr
Glu Ala Glu Phe 260 26553798DNAArtificial
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 maintainedCDS(1)..(798) 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 Ala1 5 10
15gac 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 30cgc 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 45tca 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 60att 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 Leu65
70 75 80att 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
95cgt 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 110ctg 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 125ctc 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 140cgt
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 Tyr145
150 155 160ctg 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 175aca 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 190atc
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 205ctc 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
220aac 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 His225
230 235 240atg 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 255ttc gtg ttc atc tat gag gct gag ttt taa
798Phe Val Phe Ile Tyr Glu Ala Glu Phe
260 26554265PRTArtificial SequenceSynthetic Construct
54Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala1
5 10 15Asp Glu Val Ile Ser Gly
Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20 25
30Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe
Gly Val Gly 35 40 45Ser Thr Ile
Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50
55 60Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu
Thr Ser Leu Leu65 70 75
80Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe
85 90 95Arg Gly Asp Leu Gly Arg
Trp Leu Ser Phe Ile Gly Val Val Met Lys 100
105 110Leu Phe Phe His Arg His Phe Pro Val Thr Leu Glu
Leu Leu Val Ser 115 120 125Leu Ile
Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130
135 140Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu
Val Ile Ala Cys Tyr145 150 155
160Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe
165 170 175Thr Lys Ser Asn
Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180
185 190Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe
Leu Tyr Thr Ser Leu 195 200 205Leu
Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210
215 220Asn Lys Trp Phe Asn Phe Met His Val Cys
Lys Cys Val Ser Leu His225 230 235
240Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val
Gln 245 250 255Phe Val Phe
Ile Tyr Glu Ala Glu Phe 260
26555717DNAAequorea 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 Val1 5 10
15gaa 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 30ggt 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 45act 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 60tct 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 Gln65 70
75 80cat 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
95act 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 110aag 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 125gat 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 140tat 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 Gly145
150 155 160atc 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 175caa 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 190gtc
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 205aaa 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
220aca 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 Lys225
230 23556238PRTAequorea victoria 56Met Ser Lys Gly
Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val1 5
10 15Glu Leu Asp Gly Asp Val Asn Gly Gln Lys
Phe Ser Val Ser Gly Glu 20 25
30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys
35 40 45Thr Thr Gly Lys Leu Pro Val Pro
Trp Pro Thr Leu Val Thr Thr Phe 50 55
60Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln65
70 75 80His Asp Phe Phe Lys
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85
90 95Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr
Arg Ala Glu Val 100 105 110Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115
120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu
Gly His Lys Met Glu Tyr Asn 130 135
140Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly145
150 155 160Ile Lys Val Asn
Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165
170 175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
Pro Ile Gly Asp Gly Pro 180 185
190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
195 200 205Lys Asp Pro Asn Glu Lys Arg
Asp His Met Ile Leu Leu Glu Phe Val 210 215
220Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys225
230 23557717DNAArtificial SequenceSynthetic DNA
sequence encoding Aequorea victoria GFP1 protein using codons
optimized for maize and Table 1 & Table 2 sequences are
maintainedCDS(1)..(717) 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 Val1 5 10
15gag 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 30ggt 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
45acg 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 60tct 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 Gln65 70
75 80cat 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
95act 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 110aag 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
125gac 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 140tat 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 Gly145 150
155 160ata 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
175cag 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 190gtc 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
205aag 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 220acc 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 Lys225 230
23558238PRTArtificial SequenceSynthetic Construct 58Met Ser Lys Gly Glu
Glu Leu Phe Thr Gly Val Val Pro Val Leu Val1 5
10 15Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe
Ser Val Ser Gly Glu 20 25
30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys
35 40 45Thr Thr Gly Lys Leu Pro Val Pro
Trp Pro Thr Leu Val Thr Thr Phe 50 55
60Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln65
70 75 80His Asp Phe Phe Lys
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85
90 95Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu Val 100 105
110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
115 120 125Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Met Glu Tyr Asn 130 135
140Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn
Gly145 150 155 160Ile Lys
Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175Gln Leu Ala Asp His Tyr Gln
Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185
190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu Ser 195 200 205Lys Asp Pro Asn
Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210
215 220Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu
Tyr Lys225 230 23559717DNAArtificial
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 maintainedCDS(1)..(717) 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 Val1 5 10
15gag 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 30ggt 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 45acg 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 60tct 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 Gln65
70 75 80cat 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
95act 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 110aag 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 125gac 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 140tat
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 Gly145
150 155 160ata 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 175cag 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 190gtc
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 205aag 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
220acc 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 Lys225
230 23560238PRTArtificial SequenceSynthetic Construct
60Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val1
5 10 15Glu Leu Asp Gly Asp Val
Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25
30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn
Phe Ile Cys 35 40 45Thr Thr Gly
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
His Met Lys Gln65 70 75
80His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
85 90 95Thr Ile Phe Tyr Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100
105 110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
Leu Lys Gly Ile 115 120 125Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130
135 140Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp
Lys Pro Lys Asn Gly145 150 155
160Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175Gln Leu Ala Asp
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180
185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr
Gln Ser Ala Leu Ser 195 200 205Lys
Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210
215 220Thr Ala Ala Arg Ile Thr His Gly Met Asp
Glu Leu Tyr Lys225 230
235611350DNALeptosphaeria 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 Lys1
5 10 15tcg 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
30aag 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 45gga 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 60tgg 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 Gly65 70 75
80tat 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 95cgc 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 110cga 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 125atg 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 140gga 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 Asp145 150 155
160gtc 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 175tac 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 190gcc 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 205atc 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 220ctc 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 Pro225 230 235
240cga 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 255aac 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 270tac 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 285ccc 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 300gaa 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 Arg305 310 315
320tca 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 335tgg 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 350gct 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 365cct 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 380ggg 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 Asn385 390 395
400ctg 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 415gag 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 430ttg 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 445gcg tag
1350Ala62449PRTLeptosphaeria nodorum 62Met Ala Ala Leu Asp Ser Ile Pro
Glu Asp Lys Ala Thr Ser Ser Lys1 5 10
15Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp
Tyr Lys 20 25 30Lys Ile Asn
Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35
40 45Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr
Arg Pro Thr Leu Ile 50 55 60Trp Ser
Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly65
70 75 80Tyr His Arg Leu Trp Ser His
Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85 90
95Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln
Gly Ser Ala 100 105 110Arg Trp
Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115
120 125Met Lys Asp Pro Tyr Ser Val Met Arg Gly
Leu Leu Phe Ser His Ile 130 135 140Gly
Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp145
150 155 160Val Ser Asp Leu Asp Ser
Asp Pro Val Val Val Trp Gln His Lys His 165
170 175Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe
Pro Met Ile Val 180 185 190Ala
Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195
200 205Ile Ile Arg Ala Cys Phe Val Gln Gln
Ala Thr Phe Cys Val Asn Ser 210 215
220Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro225
230 235 240Arg Asp His Val
Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245
250 255Asn Phe His His Glu Phe Pro Ser Asp Tyr
Arg Asn Ala Ile Ile Trp 260 265
270Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly
275 280 285Pro Phe Pro Leu Ala Tyr Ser
Leu Lys Thr Phe Arg Ser Asn Glu Ile 290 295
300Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys
Arg305 310 315 320Ser Gly
Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser
325 330 335Trp Asp Asp Phe Gln Ala Arg
Cys Lys Glu Ser Gly Glu Met Leu Val 340 345
350Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu
Asp His 355 360 365Pro Gly Gly Arg
Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370
375 380Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn
Ala Ala His Asn385 390 395
400Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val
405 410 415Glu Val Trp Lys Lys
Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420
425 430Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu
Gly Ala Val Ala 435 440
445Ala631350DNAArtificial SequenceSynthetic DNA sequence encoding
Leptosphaeria nodorum delta-9 fatty acid desaturase protein using
codons optimized for maize and Table 1 & Table 2 sequences are
maintainedCDS(1)..(1350) 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 Lys1 5 10
15tcg 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 30aag 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
45ggt 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 60tgg 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 Gly65 70
75 80tat 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
95cgc 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 110agg 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
125atg 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 140ggt 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 Asp145 150
155 160gtc 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
175tac 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 190gcc 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
205atc 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 220ctc 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 Pro225 230
235 240aga 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
255aac 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 270tat 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
285ccg 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 300gaa 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 Arg305 310
315 320tct 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
335tgg 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 350gct 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
365cct 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 380ggg 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 Asn385 390
395 400ctg 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
415gag 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 430ttg 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
445gcc tag
1350Ala64449PRTArtificial SequenceSynthetic Construct 64Met Ala
Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys1 5
10 15Ser Thr His Ile Gln Tyr Gln Glu
Val Thr Phe Arg Asn Trp Tyr Lys 20 25
30Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala
Leu 35 40 45Gly Leu Tyr Leu Thr
Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55
60Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr
Gly Gly65 70 75 80Tyr
His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu
85 90 95Arg Leu Phe Leu Ala Phe Thr
Gly Ala Gly Ala Ile Gln Gly Ser Ala 100 105
110Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr
Asp Thr 115 120 125Met Lys Asp Pro
Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130
135 140Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys
Gly Arg Thr Asp145 150 155
160Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His
165 170 175Tyr Gly Lys Cys Leu
Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180
185 190Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu
Val Tyr Ala Gly 195 200 205Ile Ile
Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210
215 220Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp
Asp Arg Arg Thr Pro225 230 235
240Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His
245 250 255Asn Phe His His
Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260
265 270Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr
Leu Phe Ser Leu Gly 275 280 285Pro
Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290
295 300Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys
Ala Leu Asp Lys Lys Arg305 310 315
320Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile
Ser 325 330 335Trp Asp Asp
Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu Val 340
345 350Ala Val Ala Gly Val Ile His Asp Val Ser
Gln Phe Ile Glu Asp His 355 360
365Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370
375 380Gly Met Phe Asn Gly Gly Val Tyr
Glu His Ser Asn Ala Ala His Asn385 390
395 400Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly
Gly Gln Glu Val 405 410
415Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile
420 425 430Leu Arg Gln Val Thr Arg
Val Glu Arg Leu Val Glu Gly Ala Val Ala 435 440
445Ala651350DNAArtificial 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 maintainedCDS(1)..(1350) 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 Lys1 5 10
15tcg 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 30aag 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 45ggt 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 60tgg 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 Gly65 70
75 80tat 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
95cgc 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 110agg 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 125atg 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 140ggt 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 Asp145
150 155 160gtc 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 175tac 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 190gcc
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 205atc 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
220ctc 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 Pro225
230 235 240aga 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 255aac 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
270tat 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 285ccg 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
300gaa 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 Arg305
310 315 320tct 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 335tgg 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
350gct 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 365cct 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
380ggg 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 Asn385
390 395 400ctg 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 415gag 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
430ttg 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 445gcc tag
1350Ala66449PRTArtificial
SequenceSynthetic Construct 66Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys
Ala Thr Ser Ser Lys1 5 10
15Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys
20 25 30Lys Ile Asn Trp Leu Asn Thr
Thr Leu Val Val Leu Ile Pro Ala Leu 35 40
45Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu
Ile 50 55 60Trp Ser Val Leu Tyr Tyr
Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly65 70
75 80Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser
Ala Arg Leu Pro Leu 85 90
95Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala
100 105 110Arg Trp Trp Ser Ala Asn
His Arg Ala His His Arg Trp Thr Asp Thr 115 120
125Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser
His Ile 130 135 140Gly Trp Met Val Leu
Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp145 150
155 160Val Ser Asp Leu Asp Ser Asp Pro Val Val
Val Trp Gln His Lys His 165 170
175Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val
180 185 190Ala Gly Leu Gly Trp
Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195
200 205Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe
Cys Val Asn Ser 210 215 220Leu Ala His
Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro225
230 235 240Arg Asp His Val Leu Thr Ala
Leu Val Thr Met Gly Glu Gly Tyr His 245
250 255Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn
Ala Ile Ile Trp 260 265 270Tyr
Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275
280 285Pro Phe Pro Leu Ala Tyr Ser Leu Lys
Thr Phe Arg Ser Asn Glu Ile 290 295
300Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg305
310 315 320Ser Gly Leu Asp
Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325
330 335Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu
Ser Gly Glu Met Leu Val 340 345
350Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His
355 360 365Pro Gly Gly Arg Ser Leu Ile
Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375
380Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His
Asn385 390 395 400Leu Leu
Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val
405 410 415Glu Val Trp Lys Lys Gln Arg
Val Asp Val Leu Gly Lys Ser Asp Ile 420 425
430Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala
Val Ala 435 440
445Ala67660DNAXerophyta 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 Asp1 5 10
15acc 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 30gtc 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 45gaa 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 60gtt 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 Glu65 70
75 80tgg 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
95cct 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 110gta 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 125gct 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 140tac 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 Val145
150 155 160gag 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 175tgg 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 190gag
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 205tcc aag aag gat tac ctt cgt ttt acg
aac gtc tga 660Ser Lys Lys Asp Tyr Leu Arg Phe Thr
Asn Val 210 21568219PRTXerophyta viscosa 68Met Pro Gly
Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp1 5
10 15Thr Thr Gln Gly Arg Ile Lys Ile His
Asp Tyr Val Gly Asn Gly Tyr 20 25
30Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr
35 40 45Glu Leu Gly Lys Met Ala Ala
Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55
60Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu65
70 75 80Trp Ile Lys Asp
Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85
90 95Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile
Ile Gln Gln Leu Asn Met 100 105
110Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg
115 120 125Ala Leu His Ile Ile Gly Pro
Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135
140Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala
Val145 150 155 160Glu Ser
Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn
165 170 175Trp Lys Pro Gly Glu Pro Val
Val Ile Lys Pro Asp Val Ser Ser Glu 180 185
190Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp
Leu Pro 195 200 205Ser Lys Lys Asp
Tyr Leu Arg Phe Thr Asn Val 210 21569660DNAArtificial
SequenceSynthetic DNA sequence encoding Xerophyta viscosa PER1
protein using codons optimized for maize and Table 1 & Table 2 sequences
are maintainedCDS(1)..(660) 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 Asp1 5 10
15acg 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 30gtg 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
45gaa 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 60gtg 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 Glu65 70
75 80tgg 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
95ccg 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 110gtg 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
125gca 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 140tac 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 Val145 150
155 160gag 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
175tgg 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 190gag 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
205tca aag aag gac tac ttg agg ttc acc aat gtt tga
660Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210
21570219PRTArtificial SequenceSynthetic Construct 70Met Pro Gly
Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp1 5
10 15Thr Thr Gln Gly Arg Ile Lys Ile His
Asp Tyr Val Gly Asn Gly Tyr 20 25
30Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr
35 40 45Glu Leu Gly Lys Met Ala Ala
Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55
60Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu65
70 75 80Trp Ile Lys Asp
Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85
90 95Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile
Ile Gln Gln Leu Asn Met 100 105
110Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg
115 120 125Ala Leu His Ile Ile Gly Pro
Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135
140Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala
Val145 150 155 160Glu Ser
Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn
165 170 175Trp Lys Pro Gly Glu Pro Val
Val Ile Lys Pro Asp Val Ser Ser Glu 180 185
190Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp
Leu Pro 195 200 205Ser Lys Lys Asp
Tyr Leu Arg Phe Thr Asn Val 210 21571660DNAArtificial
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 maintainedCDS(1)..(660) 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 Asp1 5 10
15acg 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 30gtg 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 45gaa 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 60gtg 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 Glu65
70 75 80tgg 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
95ccg 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 110gtg 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 125gca 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 140tac
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 Val145
150 155 160gag 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 175tgg 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 190gag
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 205tca aag aag gac tac ttg agg ttc acc
aat gtt tga 660Ser Lys Lys Asp Tyr Leu Arg Phe Thr
Asn Val 210 21572219PRTArtificial SequenceSynthetic
Construct 72Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu
Asp1 5 10 15Thr Thr Gln
Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20
25 30Val Ile Leu Phe Ser His Pro Gly Asp Phe
Thr Pro Val Cys Thr Thr 35 40
45Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50
55 60Val Lys Leu Leu Gly Leu Ser Cys Asp
Asp Val Gln Ser His Lys Glu65 70 75
80Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val
Lys Tyr 85 90 95Pro Ile
Ala Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100
105 110Val Asp Pro Asp Glu Thr Glu Ser Ser
Lys Cys Ala Val Pro Ser Arg 115 120
125Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu
130 135 140Tyr Pro Ala Ser Thr Gly Arg
Asn Met Asp Glu Val Leu Arg Ala Val145 150
155 160Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala
Thr Pro Ala Asn 165 170
175Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu
180 185 190Glu Ala Lys Lys Leu Phe
Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195 200
205Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210
215
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