Patent application title: PLANTS WITH ALTERED ROOT ARCHITECTURE, RELATED CONSTRUCTS AND METHODS INVOLVING GENES ENCODING EXOSTOSIN FAMILY POLYPEPTIDES AND HOMOLOGS THEREOF
Inventors:
Graziana Taramino (Wilmington, DE, US)
Hajime Sakai (Newark, DE, US)
Scott V. Tingey (Wilmington, DE, US)
Stephen M. Allen (Wilmington, DE, US)
Stanley Luck (Wilmington, DE, US)
Dwight Tomes (Grimes, IA, US)
Dwight Tomes (Grimes, IA, US)
Xiaomu Niu (Johnston, IA, US)
Assignees:
E.I. DU PONT AND PIONEER HI-BRED INTER
IPC8 Class: AA01H500FI
USPC Class:
800260
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of using a plant or plant part in a breeding process which includes a step of sexual hybridization
Publication date: 2009-04-30
Patent application number: 20090113570
Claims:
1. A plant comprising in its genome a recombinant DNA construct comprising
a polynucleotide operably linked to at least one regulatory element,
wherein said polynucleotide encodes a polypeptide having an amino acid
sequence of at least 50% sequence identity, based on the Clustal V method
of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29,
31, or 34 and wherein said plant exhibits altered root architecture when
compared to a control plant not comprising said recombinant DNA
construct.
2. The plant of claim 1, wherein the plant is a maize plant or a soybean plant.
3. A plant comprising in its genome:a recombinant DNA construct comprising:(a) a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or(b) a suppression DNA construct comprising at least one regulatory element operably linked to:(i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or(ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide,and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
4. The plant of claim 3, wherein the plant is a maize plant or a soybean plant.
5. The plant of claim 3, wherein said plant exhibits said alteration of said at least one agronomic characteristic when compared, under varying environmental conditions, to said control plant not comprising said recombinant DNA construct.
6. The plant of claim 5, wherein said varying environmental condition is at least one selected from drought, nitrogen, or disease.
7. The plant of claim 5, wherein the plant is a maize plant or a soybean plant.
8. The plant of claim 7, wherein the plant is a maize plant or a soybean plant.
9. The plant of claim 3, wherein said at least one agronomic characteristic is selected from the group consisting of greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, stalk lodging, plant height, ear length and harvest index.
10. The plant of claim 9, wherein the plant is a maize plant or a soybean plant.
11. The plant of claim 3, wherein said plant exhibits an increase of said at least one agronomic characteristic when compared to said control plant.
12. The plant of claim 11, wherein the plant is a maize plant or a soybean plant.
13. A method of altering root architecture in a plant, comprising:(a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34; and(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the recombinant DNA construct.
14. The method of claim 13, further comprising:(c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the recombinant DNA construct.
15. A method of evaluating root architecture in a plant, comprising:(a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and(c) evaluating root architecture of the transgenic plant compared to a control plant not comprising the recombinant DNA construct.
16. The method of claim 15, further comprising:(d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and(e) evaluating root architecture of the progeny plant compared to a control plant not comprising the recombinant DNA construct.
17. A method of evaluating root architecture in a plant, comprising:(a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct;(c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and(d) evaluating root architecture of the progeny plant compared to a control plant not comprising the recombinant DNA construct.
18. A method of determining an alteration of an agronomic characteristic in a plant, comprising:(a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and(c) determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
19. The method of claim 18, further comprising:(d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and(e) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
20. The method of claim 19, wherein said determining step comprises determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
21. The method of claim 19, wherein said determining step (e) comprises determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
22. A method of determining an alteration of an agronomic characteristic in a plant, comprising:(a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct;(c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and(d) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
23. The method of claim 22, wherein said determining step comprisesdetermining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
24. A method of determining an alteration of an agronomic characteristic in a plant, comprising:(a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to:(i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or(ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and(c) determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
25. The method of claim 24, wherein said determining step comprises determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the suppression DNA construct.
26. The method of claim 24, further comprising:(d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and(e) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
27. The method of claim 26, wherein said determining step (e) comprisesdetermining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the suppression DNA construct.
28. A method of determining an alteration of an agronomic characteristic in a plant, comprising:(a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to:(i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or(ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct and exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct;(c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and(d) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
29. The method of claim 28, wherein said determining step comprisesdetermining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
30. A method of altering root architecture in a plant, comprising:(a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to:(i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or(ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; and(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct and wherein the transgenic plant exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct.
31. The method of claim 30, further comprising:(c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and wherein the progeny plant exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct.
32. A method of evaluating root architecture in a plant, comprising:(a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to:(i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or(ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and(c) evaluating root architecture of the transgenic plant compared to a control plant not comprising the suppression DNA construct.
33. The method of claim 32, further comprising:(d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and(e) evaluating root architecture of the progeny plant compared to a control plant not comprising the suppression DNA construct.
34. A method of evaluating root architecture in a plant, comprising:(a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to:(i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or(ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;(b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct;(c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and(d) evaluating root architecture of the progeny plant compared to a control plant not comprising the suppression DNA construct.
35. An isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide having an amino acid sequence of at least 80% sequence identity, when compared to SEQ ID NO:15, or 31, or of at least 95%, when compared to SEQ ID NO: 25, based on the Clustal V method of alignment, or a full complement of said nucleic acid sequence.
36. The polynucleotide of claim 35, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:15, or 31 have at least 85% sequence identity, based on the Clustal alignment method.
37. The polynucleotide of claim 35, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:15, or 31 have at least 90% sequence identity, based on the Clustal alignment method.
38. The polynucleotide of claim 35, wherein the amino acid sequence of the polypeptide and the amino acid sequence of SEQ ID NO:15, or 31 have at least 95% sequence identity, based on the Clustal alignment method.
39. The polynucleotide of claim 35, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 15, 25, or 31.
40. The polynucleotide of claim 35, wherein the nucleic acid sequences comprises SEQ ID NO: 14, 24, or 30.
41. A vector comprising the polynucleotide of claim 35.
42. A recombinant DNA construct comprising the polynucleotide of claim 35 operably linked to at least one regulatory sequence.
43. A method for transforming a cell, comprising transforming a cell with the polynucleotide of claim 35.
44. A cell comprising the recombinant DNA construct of claim 42.
45. A method for producing a plant comprising transforming a plant cell with the polynucleotide of claim 35 and regenerating a plant from the transformed plant cell. In further embodiments, vectors and recombinant constructs comprising any of the foregoing polynucleotides and cells comprising the recombinant constructs.
46. A method of mapping genetic variations exhibiting an alteration in root architecture and/or an alteration in at least one agronomic characteristic in plants comprising:(a) crossing two plant varieties; and(b) evaluating genetic variations with respect to a nucleic acid sequence selected from the group consisting of SEQ ID 14, 16, 18, 20, 22, 24, 26, 28, 30, or 33; or(ii) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO:15, 17, 19, 21, 23, 25, 27, 29, 31, or 34;in progeny plants resulting from the cross of step (a) wherein the evaluation is made using a method selected from the group consisting of: RFLP analysis, SNP analysis, and PCR-based analysis.
47. A method of molecular breeding to alter root architecture and/or altering at least one agronomic characteristic in plants comprising:(a) crossing two plant varieties; and(b) evaluating genetic variations with respect to(i) a nucleic acid sequence selected from the group consisting of SEQ ID NO:14, 16, 18, 20, 22, 24, 26, 28, 30, or 33; or(ii) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO:15, 17, 19, 21, 23, 25, 27, 29, 31, or 34;in progeny plants resulting from the cross of step (a) wherein the evaluation is made using a method selected from the group consisting of: RFLP analysis, SNP analysis, and PCR-based analysis.
Description:
FIELD OF THE INVENTION
[0001]The field of invention relates to plant breeding and genetics and, in particular, relates to recombinant DNA constructs useful in plants for altering root architecture.
BACKGROUND OF THE INVENTION
[0002]Water and nutrient availability limit plant growth in all but a very few natural ecosystems. They limit yield in most agricultural ecosystems. Plant roots serve important functions such as water and nutrient uptake, anchorage of the plants in the soil and the establishment of biotic interactions at the rhizosphere. Elucidation of the genetic regulation of plant root development and function is therefore the subject of considerable interest in agriculture and ecology.
[0003]The root system originates from a primary root that develops during embryogenesis. The primary root produces secondary roots, which in turn produce tertiary roots. All secondary, tertiary, quaternary and further roots are referred to as lateral roots. Many plants, including maize, can also produce shoot borne roots, from consecutive under-ground nodes (crown roots) or above-ground nodes (brace roots). Three major processes affect the overall architecture of the root system. First, cell division at the primary root meristem enables indeterminate growth by adding new cells to the root. Second, lateral root formation increases the exploratory capacity of the root system. Third, root-hair formation increases the total surface of primary and lateral roots (Lopez-Bucio et al., Current Opinion in Plant Biology (2003) 6:280-287). In maize mutants have been isolated that are missing only a subset of root types. In Arabidopsis, mutations in root patterning genes such as SHORTROOT and SCARECROW, which show developmental defects in primary and lateral roots, have been identified (J. E. Malamy, Plant, Cell and Environment (2005) 28: 67-77).
[0004]A number of maize mutants affected specifically in root development have been identified (Hochholdinger et al 2004, Annals of Botany 93:359-368). The recessive mutants rtcs and rt1 forms no, or fewer, crown and brace roots, while the primary and lateral roots are not affected. In the recessive mutants des21, lateral seminal roots and root hairs are absent. Root hairs are lacking in the recessive mutant rthl-3. The mutants lrt1 and rum1 are affected before lateral root initiation and mutants slr1 and slr2 are impaired in lateral root elongation. Intrinsic response pathways that determine root system architecture include hormones, cell cycle regulators and regulatory genes. Water stress and nutrient availability belong to the environmental response pathways that determine root system architecture.
[0005]U.S. Application No. 2005-57473 filed Feb. 14, 2005 (U.S. Patent Publication No. 2005/223429 A1 published Oct. 6, 2005) concerns the use of Arabidopsis cytokinin oxidase genes to alter cytokinin levels in plants and stimulate root growth.
[0006]U.S. Pat. No. 6,344,601 (issued Feb. 5, 2002) concerns the under- or overexpression of profilin in a plant cell to alter plant growth habit, e.g. a reduced root and root hair system, delay in the onset of flowering.
[0007]WO2004/US16432 (filed May 21, 2004 (WO2004/106531 published Dec. 9, 2004) concerns the use of methods to manipulate the growth rate and/or yield and/or architecture by over expression of cis-prenyltransferase.
[0008]U.S. Application No. 2004/489500 filed Sep. 30, 2004 (U.S. Patent Publication No. 2005/059154 A1 published Mar. 13, 2005) concerns methods to modify cell number, architecture and yield using over expression of the transcription factor E2F in plants.
[0009]Activation tagging can be utilized to identify genes with the ability to affect a trait. This approach has been used in the model plant species Arabidopsis thaliana (Weigel et al., 2000, Plant Physiol. 122:1003-1013).
[0010]Insertions of transcriptional enhancer elements can dominantly activate and/or elevate the expression of nearby endogenous genes.
SUMMARY OF THE INVENTION
[0011]The present invention includes:
[0012]In one embodiment, an isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide having an amino acid sequence of at least 80% sequence identity, when compared to SEQ ID NO:15, or 31, or of at least 95%, when compared to SEQ ID NO:25, based on the Clustal V method of alignment, or a full complement of said nucleic acid sequence.
[0013]In a second embodiment, an isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide having an amino acid sequence of at least 85% sequence identity, when compared to SEQ ID NO:15, or 31 based on the Clustal V method of alignment, or a full complement of said nucleic acid sequence.
[0014]In a third embodiment, an isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide having an amino acid sequence of at least 90% sequence identity, when compared to SEQ ID NO:15, or 31 based on the Clustal V method of alignment, or a full complement of said nucleic acid sequence.
[0015]In a fourth embodiment, an isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide having an amino acid sequence of at least 95% sequence identity, when compared to SEQ ID NO:15, or 31 based on the Clustal V method of alignment, or a full complement of said nucleic acid sequence.
[0016]In a fifth embodiment, an isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO: 15, 25, or 31.
[0017]In a sixth embodiment, an isolated polynucleotide comprising a nucleic acid sequence encoding an EXST or EXST-like polypeptide, wherein the nucleic acid sequence comprises SEQ ID NO: 14, 24, or 30.
[0018]In further embodiments, vectors and recombinant constructs comprising any of the foregoing polynucleotides and cells comprising the recombinant constructs.
[0019]In additional embodiments, methods for transforming a cell with any of the foregoing the polynucleotides and for producing and regenerating a transformed plant comprising any of the foregoing polynucleotides.
[0020]In another embodiment, a plant comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, and wherein said plant exhibits altered root architecture when compared to a control plant not comprising said recombinant DNA construct.
[0021]In another embodiment, a plant comprising in its genome a recombinant DNA construct comprising:
[0022](a) a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or
[0023](b) a suppression DNA construct comprising at least one regulatory element operably linked to: (i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
[0024]In another embodiment, a method of altering root architecture in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34; and (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the recombinant DNA construct; and optionally, (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the recombinant DNA construct.
[0025]In another embodiment, a method of evaluating root architecture in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) evaluating root architecture of the transgenic plant compared to a control plant not comprising the recombinant DNA construct; and optionally, (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and optionally, (e) evaluating root architecture of the progeny plant compared to a control plant not comprising the recombinant DNA construct.
[0026]In another embodiment, a method of evaluating root architecture in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34 (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; (c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (d) evaluating root architecture of the progeny plant compared to a control plant not comprising the recombinant DNA construct.
[0027]In another embodiment, a method of determining an alteration of an agronomic characteristic in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34 (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct; and optionally, (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and optionally, (e) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
[0028]In another embodiment, a method of determining an alteration of an agronomic characteristic in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34 (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; (c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (d) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
[0029]In another embodiment, a method of determining an alteration of an agronomic characteristic in a plant, comprising:
[0030](a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: [0031](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or [0032](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;
[0033](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and
[0034](c) determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct;
[0035]and optionally, (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and optionally, (e) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
[0036]In another embodiment, a method of determining an alteration of an agronomic characteristic in a plant, comprising:
[0037](a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: [0038](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or [0039](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;
[0040](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct and exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct;
[0041](c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and
[0042](d) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
[0043]In another embodiment, a method of altering root architecture in a plant, comprising:
[0044](a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: [0045](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or [0046](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; and
[0047](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct and wherein the transgenic plant exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct; and
[0048]optionally, (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and wherein the progeny plant exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct.
[0049]In another embodiment, a method of evaluating root architecture in a plant, comprising:
[0050](a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: [0051](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or [0052](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;
[0053](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and
[0054](c) evaluating root architecture of the transgenic plant compared to a control plant not comprising the suppression DNA construct;
[0055]and optionally, (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and optionally, (e) evaluating root architecture of the progeny plant compared to a control plant not comprising the suppression DNA construct.
[0056]In another embodiment, a method of evaluating root architecture in a plant, comprising:
[0057](a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: [0058](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or [0059](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide;
[0060](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct;
[0061](c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and
[0062](d) evaluating root architecture of the progeny plant compared to a control plant not comprising the suppression DNA construct.
In another aspect, this invention also concerns a method of mapping genetic variations related to controlling embryo/endosperm size during seed development and/or altering oil phenotypes in plants comprising:
[0063](a) crossing two plant varieties; and
[0064](b) evaluating genetic variations with respect to: [0065](i) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30 or 33; or [0066](ii) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 15; 17, 19, 21, 23, 25, 27, 29, 31 or 34 [0067]in progeny plants resulting from the cross of step (a) wherein the evaluation is made using a method selected from the group consisting of: RFLP analysis, SNP analysis, and PCR-based analysis.
[0068]In another embodiment, this invention concerns a method of molecular breeding to obtain altered embryo/endosperm size during seed development and/or altered oil phenotypes in plants comprising:
[0069](a) crossing two plant varieties; and
[0070](b) evaluating genetic variations with respect to: [0071](i) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30 or 33; or [0072](ii) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 15; 17, 19, 21, 23, 25, 27, 29, 31 or 34; [0073]in progeny plants resulting from the cross of step (a) wherein the evaluation is made using a method selected from the group consisting of: RFLP analysis, SNP analysis, and PCR-based analysis.
[0074]Also included in the present invention is any progeny of the above plants, any seeds of the above plants, and cells from any of the above plants and progeny.
[0075]A method of producing seed that can be sold as a product offering with altered root architecture comprising any of the preceding preferred methods, and further comprising obtaining seeds from said progeny plant, wherein said seeds comprise in their genome said recombinant DNA construct.
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTINGS
[0076]The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.
[0077]FIG. 1 shows a map of the pHSbarENDs2 activation tagging construct (SEQ ID NO:1) used to make the Arabidopsis populations.
[0078]FIG. 2 shows a map of the vector pDONR®/Zeo (SEQ ID NO:2). The attP1 site is at nucleotides 570-801; the attP2 site is at nucleotides 2754-2985 (complementary strand).
[0079]FIG. 3 shows a map of the vector pDONR®221 (SEQ ID NO:3). The attP1 site is at nucleotides 570-801; the attP2 site is at nucleotides 2754-2985 (complementary strand).
[0080]FIG. 4 shows a map of the vector pBC-yellow (SEQ ID NO:4), a destination vector for use in construction of expression vectors for Arabidopsis. The attR1 site is at nucleotides 11276-11399 (complementary strand); the attR2 site is at nucleotides 9695-9819 (complementary strand).
[0081]FIG. 5 shows a map of PHP27840 (SEQ ID NO:5), a destination vector for use in construction of expression vectors for soybean. The attR1 site is at nucleotides 7310-7434; the attR2 site is at nucleotides 8890-9014.
[0082]FIG. 6 shows a map of PHP23236 (SEQ ID NO:6), a destination vector for use in construction of expression vectors for Gaspe Flint derived maize lines. The attR1 site is at nucleotides 2006-2130; the attR2 site is at nucleotides 2899-3023.
[0083]FIG. 7 shows a map of PHP10523 (SEQ ID NO:7), a plasmid DNA present in Agrobacterium strain LBA4404.
[0084]FIG. 8 shows a map of PHP23235 (SEQ ID NO:8), a vector used to construct the destination vector PHP23236.
[0085]FIG. 9 shows a map of the entry clone PHP20234 (SEQ ID NO:9), a vector carrying the PINII terminator. The attR2 site is at nucleotides 591-747; the attL3 site is at nucleotides 1100-1195.
[0086]FIG. 10 shows a map of PHP28529 (SEQ ID NO:10), a destination vector for use in construction of expression vectors for maize lines. The attR3 site is at nucleotides 3613-3737; the attR4 site is at nucleotides 2035-2159.
[0087]FIG. 11 shows a map of the entry clone PHP28408 (SEQ ID NO:11), a vector carrying the constitutive maize GOS2 promoter. The attL4 site is at nucleotides 160-255; the attR1 site is at nucleotides 2301-2447.
[0088]FIG. 12 shows a map of the entry clone PHP22020 (SEQ ID NO:12), a vector carrying the root maize NAS2 promoter. The attR1 site is at nucleotides 31-187; the attL4 site is at nucleotides 2578-2673.
[0089]FIG. 13 shows a map of PHP29635 (SEQ ID NO:13), a destination vector for use in construction of expression vectors for Gaspe Flint derived maize lines. The attR1 site is at nucleotides 40786-40910; the attR2 site is at nucleotides 41679-41803.
[0090]FIG. 14 shows a map of PIIOXS2a-FRT87(ni)m (SEQ ID NO:43), a vector used to construct the destination vector PHP29635.
[0091]FIGS. 15A-15I show the multiple alignment of the full length amino acid sequences of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 34, and SEQ ID NOs:35, 36, 37, and 38. Residues that match the Consensus sequence exactly are shaded. The consensus sequence is shown above each alignment. The consensus residues are determined by a straight majority.
[0092]FIG. 16 shows a chart of the percent sequence identity and the divergence values for each pair of amino acid sequences of the EXST homologs displayed in FIGS. 15A-15I.
[0093]FIG. 17 is the growth medium used for semi-hydroponics maize growth in Example 17.
[0094]FIG. 18 is a chart setting forth data relating to the effect of different nitrate concentrations on the growth and development of Gaspe Flint derived maize lines in Example 17.
[0095]The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
[0096]The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
[0097]SEQ ID NO:1 pHSbarENDs2
[0098]SEQ ID NO:2 pDONR®/Zeo
[0099]SEQ ID NO:3 pDONR®221
[0100]SEQ ID NO:4 pBC-yellow
[0101]SEQ ID NO:5 PHP27840
[0102]SEQ ID NO:6 PHP23236
[0103]SEQ ID NO:7 PHP10523
[0104]SEQ ID NO:8 PHP23235
[0105]SEQ ID NO:9 PHP20234
[0106]SEQ ID NO:10 PHP28529
[0107]SEQ ID NO:11 PHP28408
[0108]SEQ ID NO:12 PHP22020
[0109]SEQ ID NO:13 PHP29635
[0110]Contig of: lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence listing.
TABLE-US-00001 TABLE 1 EXST and EXST-like proteins SEQ ID NO: SEQ ID NO: Protein Clone Designation (Amino Acid) (Nucleotide) EXST-like Contig of: 14 15 cfp5n.pk007.k11 cfp5n.pk007.k11.f cfp6n.pk005.i1 EXST-like Contig of: 16 17 cfp3n.pk069.115 cfp3n.pk069.l15.f p0127.cntdd86ra p0127.cntdd86ra.f EXST-like my.ceb1.pk0010.e5 18 19 EXST-like cfp6n.pk002.a5:fis 20 21 EXST-like rls24.pk0026.h11:fis 22 23 EXST-like p0127.cntdd86ra:fis 24 25 EXST-like cfp5n.pk007.k11:fis 26 27 EXST-like esl1c.pk006.l19:fis 28 29 EXST-like cfp1n.pk002.o16.f:fis 30 31
[0111]SEQ ID NO:32 is the nucleotide sequence of the Arabidopsis thaliana embryo sac development arrest 5 (EDA5, Exostosin Family protein or EXST protein, NCBI GI NO: 15228598, At3g03650).
[0112]SEQ ID NO:33 is the ORF corresponding to nucleotides 245-1744 of SEQ ID NO:32.
[0113]SEQ ID NO:34 corresponds to the protein sequence (NCBI GI NO: 15228598) encoded by SEQ ID NO:33
[0114]SEQ ID NO:35 corresponds to NCBI GI NO:115476598 (Oryza sativa).
[0115]SEQ ID NO:36 corresponds to NCBI GI NO:115487106 (Oryza sativa).
[0116]SEQ ID NO:37 corresponds to NCBI GI NO:115452759 (Oryza sativa).
[0117]SEQ ID NO:38 corresponds to NCBI GI NO:115441893 (Oryza sativa).
[0118]SEQ ID NO:39 is the attB1 sequence.
[0119]SEQ ID NO:40 is the attB2 sequence.
[0120]SEQ ID NO:41 is the forward primer VC062 in Example 9.
[0121]SEQ ID NO:42 is the reverse primer VC063 in Example 9.
[0122]SEQ ID NO:43 PIIOXS2a-FRT87(ni)m.
[0123]SEQ ID NO:44 is the maize NAS2 promoter.
[0124]SEQ ID NO:45 is the GOS2 promoter.
[0125]SEQ ID NO:46 is the ubiquitin promoter.
[0126]SEQ ID NO:47 is the S2A promoter.
[0127]SEQ ID NO:48 is the PINII terminator.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0128]The disclosure of each reference set forth herein is hereby incorporated by reference in its entirety.
[0129]As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes a plurality of such plants, reference to "a cell" includes one or more cells and equivalents thereof known to those skilled in the art, and so forth.
[0130]The term "root architecture" refers to the arrangement of the different parts that comprise the root. The terms "root architecture", "root structure", "root system" or "root system architecture" are used interchangeably herewithin.
[0131]In general, the first root of a plant that develops from the embryo is called the primary root. In most dicots, the primary root is called the taproot. This main root grows downward and gives rise to branch (lateral) roots. In monocots the primary root of the plant branches, giving rise to a fibrous root system.
[0132]The term "altered root architecture" refers to aspects of alterations of the different parts that make up the root system at different stages of its development compared to a reference or control plant. It is understood that altered root architecture encompasses alterations in one or more measurable parameters, including but not limited to, the diameter, length, number, angle or surface of one or more of the root system parts, including but not limited to, the primary root, lateral or branch root, adventitious root, and root hairs, all of which fall within the scope of this invention. These changes can lead to an overall alteration in the area or volume occupied by the root. The reference or control plant does not comprise in its genome the recombinant DNA construct or heterologous construct.
[0133]An "Expressed Sequence Tag" ("EST") is a DNA sequence derived from a cDNA library and therefore is a sequence which has been transcribed. An EST is typically obtained by a single sequencing pass of a cDNA insert. The sequence of an entire cDNA insert is termed the "Full-insert Sequence" ("FIS"). A "Contig" sequence is a sequence assembled from two or more sequences that can be selected from, but not limited to, the group consisting of an EST, FIS and PCR sequence. A sequence encoding an entire or functional protein is termed a "Complete Gene Sequence" ("CGS") and can be derived from an FIS or a contig.
[0134]"Agronomic characteristics" is a measurable parameter including but not limited to greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, stalk lodging, plant height, ear length, and harvest index.
[0135]"Exostosin Family", "at-Exostosin Family, exst, at-exst are used interchangeably herewithin and refer to the Arabidopsis thaliana locus, AT3G03650 (SEQ ID NO:32).
[0136]EXST refers to the protein (SEQ ID NO:34) encoded by the ORF (SEQ ID NO:33 of AT3G03650 (SEQ ID NO:32).
[0137]"exst-like" refers to nucleotide homologs from different species, such as corn and soybean, of the Arabidopsis thaliana "exostosin family" locus, AT3G03650 (SEQ ID NO:32) and includes without limitation any of the nucleotide sequences of SEQ ID NOs:14, 16, 18, 20, 22, 24, 26, 28, and 30.
[0138]"EXST-like" refers to protein homologs from different species, such as corn and soybean, of the Arabidopsis thaliana "EXOSTOSIN FAMILY" polypeptide (SEQ ID NO:34) and includes without limitation any of the amino acid sequences of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, and 31.
[0139]"Environmental conditions" refer to conditions under which the plant is grown, such as the availability of water, availability of nutrients (for example nitrogen), or the presence of disease.
[0140]"Transgenic" refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation
[0141]"Genome" as it applies to plant cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondrial, plastid) of the cell.
[0142]"Plant" includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.
[0143]"Progeny" comprises any subsequent generation of a plant.
[0144]"Transgenic" refers to any cell, cell line, callus, tissue, plant part or plant, the genome of which has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct, including those initial transgenic events as well as those created by sexual crosses or asexual propagation from the initial transgenic event. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
[0145]"Transgenic plant" includes reference to a plant which comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.
[0146]"Heterologous" with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
[0147]"Polynucleotide", "nucleic acid sequence", "nucleotide sequence", or "nucleic acid fragment" are used interchangeably and is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
[0148]"Polypeptide", "peptide", "amino acid sequence" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
[0149]"Messenger RNA (mRNA)" refers to the RNA that is without introns and that can be translated into protein by the cell.
[0150]"cDNA" refers to a DNA that is complementary to and synthesized from a mRNA template using the enzyme reverse transcriptase. The cDNA can be single-stranded or converted into the double-stranded form using the Klenow fragment of DNA polymerase I.
[0151]"Mature" protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product have been removed.
[0152]"Precursor" protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be and are not limited to intracellular localization signals.
[0153]"Isolated" refers to materials, such as nucleic acid molecules and/or proteins, which are substantially free or otherwise removed from components that normally accompany or interact with the materials in a naturally occurring environment. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
[0154]"Recombinant" refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. "Recombinant" also includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or a cell derived from a cell so modified, but does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
[0155]"Recombinant DNA construct" refers to a combination of nucleic acid fragments that are not normally found together in nature. Accordingly, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that normally found in nature.
[0156]The terms "entry clone" and "entry vector" are used interchangeably herein.
[0157]"Regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
[0158]"Promoter" refers to a nucleic acid fragment capable of controlling transcription of another nucleic acid fragment.
[0159]"Promoter functional in a plant" is a promoter capable of controlling transcription in plant cells whether or not its origin is from a plant cell.
[0160]"Tissue-specific promoter" and "tissue-preferred promoter" are used interchangeably, and refer to a promoter that is expressed predominantly but not necessarily exclusively in one tissue or organ, but that may also be expressed in one specific cell.
[0161]"Developmentally regulated promoter" refers to a promoter whose activity is determined by developmental events.
[0162]"Operably linked" refers to the association of nucleic acid fragments in a single fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a nucleic acid fragment when it is capable of regulating the transcription of that nucleic acid fragment.
[0163]"Expression" refers to the production of a functional product. For example, expression of a nucleic acid fragment may refer to transcription of the nucleic acid fragment (e.g., transcription resulting in mRNA or functional RNA) and/or translation of mRNA into a precursor or mature protein.
[0164]"Phenotype" means the detectable characteristics of a cell or organism.
[0165]"Introduced" in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
[0166]A "transformed cell" is any cell into which a nucleic acid fragment (e.g., a recombinant DNA construct) has been introduced.
[0167]"Transformation" as used herein refers to both stable transformation and transient transformation.
[0168]"Stable transformation" refers to the introduction of a nucleic acid fragment into a genome of a host organism resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated in the genome of the host organism and any subsequent generation.
[0169]"Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus, or DNA-containing organelle, of a host organism resulting in gene expression without genetically stable inheritance.
[0170]"Allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant are the same that plant is homozygous at that locus. If the alleles present at a given locus on a pair of homologous chromosomes in a diploid plant differ that plant is heterozygous at that locus. If a transgene is present on one of a pair of homologous chromosomes in a diploid plant that plant is hemizygous at that locus.
[0171]Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain "percent identity" and "divergence" values by viewing the "sequence distances" table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.
[0172]Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook").
[0173]Turning now to preferred embodiments:
[0174]Preferred embodiments include isolated polynucleotides and polypeptides, recombinant DNA constructs, compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs.
[0175]Preferred Isolated Polynucleotides and Polypeptides
[0176]The present invention includes the following preferred isolated polynucleotides and polypeptides:
[0177]An isolated polynucleotide comprising: (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34; or (ii) a full complement of the nucleic acid sequence of (i). Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs (including suppression DNA constructs) of the present invention. The polypeptide is preferably a EXST or EXST-like protein.
[0178]An isolated polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34. The polypeptide is preferably a EXST or EXST-like protein.
[0179]An isolated polynucleotide comprising (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (i). Any of the foregoing isolated polynucleotides may be utilized in any recombinant DNA constructs (including suppression DNA constructs) of the present invention. The isolated polynucleotide encodes a EXST or EXST-like protein.
[0180]Preferred Recombinant DNA Constructs and Suppression DNA Constructs.
[0181]In one aspect, the present invention includes recombinant DNA constructs (including suppression DNA constructs).
[0182]In one preferred embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein the polynucleotide comprises (i) a nucleic acid sequence encoding an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (i).
[0183]In another preferred embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide comprises (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (i).
[0184]FIGS. 15A-15I show the multiple alignment of the amino acid sequences of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 34, and SEQ ID NOs:35, 36, 37, and 38. The multiple alignment of the sequences was performed using the Megalign® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.); in particular, using the Clustal V method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the multiple alignment default parameters of GAP PENALTY=10 and GAP LENGTH PENALTY=10, and the pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
[0185]FIG. 16 shows the percent sequence identity and the divergence values for each pair of amino acids sequences displayed in FIGS. 15A-15I.
[0186]In another preferred embodiment, a recombinant DNA construct comprises a polynucleotide operably linked to at least one regulatory sequence (e.g., a promoter functional in a plant), wherein said polynucleotide encodes a EXST or EXST-like protein.
[0187]In another aspect, the present invention includes suppression DNA constructs.
[0188]A suppression DNA construct preferably comprises at least one regulatory sequence (preferably a promoter functional in a plant) operably linked to (a) all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (a)(i); or (b) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like protein; or (c) all or part of (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (c)(i). The suppression DNA construct preferably comprises a cosuppression construct, antisense construct, viral-suppression construct, hairpin suppression construct, stem-loop suppression construct, double-stranded RNA-producing construct, RNAi construct, or small RNA construct (e.g., an siRNA construct or an miRNA construct).
[0189]It is understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences. Alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
[0190]"Suppression DNA construct" is a recombinant DNA construct which when transformed or stably integrated into the genome of the plant, results in "silencing" of a target gene in the plant. The target gene may be endogenous or transgenic to the plant. "Silencing," as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein/enzyme expressed by the target gene, and/or the level of the enzyme activity or protein functionality. The term "suppression" includes lower, reduce, decline, decrease, inhibit, eliminate or prevent. "Silencing" or "gene silencing" does not specify mechanism and is inclusive, and not limited to, anti-sense, cosuppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, and small RNA-based approaches.
[0191]A suppression DNA construct may comprise a region derived from a target gene of interest and may comprise all or part of the nucleic acid sequence of the sense strand (or antisense strand) of the target gene of interest. Depending upon the approach to be utilized, the region may be 100% identical or less than 100% identical (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to all or part of the sense strand (or antisense strand) of the gene of interest.
[0192]Suppression DNA constructs are well-known in the art, are readily constructed once the target gene of interest is selected, and include, without limitation, cosuppression constructs, antisense constructs, viral-suppression constructs, hairpin suppression constructs, stem-loop suppression constructs, double-stranded RNA-producing constructs, and more generally, RNAi (RNA interference) constructs and small RNA constructs such as siRNA (short interfering RNA) constructs and miRNA (microRNA) constructs.
[0193]"Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
"Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
[0194]"Cosuppression" refers to the production of sense RNA transcripts capable of suppressing the expression of the target protein. "Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro. Cosuppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see Vaucheret et al. (1998) Plant J. 16:651-659; and Gura (2000) Nature 404:804-808).
[0195]Another variation describes the use of plant viral sequences to direct the suppression of proximal mRNA encoding sequences (PCT Publication WO 98/36083 published on Aug. 20, 1998).
[0196]Previously described is the use of "hairpin" structures that incorporate all, or part, of an mRNA encoding sequence in a complementary orientation that results in a potential "stem-loop" structure for the expressed RNA (PCT Publication WO 99/53050 published on Oct. 21, 1999). In this case the stem is formed by polynucleotides corresponding to the gene of interest inserted in either sense or anti-sense orientation with respect to the promoter and the loop is formed by some polynucleotides of the gene of interest, which do not have a complement in the construct. This increases the frequency of cosuppression or silencing in the recovered transgenic plants. For review of hairpin suppression see Wesley, S. V. et al. (2003) Methods in Molecular Biology, Plant Functional Genomics: Methods and Protocols 236:273-286.
[0197]A construct where the stem is formed by at least 30 nucleotides from a gene to be suppressed and the loop is formed by a random nucleotide sequence has also effectively been used for suppression (PCT Publication No. WO 99/61632 published on Dec. 2, 1999).
[0198]The use of poly-T and poly-A sequences to generate the stem in the stem-loop structure has also been described (PCT Publication No. WO 02/00894 published Jan. 3, 2002).
[0199]Yet another variation includes using synthetic repeats to promote formation of a stem in the stem-loop structure. Transgenic organisms prepared with such recombinant DNA fragments have been shown to have reduced levels of the protein encoded by the nucleotide fragment forming the loop as described in PCT Publication No. WO 02/00904, published 3 Jan. 2002.
[0200]RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et al., Nature 391:806 1998). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla (Fire et al., Trends Genet. 15:358 1999). Such protection from foreign gene expression may have evolved in response to the production of double-stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single-stranded RNA of viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response through a mechanism that has yet to be fully characterized.
[0201]The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al., Nature 409:363, 2001). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes (Elbashir et al., Genes Dev. 15:188, 2001). Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., Science 293:834, 2001). The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA having sequence complementarity to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., Genes Dev. 15:188, 2001). In addition, RNA interference can also involve small RNA (e.g., miRNA) mediated gene silencing, presumably through cellular mechanisms that regulate chromatin structure and thereby prevent transcription of target gene sequences (see, e.g., Allshire, Science 297:1818-1819, 2002; Volpe et al., Science 297:1833-1837, 2002; Jenuwein, Science 297:2215-2218, 2002; and Hall et al., Science 297:2232-2237, 2002). As such, miRNA molecules of the invention can be used to mediate gene silencing via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in gene silencing either at the transcriptional or post-transcriptional level.
[0202]RNAi has been studied in a variety of systems. Fire et al. (Nature 391:806, 1998) were the first to observe RNAi in C. elegans. Wianny and Goetz (Nature Cell Biol. 2:70, 1999) describe RNAi mediated by dsRNA in mouse embryos. Hammond et al. (Nature 404:293, 2000) describe RNAi in Drosophila cells transfected with dsRNA. Elbashir et al., (Nature 411:494, 2001) describe RNAi induced by introduction of duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including human embryonic kidney and HeLa cells.
[0203]Small RNAs play an important role in controlling gene expression. Regulation of many developmental processes, including flowering, is controlled by small RNAs. It is now possible to engineer changes in gene expression of plant genes by using transgenic constructs which produce small RNAs in the plant.
[0204]Small RNAs appear to function by base-pairing to complementary RNA or DNA target sequences. When bound to RNA, small RNAs trigger either RNA cleavage or translational inhibition of the target sequence. When bound to DNA target sequences, it is thought that small RNAs can mediate DNA methylation of the target sequence. The consequence of these events, regardless of the specific mechanism, is that gene expression is inhibited.
[0205]It is thought that sequence complementarity between small RNAs and their RNA targets helps to determine which mechanism, RNA cleavage or translational inhibition, is employed. It is believed that siRNAs, which are perfectly complementary with their targets, work by RNA cleavage. Some miRNAs have perfect or near-perfect complementarity with their targets, and RNA cleavage has been demonstrated for at least a few of these miRNAs. Other miRNAs have several mismatches with their targets, and apparently inhibit their targets at the translational level. Again, without being held to a particular theory on the mechanism of action, a general rule is emerging that perfect or near-perfect complementarity causes RNA cleavage, whereas translational inhibition is favored when the miRNA/target duplex contains many mismatches. The apparent exception to this is microRNA 172 (miR172) in plants. One of the targets of miR172 is APETALA2 (AP2), and although miR172 shares near-perfect complementarity with AP2 it appears to cause translational inhibition of AP2 rather than RNA cleavage.
[0206]MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24 nucleotides (nt) in length that have been identified in both animals and plants (Lagos-Quintana et al., Science 294:853-858 2001, Lagos-Quintana et al., Curr. Biol. 12:735-739, 2002; Lau et al., Science 294:858-862, 2001; Lee and Ambros, Science 294:862-864, 2001; Llave et al., Plant Cell 14:1605-1619, 2002; Mourelatos et al., Genes. Dev. 16:720-728, 2002; Park et al., Curr. Biol. 12:1484-1495, 2002; Reinhart et al., Genes. Dev. 16:1616-1626, 2002). They are processed from longer precursor transcripts that range in size from approximately 70 to 200 nt, and these precursor transcripts have the ability to form stable hairpin structures. In animals, the enzyme involved in processing miRNA precursors is called Dicer, an RNAse III-like protein (Grishok et al., Cell 106:23-34, 2001; Hutvagner et al., Science 293:834-838, 2001; Ketting et al., Genes. Dev. 15:2654-2659, 2001). Plants also have a Dicer-like enzyme, DCL1 (previously named CARPEL FACTORY/SHORT INTEGUMENTS1/SUSPENSOR1), and recent evidence indicates that it, like Dicer, is involved in processing the hairpin precursors to generate mature miRNAs (Park et al., Curr. Biol. 12:1484-1495, 2002; Reinhart et al., Genes. Dev. 16:1616-1626, 2002). Furthermore, it is becoming clear from recent work that at least some miRNA hairpin precursors originate as longer polyadenylated transcripts, and several different miRNAs and associated hairpins can be present in a single transcript (Lagos-Quintana et al., Science 294:853-858, 2001; Lee et al., EMBO J. 21:4663-4670, 2002). Recent work has also examined the selection of the miRNA strand from the dsRNA product arising from processing of the hairpin by DICER (Schwartz, et al., Cell 115:199-208, 2003). It appears that the stability (i.e. G:C vs. A:U content, and/or mismatches) of the two ends of the processed dsRNA affects the strand selection, with the low stability end being easier to unwind by a helicase activity. The 5' end strand at the low stability end is incorporated into the RISC complex, while the other strand is degraded.
[0207]MicroRNAs appear to regulate target genes by binding to complementary sequences located in the transcripts produced by these genes. In the case of lin-4 and let-7, the target sites are located in the 3' UTRs of the target mRNAs (Lee et al., Cell 75:843-854, 1993; Wightman et al., Cell 75:855-862, 1993; Reinhart et al., Nature 403:901-906, 2000; Slack et al., Mol. Cell. 5:659-669, 2000), and there are several mismatches between the lin-4 and let-7 miRNAs and their target sites. Binding of the lin-4 or let-7 miRNA appears to cause downregulation of steady-state levels of the protein encoded by the target mRNA without affecting the transcript itself (Olsen and Ambros, Dev. Biol. 216:671-680, 1999). On the other hand, recent evidence suggests that miRNAs can in some cases cause specific RNA cleavage of the target transcript within the target site, and this cleavage step appears to require 100% complementarity between the miRNA and the target transcript (Hutvagner and Zamore, Science 297:2056-2060, 2002; Llave et al., Plant Cell 14:1605-1619, 2002). It seems likely that miRNAs can enter at least two pathways of target gene regulation: Protein downregulation when target complementarity is <100%, and RNA cleavage when target complementarity is 100%. MicroRNAs entering the RNA cleavage pathway are analogous to the 21-25 nt short interfering RNAs (siRNAs) generated during RNA interference (RNAi) in animals and posttranscriptional gene silencing (PTGS) in plants (Hamilton and Baulcombe 1999; Hammond et al., 2000; Zamore et al., 2000; Elbashir et al., 2001), and likely are incorporated into an RNA-induced silencing complex (RISC) that is similar or identical to that seen for RNAi.
[0208]Identifying the targets of miRNAs with bioinformatics has not been successful in animals, and this is probably due to the fact that animal miRNAs have a low degree of complementarity with their targets. On the other hand, bioinformatic approaches have been successfully used to predict targets for plant miRNAs (Llave et al., Plant Cell 14:1605-1619 2002; Park et al., Curr. Biol. 12:1484-1495 2002; Rhoades et al., Cell 110:513-520 2002), and thus it appears that plant miRNAs have higher overall complementarity with their putative targets than do animal miRNAs. Most of these predicted target transcripts of plant miRNAs encode members of transcription factor families implicated in plant developmental patterning or cell differentiation.
[0209]A recombinant DNA construct (including a suppression DNA construct) of the present invention preferably comprises at least one regulatory sequence.
[0210]A preferred regulatory sequence is a promoter.
[0211]A number of promoters can be used in recombinant DNA constructs (and suppression DNA constructs) of the present invention. The promoters can be selected based on the desired outcome, and may include constitutive, tissue-specific, cell specific, inducible, or other promoters for expression in the host organism.
[0212]High level, constitutive expression of the candidate gene under control of the 35S or UBI promoter may have pleiotropic effects, although Candidate gene efficacy may be estimated when driven by a constitutive promoter.
[0213]Use of tissue-specific and/or stress-specific expression may eliminate undesirable effects but retain the ability to alter root architecture. This effect has been observed in Arabidopsis (Kasuga et al. (1999) Nature Biotechnol. 17:287-291).
[0214]Suitable constitutive promoters for use in a plant host cell include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (UBI) (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), the maize GOS2 promoter (WO0020571 A2, published Apr. 1, 2000) and the like. Other constitutive promoters include, for example, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
[0215]In choosing a promoter to use in the methods of the invention, it may be desirable to use a tissue-specific or developmentally regulated promoter.
[0216]A preferred tissue-specific or developmentally regulated promoter is a DNA sequence which regulates the expression of a DNA sequence selectively in the cells/tissues of a plant critical to tassel development, seed set, or both, and limits the expression of such a DNA sequence to the period of tassel development or seed maturation in the plant. Any identifiable promoter may be used in the methods of the present invention which causes the desired temporal and spatial expression.
[0217]Promoters which are seed or embryo specific and may be useful in the invention include soybean Kunitz trysin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)), patatin (potato tubers) (Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29), convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al. (1991) Mol. Gen. Genet. 259:149-157; Newbigin, E. J., et al. (1990) Planta 180:461-470; Higgins, T. J. V., et al. (1988) Plant. Mol. Biol. 11:683-695), zein (maize endosperm) (Schemthaner, J. P., et al. (1988) EMBO J. 7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324), phytohemagglutinin (bean cotyledon) (Voelker, T. et al. (1987) EMBO J. 6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al. (1988) EMBO J. 7:297-302), glutelin (rice endosperm), hordein (barley endosperm) (Marris, C., et al. (1988) Plant Mol. Biol. 10:359-366), glutenin and gliadin (wheat endosperm) (Colot, V., et al. (1987) EMBO J. 6:3559-3564), and sporamin (sweet potato tuberous root) (Hattori, T., et al. (1990) Plant Mol. Biol. 14:595-604). Promoters of seed-specific genes operably linked to heterologous coding regions in chimeric gene constructions maintain their temporal and spatial expression pattern in transgenic plants. Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and Brassica napus seeds (Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)), bean lectin and bean beta-phaseolin promoters to express luciferase (Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to express chloramphenicol acetyl transferase (Colot et al., EMBO J. 6:3559-3564 (1987)).
[0218]Inducible promoters selectively express an operably linked DNA sequence in response to the presence of an endogenous or exogenous stimulus, for example by chemical compounds (chemical inducers) or in response to environmental, hormonal, chemical, and/or developmental signals. Inducible or regulated promoters include, for example, promoters regulated by light, heat, stress, flooding or drought, phytohormones, wounding, or chemicals such as ethanol, jasmonate, salicylic acid, or safeners.
[0219]Preferred promoters include the following: 1) the stress-inducible RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91); 2) the barley promoter, B22E; expression of B22E is specific to the pedicel in developing maize kernels ("Primary Structure of a Novel Barley Gene Differentially Expressed in Immature Aleurone Layers". Klemsdal, S. S. et al., Mol. Gen. Genet. 228(1/2):9-16 (1991)); and 3) maize promoter, Zag2 ("Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS", Schmidt, R. J. et al., Plant Cell 5(7):729-737 (1993)). "Structural characterization, chromosomal localization and phylogenetic evaluation of two pairs of AGAMOUS-like MADS-box genes from maize", Theissen et al., Gene 156(2): 155-166 (1995); NCBI GenBank Accession No. X80206)). Zag2 transcripts can be detected 5 days prior to pollination to 7 to 8 days after pollination (DAP), and directs expression in the carpel of developing female inflorescences and Ciml which is specific to the nucleus of developing maize kernels. Ciml transcript is detected 4 to 5 days before pollination to 6 to 8 DAP. Other useful promoters include any promoter which can be derived from a gene whose expression is maternally associated with developing female florets.
[0220]Additional preferred promoters for regulating the expression of the nucleotide sequences of the present invention in plants are vascular element specific or stalk-preferred promoters. Such stalk-preferred promoters include the alfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al., Plant Mol. Biol. 27:513-528 (1995)) and S2B promoter (GenBank Accession No. EF030817) and the like, herein incorporated by reference.
[0221]Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro, J. K., and Goldberg, R. B., Biochemistry of Plants 15:1-82 (1989). (Put this with the other constitutive promoter description.)
[0222]Preferred promoters may include: RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin (SEQ ID NO:46), CaMV 19S, nos, Adh, sucrose synthase, R-allele, root cell promoter, the vascular tissue specific promoters S2A (Genbank accession number EF030816; SEQ ID NO:47) and S2B (Genbank accession number EF030817) and the constitutive promoter GOS2 (SEQ ID NO:45) from Zea mays. Other preferred promoters include root preferred promoters, such as the maize NAS2 promoter (SEQ ID NO:44), the maize Cyclo promoter (US 2006/0156439, published Jul. 13, 2006), the maize ROOTMET2 promoter (WO05063998, published Jul. 14, 2005), the CR1BIO promoter (WO06055487, published May 26, 2006), the CRWAQ81 (WO05035770, published Apr. 21, 2005) and the maize ZRP2.47 promoter (NCBI accession number: U38790, gi: 1063664).
[0223]A "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that is sufficient to afford putative identification of the promoter that the nucleotide sequence comprises. Nucleotide sequences can be evaluated either manually, by one skilled in the art, or using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, a sequence of thirty or more contiguous nucleotides is necessary in order to putatively identify a promoter nucleic acid sequence as homologous to a known promoter. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
[0224]Recombinant DNA constructs (and suppression DNA constructs) of the present invention may also include other regulatory sequences, including but not limited to, translation leader sequences, introns, and polyadenylation recognition sequences. In another preferred embodiment of the present invention, a recombinant DNA construct of the present invention further comprises an enhancer or silencer.
[0225]An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. Cell. Biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).
[0226]If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
[0227]A translation leader sequence is a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D. Molecular Biotechnology 3:225 (1995)).
[0228]In another preferred embodiment of the present invention, a recombinant DNA construct of the present invention further comprises an enhancer or silencer.
[0229]Any plant can be selected for the identification of regulatory sequences and genes to be used in creating recombinant DNA constructs and suppression DNA constructs of the present invention. Examples of suitable plant targets for the isolation of genes and regulatory sequences would include but are not limited to alfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrot, cassaya, castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus, clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine, watermelon, wheat, yams, and zucchini. Particularly preferred plants for the identification of regulatory sequences are Arabidopsis, corn, wheat, soybean, and cotton.
[0230]Preferred Compositions
[0231]A preferred composition of the present invention is a plant comprising in its genome any of the recombinant DNA constructs (including any of the suppression DNA constructs) of the present invention (such as those preferred constructs discussed above). Preferred compositions also include any progeny of the plant, and any seed obtained from the plant or its progeny, wherein the progeny or seed comprises within its genome the recombinant DNA construct (or suppression DNA construct). Progeny includes subsequent generations obtained by self-pollination or out-crossing of a plant. Progeny also includes hybrids and inbreds.
[0232]Preferably, in hybrid seed propagated crops, mature transgenic plants can be self-pollinated to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced recombinant DNA construct (or suppression DNA construct). These seeds can be grown to produce plants that would exhibit altered root (or plant) architecture, or used in a breeding program to produce hybrid seed, which can be grown to produce plants that would exhibit altered root (or plant) architecture. Preferably, the seeds are maize.
[0233]Preferably, the plant is a monocotyledonous or dicotyledonous plant, more preferably, a maize or soybean plant, even more preferably a maize plant, such as a maize hybrid plant or a maize inbred plant. The plant may also be sunflower, sorghum, castor bean, grape, canola, wheat, alfalfa, cotton, rice, barley or millet.
[0234]Preferably, the recombinant DNA construct is stably integrated into the genome of the plant.
[0235]Particularly preferred embodiments include but are not limited to the following preferred embodiments:
[0236]1. A plant (preferably a maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, and wherein said plant exhibits an altered root architecture when compared to a control plant not comprising said recombinant DNA construct. Preferably, the plant further exhibits an alteration of at least one agronomic characteristic when compared to the control plant.
[0237]2. A plant (preferably a maize or soybean plant) comprising in its genome:
[0238]a recombinant DNA construct comprising:
[0239](a) a polynucleotide operably linked to at least one regulatory element, wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or
[0240](b) a suppression DNA construct comprising at least one regulatory element operably linked to: [0241](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (b)(i)(A); or [0242](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
[0243]3. A plant (preferably a maize or soybean plant) comprising in its genome a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein said polynucleotide encodes a EXST or EXST-like protein, and wherein said plant exhibits an altered root architecture when compared to a control plant not comprising said recombinant DNA construct. Preferably, the plant further exhibits an alteration of at least one agronomic characteristic.
[0244]Preferably, the EXST protein is from Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja or Glycine tomentella.
[0245]4. A plant (preferably a maize or soybean plant) comprising in its genome a suppression DNA construct comprising at least one regulatory element operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like protein, and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
[0246]5. A plant (preferably a maize or soybean plant) comprising in its genome a suppression DNA construct comprising at least one regulatory element operably linked to all or part of (a) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (b) a full complement of the nucleic acid sequence of (a), and wherein said plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising said recombinant DNA construct.
[0247]6. Any progeny of the above plants in preferred embodiments 1-5, any seeds of the above plants in preferred embodiments 1-5, any seeds of progeny of the above plants in preferred embodiments 1-5, and cells from any of the above plants in preferred embodiments 1-5 and progeny thereof.
[0248]In any of the foregoing preferred embodiments 1-6 or any other embodiments of the present invention, the recombinant DNA construct (or suppression DNA construct) preferably comprises at least a promoter that is functional in a plant as a preferred regulatory sequence.
[0249]In any of the foregoing preferred embodiments 1-6 or any other embodiments of the present invention, the alteration of at least one agronomic characteristic is either an increase or decrease, preferably an increase.
[0250]In any of the foregoing preferred embodiments 1-6 or any other embodiments of the present invention, the at least one agronomic characteristic is preferably selected from the group consisting of greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, stalk lodging, plant height, ear length and harvest index. Yield, greenness, biomass and root lodging are particularly preferred agronomic characteristics for alteration (preferably an increase).
[0251]In any of the foregoing preferred embodiments 1-6 or any other embodiments of the present invention, the plant preferably exhibits the alteration of at least one agronomic characteristic irrespective of the environmental conditions, for example, water and nutrient availability, when compared to a control plant.
[0252]One of ordinary skill in the art is familiar with protocols for determining alteration in plant root architecture. For example, transgenic maize plants can be assayed for changes in root architecture at seedling stage, flowering time or maturity. Alterations in root architecture can be determined by counting the nodal root numbers of the top 3 or 4 nodes of the greenhouse grown plants or the width of the root band. "Root band" refers to the width of the mat of roots at the bottom of a pot at plant maturity. Other measures of alterations in root architecture include, but are not limited to, the number of lateral roots, average root diameter of nodal roots, average root diameter of lateral roots, number and length of root hairs. The extent of lateral root branching (e.g. lateral root number, lateral root length) can be determined by sub-sampling a complete root system, imaging with a flat-bed scanner or a digital camera and analyzing with WinRHIZO® software (Regent Instruments Inc.).
Data taken on root phenotype are subjected to statistical analysis, normally a t-test to compare the transgenic roots with that of non-transgenic sibling plants. One-way ANOVA may also be used in cases where multiple events and/or constructs are involved in the analysis.
[0253]The Examples below describe some representative protocols and techniques for detecting alterations in root architecture.
[0254]One can also evaluate alterations in root architecture by the ability of the plant to increase yield in field testing when compared, under the same conditions, to a control or reference plant.
[0255]One can also evaluate alterations in root architecture by the ability of the plant to maintain substantial yield (preferably at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% yield) in field testing under stress conditions (e.g., nutrient over-abundance or limitation, water over-abundance or limitation, presence of disease), when compared to the yield of a control or reference plant under non-stressed conditions.
[0256]Alterations in root architecture can also be measured by determining the resistance to root lodging of the transgenic plants compared to reference or control plants.
[0257]One of ordinary skill in the art would readily recognize a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant in any embodiment of the present invention in which a control or reference plant is utilized (e.g., compositions or methods as described herein). For example, by way of non-limiting illustrations:
[0258]1. Progeny of a transformed plant which is hemizygous with respect to a recombinant DNA construct (or suppression DNA construct), such that the progeny are segregating into plants either comprising or not comprising the recombinant DNA construct (or suppression DNA construct): the progeny comprising the recombinant DNA construct (or suppression DNA construct) would be typically measured relative to the progeny not comprising the recombinant DNA construct (or suppression DNA construct) (i.e., the progeny not comprising the recombinant DNA construct (or suppression DNA construct) is the control or reference plant).
[0259]2. Introgression of a recombinant DNA construct (or suppression DNA construct) into an inbred line, such as in maize, or into a variety, such as in soybean: the introgressed line would typically be measured relative to the parent inbred or variety line (i.e., the parent inbred or variety line is the control or reference plant).
[0260]3. Two hybrid lines, where the first hybrid line is produced from two parent inbred lines, and the second hybrid line is produced from the same two parent inbred lines except that one of the parent inbred lines contains a recombinant DNA construct (or suppression DNA construct): the second hybrid line would typically be measured relative to the first hybrid line (i.e., the parent inbred or variety line is the control or reference plant).
[0261]4. A plant comprising a recombinant DNA construct (or suppression DNA construct): the plant may be assessed or measured relative to a control plant not comprising the recombinant DNA construct (or suppression DNA construct) but otherwise having a comparable genetic background to the plant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of nuclear genetic material compared to the plant comprising the recombinant DNA construct (or suppression DNA construct). There are many laboratory-based techniques available for the analysis, comparison and characterization of plant genetic backgrounds; among these are Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLP®s), and Simple Sequence Repeats (SSRs) which are also referred to as Microsatellites.
[0262]Furthermore, one of ordinary skill in the art would readily recognize that a suitable control or reference plant to be utilized when assessing or measuring an agronomic characteristic or phenotype of a transgenic plant would not include a plant that had been previously selected, via mutagenesis or transformation, for the desired agronomic characteristic or phenotype.
Preferred Methods
[0263]Preferred methods include but are not limited to methods for altering root architecture in a plant, methods for evaluating alteration of root architecture in a plant, methods for altering an agronomic characteristic in a plant, methods for determining an alteration of an agronomic characteristic in a plant, and methods for producing seed. Preferably, the plant is a monocotyledonous or dicotyledonous plant, more preferably, a maize or soybean plant, even more preferably a maize plant. The plant may also be sunflower, sorghum, castor bean, canola, wheat, alfalfa, cotton, rice, barley or millet. The seed is preferably a maize or soybean seed, more preferably a maize seed, and even more preferably, a maize hybrid seed or maize inbred seed.
[0264]Particularly preferred methods include but are not limited to the following:
[0265]A method of altering root architecture of a plant, comprising: (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (preferably a promoter functional in a plant), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34; and (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the recombinant DNA construct. The method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the recombinant DNA construct.
[0266]A method of altering root architecture in a plant, comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory sequence (preferably a promoter functional in a plant) operably linked to:
[0267](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or
[0268](ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; and
[0269](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct and exhibits an altered root architecture when compared to a control plant not comprising the suppression DNA construct. The method may further comprise (c) obtaining a progeny plant derived from the transgenic plant, wherein said progeny plant comprises in its genome the recombinant DNA construct and exhibits altered root architecture when compared to a control plant not comprising the suppression DNA construct.
[0270]A method of evaluating altered root architecture in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least on regulatory sequence (preferably a promoter functional in a plant), wherein the polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; and (c) evaluating root architecture of the transgenic plant compared to a control plant not comprising the recombinant DNA construct. The method may further comprise (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (e) evaluating root architecture of the progeny plant compared to a control plant not comprising the recombinant DNA construct.
[0271]A method of evaluating altered root architecture in a plant, comprising (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory sequence (preferably a promoter functional in a plant) operably linked to:
[0272](i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; and
[0273](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and (c) evaluating the transgenic plant for altered root architecture compared to a control plant not comprising the suppression DNA construct. The method may further comprise (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (e) evaluating the progeny plant for altered root architecture compared to a control plant not comprising the suppression DNA construct.
[0274]A method of evaluating altered root architecture in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (preferably a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34 (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the recombinant DNA construct; (c) obtaining a progeny plant derived from said transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (d) evaluating the progeny plant for altered root architecture compared to a control plant not comprising the recombinant DNA construct.
[0275]A method of evaluating root architecture in a plant, comprising:
(a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to: (i) all or part of: (A) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (B) a full complement of the nucleic acid sequence of (a)(i)(A); or (ii) a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; (c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (d) evaluating root architecture of the progeny plant compared to a control plant not comprising the suppression DNA construct.
[0276]A method of determining an alteration of an agronomic characteristic in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least on regulatory sequence (preferably a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34 (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome said recombinant DNA construct; and (c) determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct. The method may further comprise (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (e) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
[0277]A method of determining an alteration of an agronomic characteristic in a plant, comprising (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory sequence (preferably a promoter functional in a plant) operably linked to all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (i); (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and (c) determining whether the transgenic plant exhibits an alteration in at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct. The method may further comprise (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (e) determining whether the progeny plant exhibits an alteration in at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
[0278]A method of determining an alteration of an agronomic characteristic in a plant, comprising (a) introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence (preferably a promoter functional in a plant), wherein said polynucleotide encodes a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34 (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome said recombinant DNA construct; (c) obtaining a progeny plant derived from said transgenic plant, wherein the progeny plant comprises in its genome the recombinant DNA construct; and (d) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct. The method of determining an alteration of an agronomic characteristic in a plant may further comprise determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
[0279]A method of determining an alteration of an agronomic characteristic in a plant, comprising (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory sequence (preferably a promoter functional in a plant) operably linked to all or part of (i) a nucleic acid sequence encoding a polypeptide having an amino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, or 34, or (ii) a full complement of the nucleic acid sequence of (i);
[0280](b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; (c) obtaining a progeny plant derived from said transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (d) determining whether the progeny plant exhibits an alteration in at least one agronomic characteristic when compared to a control plant not comprising the recombinant DNA construct.
[0281]A method of determining an alteration of an agronomic characteristic in a plant, comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; and (c) determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct. The method may further comprise: (d) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (e) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
[0282]A method of determining an alteration of an agronomic characteristic in a plant, comprising: (a) introducing into a regenerable plant cell a suppression DNA construct comprising at least one regulatory element operably linked to a region derived from all or part of a sense strand or antisense strand of a target gene of interest, said region having a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 56%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal V method of alignment, when compared to said all or part of a sense strand or antisense strand from which said region is derived, and wherein said target gene of interest encodes a EXST or EXST-like polypeptide; (b) regenerating a transgenic plant from the regenerable plant cell after step (a), wherein the transgenic plant comprises in its genome the suppression DNA construct; (c) obtaining a progeny plant derived from the transgenic plant, wherein the progeny plant comprises in its genome the suppression DNA construct; and (d) determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared to a control plant not comprising the suppression DNA construct.
[0283]A method of producing seed (preferably seed that can be sold as a product offering with altered root architecture) comprising any of the preceding preferred methods, and further comprising obtaining seeds from said progeny plant, wherein said seeds comprise in their genome said recombinant DNA construct (or suppression DNA construct).
[0284]In any of the foregoing preferred methods or any other embodiments of methods of the present invention, the step of determining an alteration of an agronomic characteristic in a transgenic plant, if applicable, may preferably comprise determining whether the transgenic plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
[0285]In any of the foregoing preferred methods or any other embodiments of methods of the present invention, the step of determining an alteration of an agronomic characteristic in a progeny plant, if applicable, may preferably comprise determining whether the progeny plant exhibits an alteration of at least one agronomic characteristic when compared, under varying environmental conditions, to a control plant not comprising the recombinant DNA construct.
[0286]In any of the preceding preferred methods or any other embodiments of methods of the present invention, in said introducing step said regenerable plant cell preferably comprises a callus cell (preferably embryogenic), a gametic cell, a meristematic cell, or a cell of an immature embryo. The regenerable plant cells are preferably from an inbred maize plant.
[0287]In any of the preceding preferred methods or any other embodiments of methods of the present invention, said regenerating step preferably comprises: (i) culturing said transformed plant cells in a media comprising an embryogenic promoting hormone until callus organization is observed; (ii) transferring said transformed plant cells of step (i) to a first media which includes a tissue organization promoting hormone; and (iii) subculturing said transformed plant cells after step (ii) onto a second media, to allow for shoot elongation, root development or both.
[0288]In any of the preceding preferred methods or any other embodiments of methods of the present invention, alternatives exist for introducing into a regenerable plant cell a recombinant DNA construct comprising a polynucleotide operably linked to at least one regulatory sequence. For example, one may introduce into a regenerable plant cell a regulatory sequence (such as one or more enhancers, preferably as part of a transposable element), and then screen for an event in which the regulatory sequence is operably linked to an endogenous gene encoding a polypeptide of the instant invention.
[0289]The introduction of recombinant DNA constructs of the present invention into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector mediated DNA transfer, bombardment, or Agrobacterium mediated transformation.
[0290]In any of the preceding preferred methods or any other embodiments of methods of the present invention, the at least one agronomic characteristic is preferably selected from the group consisting of greenness, yield, growth rate, biomass, fresh weight at maturation, dry weight at maturation, fruit yield, seed yield, total plant nitrogen content, fruit nitrogen content, seed nitrogen content, nitrogen content in a vegetative tissue, total plant free amino acid content, fruit free amino acid content, seed free amino acid content, free amino acid content in a vegetative tissue, total plant protein content, fruit protein content, seed protein content, protein content in a vegetative tissue, drought tolerance, nitrogen uptake, root lodging, stalk lodging, plant height, ear length, stalk lodging and harvest index. Yield, greenness, biomass and root lodging are particularly preferred agronomic characteristics for alteration (preferably an increase).
In any of the preceding preferred methods or any other embodiments of methods of the present invention, the plant preferably exhibits the alteration of at least one agronomic characteristic irrespective of the environmental conditions when compared to a control.
[0291]The introduction of recombinant DNA constructs of the present invention into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector mediated DNA transfer, bombardment, or Agrobacterium mediated transformation.
[0292]Preferred techniques are set forth below in the Examples below for transformation of maize plant cells and soybean plant cells.
[0293]Other preferred methods for transforming dicots, primarily by use of Agrobacterium tumefaciens, and obtaining transgenic plants include those published for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135, U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011, McCabe et. al., Bio/Technology 6:923 (1988), Christou et al., Plant Physiol. 87:671 674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653 657 (1996), McKently et al., Plant Cell Rep. 14:699 703 (1995)); papaya; and pea (Grant et al., Plant Cell Rep. 15:254 258, (1995)).
[0294]Transformation of monocotyledons using electroporation, particle bombardment, and Agrobacterium have also been reported and are included as preferred methods, for example, transformation and plant regeneration as achieved in asparagus (Bytebier et al., Proc. Natl. Acad. Sci. U.S.A. 84:5354, (1987)); barley (Wan and Lemaux, Plant Physiol. 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204 (1988), Gordon-Kamm et al., Plant Cell 2:603 618 (1990), Fromm et al., Bio/Technology 8:833 (1990), Koziel et al., Bio/Technology 11:194, (1993), Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somers et al., Bio/Technology 10:1589 (1992)); orchard grass (Horn et al., Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., Theor. Appl. Genet. 205:34, (1986); Part et al., Plant Mol. Biol. 32:1135 1148, (1996); Abedinia et al., Aust. J. Plant Physiol. 24:133 141 (1997); Zhang and Wu, Theor. Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell Rep. 7:379, (1988); Battraw and Hall, Plant Sci. 86:191 202 (1992); Christou et al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature 325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992)); tall fescue (Wang et al., Bio/Technology 10:691 (1992)), and wheat (Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).
[0295]There are a variety of methods for the regeneration of plants from plant tissue. The particular method of regeneration will depend on the starting plant tissue and the particular plant species to be regenerated.
[0296]The regeneration, development, and cultivation of plants from single plant protoplast transformants or from various transformed explants is well known in the art (Weissbach and Weissbach, In: Methods for Plant Molecular Biology, (Eds.), Academic Press, Inc. San Diego, Calif., (1988)). This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those individualized cells through the usual stages of embryonic development through the rooted plantlet stage. Transgenic embryos and seeds are similarly regenerated. The resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
[0297]The development or regeneration of plants containing the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in the art. Preferably, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plant of the present invention containing a desired polypeptide is cultivated using methods well known to one skilled in the art.
[0298]In another aspect, this invention also concerns a method of mapping genetic variations related to altering root architecture and/or altering at least one agronomic characteristic in plants comprising:
[0299](a) crossing two plant varieties; and
[0300](c) evaluating genetic variations with respect to: [0301](i) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30 or 33; or [0302](iii) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 15; 17, 19, 21, 23, 25, 27, 29, 31 or 34 [0303]in progeny plants resulting from the cross of step (a) wherein the evaluation is made using a method selected from the group consisting of: RFLP analysis, SNP analysis, and PCR-based analysis.
[0304]In another embodiment, this invention concerns a method of molecular breeding to obtain an altered root architecture and/or at least one altered agronomic characteristic in plants comprising:
[0305](a) crossing two plant varieties; and
[0306](b) evaluating genetic variations with respect to: [0307](i) a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28, 30 or 33; or [0308](iii) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 15; 17, 19, 21, 23, 25, 27, 29, 31 or 34; [0309]in progeny plants resulting from the cross of step (a) wherein the evaluation is made using a method selected from the group consisting of: RFLP analysis, SNP analysis, and PCR-based analysis.
[0310]The terms "mapping genetic variation" or "mapping genetic variability" are used interchangeably and define the process of identifying changes in DNA sequence, whether from natural or induced causes, within a genetic region that differentiates between different plant lines, cultivars, varieties, families, or species. The genetic variability at a particular locus (gene) due to even minor base changes can alter the pattern of restriction enzyme digestion fragments that can be generated. Pathogenic alterations to the genotype can be due to deletions or insertions within the gene being analyzed or even single nucleotide substitutions that can create or delete a restriction enzyme recognition site. RFLP analysis takes advantage of this and utilizes Southern blotting with a probe corresponding to the isolated nucleic acid fragment of interest.
[0311]Thus, if a polymorphism (i.e., a commonly occurring variation in a gene or segment of DNA; also, the existence of several forms of a gene (alleles) in the same species) creates or destroys a restriction endonuclease cleavage site, or if it results in the loss or insertion of DNA (e.g., a variable nucleotide tandem repeat (VNTR) polymorphism), it will alter the size or profile of the DNA fragments that are generated by digestion with that restriction endonuclease. As such, individuals that possess a variant sequence can be distinguished from those having the original sequence by restriction fragment analysis. Polymorphisms that can be identified in this manner are termed "restriction fragment length polymorphisms: ("RFLPs"). RFLPs have been widely used in human and plant genetic analyses (Glassberg, UK Patent Application 2135774; Skolnick et al, Cytogen. Cell Genet. 32:58-67 (1982); Botstein et al, Ann. J. Hum. Genet. 32:314-331 (1980); Fischer et al (PCT Application WO 90/13668; Uhlen, PCT Application WO 90/11369).
[0312]A central attribute of "single nucleotide polymorphisms" or "SNPs" is that the site of the polymorphism is at a single nucleotide. SNPs have certain reported advantages over RFLPs or VNTRs. First, SNPs are more stable than other classes of polymorphisms. Their spontaneous mutation rate is approximately 10-9 (Kornberg, DNA Replication, W.H. Freeman & Co., San Francisco, 1980), approximately, 1,000 times less frequent than VNTRs (U.S. Pat. No. 5,679,524). Second, SNPs occur at greater frequency, and with greater uniformity than RFLPs and VNTRs. As SNPs result from sequence variation, new polymorphisms can be identified by sequencing random genomic or cDNA molecules. SNPs can also result from deletions, point mutations and insertions. Any single base alteration, whatever the cause, can be a SNP. The greater frequency of SNPs means that they can be more readily identified than the other classes of polymorphisms.
[0313]SNPs can be characterized using any of a variety of methods. Such methods include the direct or indirect sequencing of the site, the use of restriction enzymes where the respective alleles of the site create or destroy a restriction site, the use of allele-specific hybridization probes, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism or by other biochemical interpretation. SNPs can be sequenced by a number of methods. Two basic methods may be used for DNA sequencing, the chain termination method of Sanger et al, Proc. Natl. Acad. Sci. (U.S.A.) 74:5463-5467 (1977), and the chemical degradation method of Maxam and Gilbert, Proc. Natl. Acad. Sci. (U.S.A.) 74: 560-564 (1977).
[0314]Furthermore, single point mutations can be detected by modified PCR techniques such as the ligase chain reaction ("LCR") and PCR-single strand conformational polymorphisms ("PCR-SSCP") analysis. The PCR technique can also be used to identify the level of expression of genes in extremely small samples of material, e.g., tissues or cells from a body. The technique is termed reverse transcription-PCR ("RT-PCR").
[0315]The term "molecular breeding" defines the process of tracking molecular markers during the breeding process. It is common for the molecular markers to be linked to phenotypic traits that are desirable. By following the segregation of the molecular marker or genetic trait, instead of scoring for a phenotype, the breeding process can be accelerated by growing fewer plants and eliminating assaying or visual inspection for phenotypic variation. The molecular markers useful in this process include, but are not limited to, any marker useful in identifying mapable genetic variations previously mentioned, as well as any closely linked genes that display synteny across plant species. The term "synteny" refers to the conservation of gene placement/order on chromosomes between different organisms. This means that two or more genetic loci, that may or may not be closely linked, are found on the same chromosome among different species. Another term for synteny is "genome colinearity".
[0316]The goal of gene mapping is to identify genes which contribute to phenotypes of interest. The first stage of mapping is usually to locate a general region of a chromosome which is associated with transmission of the phenotypes of interest. Next, the gene and ultimately, particular alleles, are identified as having a causative role.
[0317]Association mapping generally falls into two broad categories: 1) candidate-gene association mapping, which relates polymorphisms in selected candidate genes that have purported roles in controlling phenotypic variation for specific traits; and 2) genome-wide association mapping, or genome scan, which surveys genetic variation in the whole genome to find signals of association for various complex traits.
[0318]In candidate-gene association mapping, candidate genes are selected based on prior knowledge from mutational analysis, biochemical pathway, or linkage analysis of the trait of interest. An independent set of random markers need to be scored to infer genetic relationships.
[0319]Genome-wide association mapping is a comprehensive approach to systematically search the genome for causal genetic variation. A large number of markers are tested for association with various complex traits, and prior information regarding candidate genes is not required. (Zhu et al. (2008) The Plant Genome, 1: 5-20).
EXAMPLES
[0320]The present invention is further illustrated in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, 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 invention to adapt it to various usages and conditions. Thus, various modifications 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.
Example 1
Creation of an Arabidopsis Population with Activation-Tagged Genes
[0321]A 18.5 kb T-DNA based binary construct was created, pHSbarENDs2 (FIG. 1; SEQ ID NO:1;) containing four multimerized enhancer elements derived from the Cauliflower Mosaic Virus 35S promoter, corresponding to sequences -341 to -64, as defined by Odell et al. (1985) Nature 313:810-812. The construct also contains vector sequences (pUC9) to allow plasmid rescue, transposon sequences (Ds) to remobilize the T-DNA, and the bar gene to allow for glufosinate selection of transgenic plants. Only the 10.8 kb segment from the right border (RB) to left border (LB) inclusive will be transferred into the host plant genome. Since the enhancer elements are located near the RB, they can induce cis-activation of genomic loci following T-DNA integration.
[0322]The pHSbarENDs2 construct was transformed into Agrobacterium tumefaciens strain C58, grown in LB at 25° C. to OD600 ˜1.0. Cells were then pelleted by centrifugation and resuspended in an equal volume of 5% sucrose/0.05% Silwet L-77 (OSI Specialties, Inc). At early bolting, soil grown Arabidopsis thaliana ecotype Col-0 were top watered with the Agrobacterium suspension. A week later, the same plants were top watered again with the same Agrobacterium strain in sucrose/Silwet. The plants were then allowed to set seed as normal. The resulting T1 seed were sown on soil, and transgenic seedlings were selected by spraying with glufosinate (Finale®; AgrEvo; Bayer Environmental Science). T2 seed was collected from approximately 35,000 individual glufosinate resistant T1 plants. T2 plants were grown and equal volumes of T3 seed from 96 separate T2 lines were pooled. This constituted 360 sub-populations.
[0323]A total of 100,000 glufosinate resistant T1 seedlings were selected. T2 seeds from each line were kept separate.
Example 2A
Screens to Identify Lines with Altered Root Architecture
Non-Limiting Nitrogen Conditions
[0324]Activation-tagged Arabidopsis seedlings, grown under non-limiting nitrogen conditions, can be analyzed for altered root system architecture when compared to control seedlings during early development from the population described in Example 1.
[0325]From each of 96,000 separate T1 activation-tagged lines, ten T2 seeds can be sterilized with chlorine gas and planted on petri plates containing the following medium: 0.5×N-Free Hoagland's, 60 mM KNO3, 0.1% sucrose, 1 mM MES and 1% Phytagel®. Typically 10 plates are placed in a rack. Plates are kept for three days at 4° C. to stratify seeds and then held vertically for 11 days at 22° C. light and 20° C. dark. Photoperiod is 16 h; 8 h dark, average light intensity was ˜180 μmol/m2/s. Racks (typically holding 10 plates each) are rotated daily within each shelf. At day 14, plates are evaluated for seedling status, whole plate digital images were taken, and analyzed for root area. Plates are arbitrarily divided in 10 horizontal areas. The root area in each of 10 horizontal zones on the plate is expressed as a percentage of the total area. Only areas in zones 3 to 9 are used to calculate the total root area of the line. Rootbot image analysis tool (proprietary) developed by ICORIA can be used to assess root area. Total root area is expressed in mm2.
[0326]Lines with enhanced root growth characteristics are expected to lie at the upper extreme of the root area distributions. A sliding window approach can be used to estimate the variance in root area for a given rack with the assumption that there could be up to two outliers in the rack. Environmental variations in various factors including growth media, temperature, and humidity can cause significant variation in root growth, especially between sow dates. Therefore the lines are grouped by sow date and shelf for the data analysis. The racks in a particular sow date/shelf group are then sorted by mean root area. Root area distributions for sliding windows is performed by combining data for a rack, ri, with data from the rack with the next lowest, (ri-1, and the next highest mean root area, ri+1. The variance of the combined distribution is then analyzed to identify outliers in ri using a Grubbs-type approach (Barnett et al., Outliers in Statistical Data, John Wiley & Sons, 3rd edition (1994).
[0327]Lines with significant enhanced root growth as determined by the method outlined above, are designated as Phase 1 hits. Phase 1 hits are re-screened in duplicate under the same assay conditions. When either or both of the Phase 2 replicates shows a significant difference from the mean, the line is then considered a validated root architecture line.
[0328]Those lines that are again found to be outliers in at least one plate in Phase 2 are subjected to a Phase 3 screening performed in house, to validate the results obtained in Phase 1 and Phase 2. The results are validated in Phase 3 using both the Rootboot image analysis (as described above) and WinRHIZO® as described below. The confirmation is performed in the same fashion as in the first round of screening. T2 seeds are sterilized using 50% household bleach 0.01% triton X-100 solution and plated onto the same plate medium as described in the first round of screening at a density of 10 seeds/plate. Plates are kept for three days at 4° C. to stratify seeds, and grown in the same temperature and photoperiod as the first experiment with the light intensity ˜160 μmol/m2/s. Plates are placed vertically into the eight center positions of a 10 plate rack with the first and last position holding blank plates. The racks and the plates within a rack are rotated every other day. Two sets of pictures are taken for each plate. The first set taking place at day 14-16 when the primary roots for most lines have reached the bottom of the plate, the second set of pictures two days later after more lateral roots have developed. The latter set of picture is usually used for data analysis. These seedlings grown on vertical plates are analyzed for root growth with the software WinRHIZO® (Regent Instruments Inc), an image analysis system specifically designed for root measurement. WinRHIZO® uses the contrast in pixels to distinguish the light root from the darker background. To identify the maximum amount of roots without picking up background, the pixel classification is 150-170 and the filter feature is used to remove objects that have a length/width ratio less then 10.0. The area on the plates analyzed is from the edge of the plant's leaves to about 1 cm from the bottom of the plate. The exact same WinRHIZO® settings and area of analysis are used to analyze all plates within a batch. The total root length score given by WinRHIZO® for a plate is divided by the number of plants that has germinated and has grown halfway down the plate. Three plates for every line are grown and their scores are averaged. This average is then compared to the average of three plates containing wild type seeds that are grown at the same time.
[0329]Arabidopsis activation tagged lines re-confirmed by having a higher value of root growth compared to wild type are then used for the molecular identification of the DNA flanking the T-DNA insertion.
Example 2B
Identification of Mutant Lines with an Altered Root Phenotype in a Mutant Population
Limiting Nitrogen Conditions
A Two-Step Screening Procedure can be Used, Comprising:
[0330](1) Identification of an altered root growth phenotype in a vertical plate assay;
[0331](2) Confirm herbicide resistance and root phenotype in rescued mutant lines; The primary screen is based on vertical plates containing Nitrogen-free Hoagland salts, 0.3% sucrose and 1 mM KNO3. The media also contains 0.8%-1.0% PhytaGel as a gelling agent. Media with Phytagel at 1.0% is sometimes difficult to pour as it solidifies quickly, however, at below 0.8% the media will slide off plates when placed vertically. Mutants from an activation-tagged population where pools of 100 lines each are available for a total of 36000 lines are being screened. On each plate, 12 mutant and 2 wild type Columbia seeds are seeded. Plates are placed in a growth room with a constant temperature of 26° C., 16 hr-day cycle with an average of 110 μE/m2s light intensity at the top of the plates. These plates are photographed 3-4 times in a 2.5 week time frame. Individual seedlings are rescued when a clear root phenotype is observed. Rescued seedlings are grown to maturity in a growth chamber (24° c., 16 hr day, 250-300 μE/m2s) for seed collection.
[0332]For the secondary screening, seeds from putative hits identified in the primary screen are sowed on plates containing the same media as above plus 6 mg/L bialaphos. Wild type Columbia seeds are sown at the same time on the same media but without bialaphos. Each plate has 10 seeds. There are 3 plates for each mutant line, and 2 plates for wild type Columbia, as replication. These plates are placed under the same growth conditions as described above in a growth room. Those lines that do not have herbicide resistance or no obvious root phenotype are discarded as false positives. Lines validated by the second screen are saved for further study.
Example 3
Identification of Activation-Tagged Genes
[0333]Genes flanking the T-DNA insert in lines with altered root architecture are identified using one, or both, of the following two standard procedures: (1) thermal asymmetric interlaced (TAIL) PCR (Liu et al., (1995), Plant J. 8:457-63); and (2) SAIFF PCR (Siebert et al., (1995) Nucleic Acids Res. 23:1087-1088). In lines with complex multimerized T-DNA inserts, TAIL PCR and SAIFF PCR may both prove insufficient to identify candidate genes. In these cases, other procedures, including inverse PCR, plasmid rescue and/or genomic library construction, can be employed.
[0334]A successful result is one where a single TAIL or SAIFF PCR fragment contains a T-DNA border sequence and Arabidopsis genomic sequence.
[0335]Once a tag of genomic sequence flanking a T-DNA insert is obtained, candidate genes are identified by alignment to publicly available Arabidopsis genome sequence.
[0336]Specifically, the annotated gene nearest the 35S enhancer elements/T-DNA RB are candidates for genes that are activated.
[0337]To verify that an identified gene is truly near a T-DNA and to rule out the possibility that the TAIL/SAIFF fragment is a chimeric cloning artifact, a diagnostic PCR on genomic DNA is done with one oligo in the T-DNA and one oligo specific for the candidate gene. Genomic DNA samples that give a PCR product are interpreted as representing a T-DNA insertion. This analysis also verifies a situation in which more than one insertion event occurs in the same line, e.g., if multiple differing genomic fragments are identified in TAIL and/or SAIFF PCR analyses.
Example 4
Identification of Activation-Tagged exst Gene
[0338]The exst gene was obtained by the screening procedure as described in Example 2A. Identification of the activation-tagged gene was performed as described in Example 3.
[0339]One line (112299) displaying altered root architecture was further analyzed. DNA from the line was extracted and the T-DNA insertion was found by ligation mediated PCR (Siebert et al., (1995) Nucleic Acids Res. 23:1087-1088) using primers within the LeftBorder of the T-DNA. Once a tag of genomic sequence flanking a T-DNA insert was obtained, the candidate gene was identified by sequence alignment to the completed Arabidopsis genome. One of the insertion sites identified was identified as a chimeric insertion; Left Border T-DNA sequence was determined to be at both ends of the T-DNA insertion. It is still possible that the enhancer elements located near the Right Border of the T-DNA are close enough to have an effect on the nearby candidate gene. In this case the location of the Right Border was assumed to be present at the insertion site, and the two genes that flank the insertion site were chosen as candidates. One of the genes nearest the 35S enhancers of the chimeric insertion was AT3G03650 (nucleotides 245-1744 corresponding to the ORF (SEQ ID NO:33), encoding the EXST protein (SEQ ID NO:34), referred herein as EXOSTOSIN FAMILY or EXST.
Example 5A
Validation of a Candidate Arabidopsis Gene (AT3G03650) for its Ability to Enhance Root Architecture in Plants Via Transformation into Arabidopsis
[0340]Candidate genes can be transformed into Arabidopsis and overexpressed under the 35S promoter. If the same or similar phenotype is observed in the transgenic line as in the parent activation-tagged line, then the candidate gene is considered to be a validated "lead gene" in Arabidopsis.
[0341]The Arabidopsis AT3G03650 Gene can be directly tested for its ability to enhance Root Architecture in Arabidopsis.
[0342]The Arabidopsis AT3G03650 cDNA was PCR amplified with oligos that introduce the attB1 (SEQ ID NO:39) sequence, a consensus start sequence (CAACA) upstream of the ATG start codon and the first 25 nucleotides of the protein coding-region of the AT3G03650 DNA (SEQ ID NO:57) and the attB2 (SEQ ID NO:40) sequence and the last 25 nucleotides of the protein-coding region including the stop codon of said cDNA. Using Invitrogen® Gateway® technology a MultiSite Gateway® BP Recombination Reaction was performed with pDONR®/Zeo (Invitrogen®, FIG. 2; SEQ ID NO:2). This process removes the bacteria lethal ccdB gene, as well as the chloramphenicol resistance gene (CAM) from pDONR®/Zeo and directionally clones the PCR product with flanking attB1 (SEQ ID NO:39) and attB2 (SEQ ID NO:40) sites creating entry clone PHP28732.
[0343]A 16.8-kb T-DNA based binary vector, called pBC-yellow (FIG. 4, SEQ ID NO:4), was constructed with the 1.3-kb 35S promoter immediately upstream of the Invitrogen® Gateway® C1 conversion insert containing the ccdB gene and the chloramphenicol resistance gene (CAM) flanked by attR1 and attR2 sequences. The vector also contains a YFP marker under the control of the Rd29a promoter for the selection of transformed seeds.
[0344]Using Invitrogen® Gateway® technology a MultiSite Gateway® LR Recombination Reaction was performed on the entry clone containing the directionally cloned PCR product and pBC-yellow. This allowed rapid and directional cloning of the AT3G03650 gene behind the 35S promoter in pBC-yellow.
[0345]The 35S-AT3G03650 gene construct was introduced into wild-type Arabidopsis ecotype Col-0, using the same Agrobacterium-mediated transformation procedure described in Example 1.
[0346]Transgenic T1 seeds were selected by the presence of the fluorescent YFP marker. Fluorescent seeds were subjected to the Root Architecture Assay following the procedure described in Example 2A. Transgenic T1 seeds were re-screened using 6 plates per construct. Two plates per rack containing non-transformed Columbia seed discarded from fluorescent seed sorting served as a control.
[0347]Six plates per construct were analyzed statistically and a trend was detected between the number of plants growing on a plate and their average WinRHIZO® score. WinRHIZO® scores were normalized for this trend and the root score corresponding to the construct was divided by the wild-type root score.
Example 5B
Screen of Candidate Genes Under Nitrogen Limiting Conditions
[0348]Transgenic T1 seed selected by the presence of the fluorescent marker YFP as described above in Example 5A can also be screened for their tolerance to grow under nitrogen limiting conditions. For this purpose 32 transgenic individuals can be grown next to 32 wild-type individuals on one plate with either 0.4 mM KNO3 or 60 mM KNO3. If a line shows a statistically significant difference from the controls, the line is considered a validated nitrogen-deficiency tolerant line. After masking the plate image to remove background color, two different measurements are collected for each individual: total rosetta area, and the percentage of color that falls into a green color bin. Using hue, saturation and intensity data (HIS), the green color bin consists of hues 50-66. Total rosetta area is used as a measure of plant biomass, whereas the green color bin has been shown by dose-response studies to be an indicator of nitrogen assimilation.
Example 5C
Validation of a Candidate Arabidopsis Gene (At3G03650) for its Ability to Improve Nitrogen Utilization in Plants Via Transformation into Arabidopsis
[0349]Transgenic seeds were screened for their ability to grow under nitrogen limiting conditions as described in Example 5B.
[0350]Plants were evaluated at 10, 11, 12 and 13 days. Transgenic individuals expressing the Arabidopsis Candidate gene (AT3G03650) did not validate as nitrogen-deficient tolerant compared to the wild type plants, when grown on media containing limiting concentrations of nitrogen (0.4 mM KNO3).
Example 5D
Screen to Identify Lines with Improved Nitrate Uptake
[0351]For each overexpressor line, twelve T2 plants are sown on 96 well micro titer plates containing 2 mM MgSO4, 0.5 mM KH2PO4, 1 mM CaCl2, 2.5 mM KCl, 0.15 mM Sprint 330, 0.06 mM FeSO4, 1 μM MnCl2.4H2O, 1 μM ZnSO4.7H2O, 3 μM H3BO3, 0.1 μM NaMoO4, 0.1 μM CuSO4.5H2O,
0.8 mM potassium nitrate, 0.1% sucrose, 1 mM MES, 200 μM bromophenol red and 0.40% Phytagel® (pH assay medium). The pH of the medium is so that the color of bromophenol is red, the pH indicator dye, is yellow.
[0352]Four lines are plated per plate, and the inclusion of 12 wild-type individuals and 12 individuals from a line that has shown an improvement in nitrate uptake (positive control) on each plate makes for a total of 72 individuals on each 96 well micro titer plate A web-based random sequence generator can be used to determine the order of the lines on each plate. Seeds are not plated in Row A or Row H on the 96 well micro titer plate. Four plates are plated for each experiment, resulting in a maximum of 48 plants per line analyzed. Plates are kept for three days in the dark at 4° C. to stratify seeds, and then placed horizontally for six days at 22° C. light and dark. Photoperiod is sixteen hours light; eight hours dark, with an average light intensity of ˜200 mmol/m2/s. Plates are rotated and shuffled within each shelf. At day eight or nine (five or six days of growth), seedling status is evaluated by recording the color of the medium as pink, peach, yellow or no germination. Then the plants and/or seeds are removed from each well. Each medium plug is transferred to 1.2 ml micro titer tubes and placed in the corresponding well in a 96 well deep micro titer plate. An equal volume of water containing 2 μM flourescein is added to each 1.2 ml micro titer tube. The plate is covered with foil and autoclaved on liquid cycle. Each tube is mixed well, and an aliquot is removed from each tube and analyzed for amount of nitrate remaining in the medium. If t-test shows that a line is significantly different (p<0.05) from wild-type control, the line is then considered a validated improved nitrate uptake line.
Example 5E
Validation of Increased Nitrate Uptake by Transgenic Lines Containing the Candidate Arabidopsis Gene (AT3G03650)
[0353]Transgenic seeds were screened for increased nitrate uptake as described in Example 5D.
[0354]Transgenic individuals overexpressing the Arabidopsis Candidate gene (AT3G03650) did not validate as an improved nitrate uptake line compared to wild type plants not overexpressing the Arabidopsis candidate gene.
Example 6
Composition of cDNA Libraries
Isolation and Sequencing of cDNA Clones
[0355]cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP® XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP® XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
[0356]Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.
[0357]Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI Prism dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.
[0358]Sequence data is collected (ABI Prism Collections) and assembled using Phred and Phrap (Ewing et al. (1998) Genome Res. 8:175-185; Ewing and Green (1998) Genome Res. 8:186-194). Phred is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (Gordon et al. (1998) Genome Res. 8:195-202).
[0359]In some of the clones the cDNA fragment corresponds to a portion of the 3'-terminus of the gene and does not cover the entire open reading frame. In order to obtain the upstream information one of two different protocols are used. The first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame. Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries some times are chosen based on previous knowledge that the specific gene should be found in a certain tissue and some times are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries. Library pools are normally prepared using from 3 to 5 different libraries and normalized to a uniform dilution. In the first round of amplification both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5'-terminus of the clone coupled with a gene-specific (reverse) primer. The first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3'-untranslated region (also referred to as UTR). In the second round of amplification a nested set of primers is used for both methods. The resulting DNA fragment is ligated into a pBluescript vector using a commercial kit and following the manufacturer's protocol. This kit is selected from many available from several vendors including Invitrogen® (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA is isolated by alkaline lysis method and submitted for sequencing and assembly using Phred/Phrap, as above.
Example 7
Identification of cDNA Clones
[0360]cDNA clones encoding EXST-like polypeptides can be identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained as described in Example 6 can be analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as "pLog" values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST "hit" represent homologous proteins.
[0361]ESTs submitted for analysis are compared to the Genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402) against the Du Pont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 6. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.
Example 8
Preparation of cDNA Libraries and Characterization of cDNA Clones Encoding EXST-Like Polypeptides
[0362]cDNA libraries representing mRNAs from various tissues of Maize, Soybean, Rice, Sunflower, Guar, Wheat, Florida bitterbush, Oat, Cotton, Amaranth and Canola were prepared as described in Example 6 of the libraries are described below. The characteristics of the libraries are described below.
TABLE-US-00002 TABLE 2 cDNA Libraries from Maize, Soybean, Rice, Sunflower, Guar, Wheat, Florida bitterbush, Oat, Cotton, Amaranth and Canola Library Tissue Clone cfp5n Maize Kernel, pooled stages, Full-length enriched, cfp5n.pk007.k11 normalized p0127 Nucellus tissue, 5 days after silking, screened 1. p0127.cntdd86ra cfp6n Maize Leaf and Seed pooled, Full-length enriched cfp6n.pk002.a5 normalized cfp6n.pk002.a5:fis Ctn1c Corn (Zea mays L., B73) night harvested tassel (v12 ctn1c.pk002.p16 stage). rls24 Rice Leaf 15 Days After Germination, 24 Hours After rls24.pk0026.h11 Infection of Strain Magnaporthe grisea 4360-R-67 (AVR2- rls24.pk0026.h11:fis YAMO); Susceptible esl1c rye leaves, chilled, to induce cold-responsive gene esl1c.pk006.l19 sequences that can be used to transform corn for esl1c.pk006.l19:fis tolerance to cold or dehydration stress.. cfp1n Maize Tassel V7 to V12 pooled, Full-length enriched cfp1n.pk002.o16.f:fis normalized ebb2c Immature buds of Canola Rf gene knock out mutant line, ebb2c.pk005.f9 02SM5. ebb2c.pk005.f9:fis lds1c Guar (Cyamopsis tetragonoloba) seeds harvested at 15 lds1c.pk008.m15 DAF lds1c.pk008.m15:fis eas1c Amaranthus retroflexus young seeds eas1c.pk002.p14 eas1c.pk002.p14:fis egh1c Upland Cotton (Gossypium hirsutum) germinating seeds. egh1c.pk005.b21 egh1c.pk005.b21:fis ort1f Oat (Avena strigosa) full length oat root tip ort1f.pk014.e9 ort1f.pk014.e9:fis pps Developing Seeds of Picramnia pentandra (Florida pps.pk007.b3 bitterbush) pps.pk0007.b3:fis hso1c Oxalate oxidase-transgenic sunflower plants hso1c.pk001.n10 hso1c.pk001.n10:fis sgs1c Soybean Seeds 4 Hours After Germination sgs1c.pk004.m16 sgs1c.pk004.m14 scn1c Soybean (Glycine max L., 6705) Embryogenic scn1c.pk001.m7 suspension culture 10 months old (necrotic tissue). scn1c.pk001.m7:fis wpa1c Wheat (Triticum aestivum) pre-meiotic anthers JIC wpa1c.pk011.n19 wpa1c.pk011.n19:fis rdi2c Rice (Oryza sativa, Nipponbare) developing inflorescence rdi2c.pk011.p5 at rachis branch-floral organ primordia formation rdi2c.pk011.p5:fis smj1c Characterization of IPT transcripts from transgenic smj1c.pk006.c12.f soybean. The lead Yield Enhancement (Soy YE2.1) smj1c.pk006.c12.f:fis construct is expressing Agrobacterium isopentenyl transferase gene.
[0363]The BLASTX search using the EST sequences from clones listed in Table 1 revealed similarity of the polypeptides to EXST-like polypeptides from rice (GI No. 115476598, 115487106, 115452759 and 115441893 corresponding to SEQ ID NO's:35, 36, 37, and 38, respectively. Shown in Table 3 are the BLASTP results for the sequences of the entire cDNA inserts ("Full-insert Sequence" or "FIS") of the clones listed in Table 2. Each cDNA insert encodes an entire or functional protein ("Complete Gene Sequence" or "CGS"). Also shown in Tables 3 and 4 are the percent sequence identity values for each pair of amino acid sequences using the Clustal V method of alignment with default parameters:
TABLE-US-00003 TABLE 3 BLASTP Results and Percent Identity for Sequences Encoding Polypeptides Homologous to EXST-like Polypeptides BLAST pLog % Sequence Status NCBI GI No. Score identity Contig of: Contig 115487106 >180 77.5 cfp5n.pk007.k11 (Oryza sativa) cfp5n.pk007.k11.f SEQ ID NO: 36 cfp6n.pk005.i1 SEQ ID NO: 14 Contig of: Contig 115452759 (Oryza 176 70.0 cfp3n.pk069.l15 sativa) cfp3n.pk069.l15.f SEQ ID NO: 37 p0127.cntdd86ra p0127.cntdd86ra.f SEQ ID NO: 16 my.ceb1.pk0010.e5 FIS 115441893 (Oryza 160 90.1 SEQ ID NO: 18 sativa) SEQ ID NO: 38 cfp6n.pk002.a5:fis CGS 115452759 (Oryza >180 82.4 SEQ ID NO: 20 sativa) SEQ ID NO: 37 rls24.pk0026.h11:fis CGS 115476598 (Oryza >180 99.8 SEQ ID NO: 22 sativa) SEQ ID NO: 35 p0127.cntdd86ra:fis CGS 11542759 (Oryza >180 80.8 SEQ ID NO: 24 sativa) SEQ ID NO: 37 cfp5n.pk007.k11:fis FIS 115487106 (Oryza 85 36.2 SEQ ID NO: 26 sativa) SEQ ID NO: 36 esl1c.pk006.l19:fis CGS 115487106 (Oryza >180 78.9 SEQ ID NO: 28 sativa) SEQ ID NO: 36 cfp1n.pk002.o16.f:fis CGS 115476598 (Oryza >180 63.5 SEQ ID NO: 30 sativa) SEQ ID NO: 35
[0364]FIGS. 15A-15I show the multiple alignment of the full length amino acid sequences of SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27, 29, 31, 34, and SEQ ID NOs:35, 36, 37, and 38. FIG. 16 presents the percent sequence identities and divergence values for each sequence pair presented in FIGS. 15A-15I.
[0365]Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
[0366]Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode EXST-like polypeptides.
TABLE-US-00004 TABLE 4 BLASTP Results for Sequences Encoding Polypeptides Homologous to EXST and EXST-like polypeptides BLAST pLog % Sequence Status Reference Score identity Contig of: CGS SEQ ID 49101 in >180 77.5 cfp5n.pk007.k11 JP2005185101 cfp5n.pk007.k11.f cfp6n.pk005.i1 SEQ ID NO: 14 Contig of: EST SEQ ID 345741 in >180 80.1 cfp3n.pk069.l15 US2004214272 cfp3n.pk069.l15.f p0127.cntdd86ra p0127.cntdd86ra.f SEQ ID NO: 16 my.ceb1.pk0010.e5 CGS SEQ ID 7611 in 175 99.6 SEQ ID NO: 18 US2004216190 cfp6n.pk002.a5:fis CGS SEQ ID 345741 in >180 98.9 SEQ ID NO: 20 US2004214272 rls24.pk0026.h11:fis CGS SEQ ID NO 54370 >180 99.8 SEQ ID NO: 22 in JP2005185101 p0127.cntdd86ra:fis CGS SEQ ID 345741 in >180 91.4 SEQ ID NO: 24 US20042 cfp5n.pk007.k11:fis CGS SEQ ID 361954 in 94 75.1 SEQ ID NO: 26 US2004214272 esl1c.pk006.l19:fis CGS SEQ ID 10067 in >180 98.3 SEQ ID NO: 28 US2004216190- A1 cfp1n.pk002.o16.f:fis CGS SEQ ID 54370 in >180 63.5 SEQ ID NO: 30 JP2005185101
Example 9
Preparation of a Plant Expression Vector Containing a Homolog of the Arabidopsis Lead Gene (AT3G03650)
[0367]Sequences homologous to the lead EXST gene can be identified using sequence comparison algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410 (1993); see also the explanation of the BLAST algorithm on the world wide web site for the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health). Homologous EXST-like sequences, such as the ones described in Example 8, can be PCR-amplified by either of the following methods.
[0368]Method 1 (RNA-based): If the 5' and 3' sequence information for the protein-coding region of a EXST homolog is available, gene-specific primers can be designed as outlined in Example 5A. RT-PCR can be used with plant RNA to obtain a nucleic acid fragment containing the EXST protein-coding region flanked by attB1 (SEQ ID NO:39) and attB2 (SEQ ID NO:40) sequences. The primer may contain a consensus Kozak sequence (CAACA) upstream of the start codon.
[0369]Method 2 (DNA-based): Alternatively, if a cDNA clone is available for a gene encoding an EXST polypeptide homolog, the entire cDNA insert (containing 5' and 3' non-coding regions) can be PCR amplified. Forward and reverse primers can be designed that contain either the attB1 sequence and vector-specific sequence that precedes the cDNA insert or the attB2 sequence and vector-specific sequence that follows the cDNA insert, respectively. For a cDNA insert cloned into the vector pBluescript SK+, the forward primer VC062 (SEQ ID NO:41) and the reverse primer VC063 (SEQ ID NO:42) can be used.
[0370]Methods 1 and 2 can be modified according to procedures known by one skilled in the art. For example, the primers of method 1 may contain restriction sites instead of attB1 and attB2 sites, for subsequent cloning of the PCR product into a vector containing attB1 and attB2 sites. Additionally, method 2 can involve amplification from a cDNA clone, a lambda clone, a BAC clone or genomic DNA.
[0371]A PCR product obtained by either method above can be combined with the Gateway® donor vector, such as pDONR®/Zeo (Invitrogen®, FIG. 2; SEQ ID NO:2) or pDONR®221 (Invitrogen®, FIG. 3; SEQ ID NO:3) using a BP Recombination Reaction. This process removes the bacteria lethal ccdB gene, as well as the chloramphenicol resistance gene (CAM) from pDONR®221 and directionally clones the PCR product with flanking attB1 and attB2 sites to create an entry clone. Using the Invitrogen® Gateway® Clonase® technology, the homologous EXST-like gene from the entry clone can then be transferred to a suitable destination vector to obtain a plant expression vector for use with Arabidopsis, corn and soy, such as pBC-Yellow (FIG. 4; SEQ ID NO:4), PHP27840 (FIG. 5; SEQ ID NO:5) or PHP23236 (FIG. 6; SEQ ID NO:6), to obtain a plant expression vector for use with Arabidopsis, soybean and corn, respectively.
[0372]Alternatively a MultiSite Gateway® LR recombination reaction between multiple entry clones and a suitable destination vector can be performed to create an expression vector. An Example of this procedure is outlined in Example 14A, describing the construction of maize expression vectors for transformation of maize lines.
Example 10
Preparation of Soybean Expression Vectors and Transformation of Soybean with Validated Arabidopsis Lead Genes and Homologs Thereof
[0373]Soybean plants can be transformed to overexpress the validated Arabidopsis gene (AT3G03650) and the corresponding homologs from various species in order to examine the resulting phenotype.
[0374]The entry clones described in Example 5A and 9 can be used to directionally clone each gene into PHP27840 vector (FIG. 5, SEQ ID NO:5) such that expression of the gene is under control of the SCP1 promoter.
[0375]Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides.
[0376]To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos, which produce secondary embryos, are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiply as early, globular staged embryos, the suspensions are maintained as described below.
[0377]Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic® PDS1000/HE instrument (helium retrofit) can be used for these transformations.
[0378]A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from cauliflower mosaic virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. Another selectable marker gene which can be used to facilitate soybean transformation is an herbicide-resistant acetolactate synthase (ALS) gene from soybean or Arabidopsis. ALS is the first common enzyme in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. Mutations in ALS have been identified that convey resistance to some or all of three classes of inhibitors of ALS (U.S. Pat. No. 5,013,659; the entire contents of which are herein incorporated by reference). Expression of the herbicide-resistant ALS gene can be under the control of a SAM synthetase promoter (U.S. Patent Application No. US-2003-0226166-A1; the entire contents of which are herein incorporated by reference).
[0379]To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.
[0380]Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
[0381]Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
[0382]Enhanced root architecture can be measured in soybean by growing the plants in soil and wash the roots before analysis of the total root mass with WinRHIZO®.
[0383]Soybean plants transformed with validated genes can then be assayed to study agronomic characteristics relative to control or reference plants. For example, nitrogen utilization efficacy, yield enhancement and/or stability under various environmental conditions (e.g. nitrogen limiting conditions, drought etc.).
Example 11
Transformation of Maize with Validated Arabidopsis Lead Genes Using Particle Bombardment
[0384]Maize plants can be transformed to overexpress a validated Arabidopsis lead gene or the corresponding homologs from various species in order to examine the resulting phenotype.
[0385]The Gateway® entry clones described in Example 5A can be used to directionally clone each gene into a maize transformation vector. Expression of the gene in maize can be under control of a constitutive promoter such as the maize ubiquitin promoter (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992))
[0386]The recombinant DNA construct described above can then be introduced into maize cells by the following procedure. Immature maize embryos can be dissected from developing caryopses derived from crosses of the inbred maize lines H99 and LH132. The embryos are isolated ten to eleven days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al., Sci. Sin. Peking 18:659-668 (1975)). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every two to three weeks.
[0387]The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from cauliflower mosaic virus (Odell et al., Nature 313:810-812 (1985)) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
[0388]The particle bombardment method (Klein et al., Nature 327:70-73 (1987)) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After ten minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton® flying disc (Bio-Rad Labs). The particles are then accelerated into the maize tissue with a Biolistic® PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
[0389]For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
[0390]Seven days after bombardment the tissue can be transferred to N6 medium that contains bialaphos (5 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional two weeks the tissue can be transferred to fresh N6 medium containing bialaphos. After six weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialaphos-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.
[0391]Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).
Transgenic T0 plants can be regenerated and their phenotype determined following HTP procedures. T1 seed can be collected.
[0392]T1 plants can be grown and analyzed for phenotypic changes. The following parameters can be quantified using image analysis: plant area, volume, growth rate and color analysis can be collected and quantified. Expression constructs that result in an alteration of root architecture or any one of the agronomic characteristics listed above compared to suitable control plants, can be considered evidence that the Arabidopsis lead gene functions in maize to alter root architecture or plant architecture.
[0393]Furthermore, a recombinant DNA construct containing a validated Arabidopsis gene can be introduced into a maize line either by direct transformation or introgression from a separately transformed line.
[0394]Transgenic plants, either inbred or hybrid, can undergo more vigorous field-based experiments to study root or plant architecture, yield enhancement and/or resistance to root lodging under various environmental conditions (e.g. variations in nutrient and water availability).
[0395]Subsequent yield analysis can also be done to determine whether plants that contain the validated Arabidopsis lead gene have an improvement in yield performance, when compared to the control (or reference) plants that do not contain the validated Arabidopsis lead gene. Plants containing the validated Arabidopsis lead gene would improved yield relative to the control plants, preferably 50% less yield loss under adverse environmental conditions or would have increased yield relative to the control plants under varying environmental conditions (e.g. increased yield under non limiting nitrogen conditions compared to control.
Example 12
Electroporation of Agrobacterium tumefaciens LBA4404
[0396]Electroporation competent cells (40 μl), such as Agrobacterium tumefaciens LBA4404 (containing PHP10523), are thawn on ice (20-30 min). PHP10523 contains VIR genes for T-DNA transfer, an Agrobacterium low copy number plasmid origin of replication, a tetracycline resistance gene, and a cos site for in vivo DNA biomolecular recombination. Meanwhile the electroporation cuvette is chilled on ice. The electroporator settings are adjusted to 2.1 kV.
[0397]A DNA aliquot (0.5 μL JT (U.S. Pat. No. 7,087,812) parental DNA at a concentration of 0.2 μg-1.0 μg in low salt buffer or twice distilled H2O) is mixed with the thawn Agrobacterium cells while still on ice. The mix is transferred to the bottom of electroporation cuvette and kept at rest on ice for 1-2 min. The cells are electroporated (Eppendorf electroporator 2510) by pushing "Pulse" button twice (ideally achieving a 4.0 msec pulse). Subsequently 0.5 ml 2×YT medium (or SOCmedium) are added to cuvette and transferred to a 15 ml Falcon tube. The cells are incubated at 28-30° C., 200-250 rpm for 3 h.
[0398]Aliquots of 250 μl are spread onto #30B (YM+50 μg/mL Spectinomycin) plates and incubated 3 days at 28-30° C. To increase the number of transformants one of two optional steps can be performed:
Option 1: overlay plates with 30 μl of 15 mg/ml Rifampicin. LBA4404 has a chromosomal resistance gene for Rifampicin. This additional selection eliminates some contaminating colonies observed when using poorer preparations of LBA4404 competent cells.Option 2: Perform two replicates of the electroporation to compensate for poorer electrocompetent cells.
Identification of Transformants:
[0399]Four independent colonies are picked and streaked on AB minimal medium plus 50 mg/mL Spectinomycin plates (#12S medium) for isolation of single colonies. The plated are incubate at 28° C. for 2-3 days.
[0400]A single colony for each putative co-integrate is picked and inoculated with 4 ml #60A with 50 mg/l Spectinomycin. The mix is incubated for 24 h at 28° C. with shaking. Plasmid DNA from 4 ml of culture is isolated using Qiagen Miniprep+optional PB wash. The DNA is eluted in 30 μl. Aliquots of 2 μl are used to electroporate 20 μl of DH10b+20 μl of ddH2O as per above.
Optionally a 15 μl aliquot can be used to transform 75-100 μl of Invitrogen®-Library Efficiency DH5α. The cells are spread on LB medium plus 50 mg/mL Spectinomycin plates (#34T medium) and incubated at 37° C. overnight.
[0401]Three to four independent colonies are picked for each putative co-integrate and inoculated 4 ml of 2×YT (#60A) with 50 μg/ml Spectinomycin. The cells are incubated at 37° C. overnight with shaking.
[0402]The plasmid DNA is isolated from 4 ml of culture using QIAprep® Miniprep with optional PB wash (elute in 50 μl) and 8 μl are used for digestion with SaII (using JT parent and PHP10523 as controls).
[0403]Three more digestions using restriction enzymes BamHI, EcoRI, and HindIII are performed for 4 plasmids that represent 2 putative co-integrates with correct SaII digestion pattern (using parental DNA and PHP10523 as controls). Electronic gels are recommended for comparison.
[0404]Alternatively, for high throughput applications, such as described for Gaspe Flint Derived Maize Lines (Examples 15-17), instead of evaluating the resulting co-integrate vectors by restriction analysis, three colonies can be simultaneously used for the infection step as described in Example 13.
Example 13
Agrobacterium Mediated Transformation into Maize
[0405]Maize plants can be transformed to overexpress a validated Arabidopsis lead gene or the corresponding homologs from various species in order to examine the resulting phenotype.
[0406]Agrobacterium-mediated transformation of maize is performed essentially as described by Zhao et al., in Meth. Mol. Biol. 318:315-323 (2006) (see also Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No. 5,981,840 issued Nov. 9, 1999, incorporated herein by reference). The transformation process involves bacterium innoculation, co-cultivation, resting, selection and plant regeneration.
1. Immature Embryo Preparation
[0407]Immature embryos are dissected from caryopses and placed in a 2 mL microtube containing 2 mL PHI-A medium.
2. Agrobacterium Infection and Co-Cultivation of Embryos
2.1 Infection Step
[0408]PHI-A medium is removed with 1 mL micropipettor and 1 mL Agrobacterium suspension is added. Tube is gently inverted to mix. The mixture is incubated for 5 min at room temperature.
2.2 Co-Culture Step
[0409]The Agrobacterium suspension is removed from the infection step with a 1 mL micropipettor. Using a sterile spatula the embryos are scraped from the tube and transferred to a plate of PHI-B medium in a 100×15 mm Petri dish. The embryos are oriented with the embryonic axis down on the surface of the medium. Plates with the embryos are cultured at 20° C., in darkness, for 3 days. L-Cysteine can be used in the co-cultivation phase. With the standard binary vector, the co-cultivation medium supplied with 100-400 mg/L L-cysteine is critical for recovering stable transgenic events.
3. Selection of Putative Transgenic Events
[0410]To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos are transferred, maintaining orientation and the dishes are sealed with Parafilm. The plates are incubated in darkness at 28° C. Actively growing putative events, as pale yellow embryonic tissue are expected to be visible in 6-8 weeks. Embryos that produce no events may be brown and necrotic, and little friable tissue growth is evident. Putative transgenic embryonic tissue is subcultured to fresh PHI-D plates at 2-3 week intervals, depending on growth rate. The events are recorded.
4. Regeneration of T0 Plants
[0411]Embryonic tissue propagated on PHI-D medium is subcultured to PHI-E medium (somatic embryo maturation medium); in 100×25 mm Petri dishes and incubated at 28° C., in darkness, until somatic embryos mature, for about 10-18 days. Individual, matured somatic embryos with well-defined scutellum and coleoptile are transferred to PHI-F embryo germination medium and incubated at 28° C. in the light (about 80 μE from cool white or equivalent fluorescent lamps). In 7-10 days, regenerated plants, about 10 cm tall, are potted in horticultural mix and hardened-off using standard horticultural methods.
Media for Plant Transformation
[0412]1. PHI-A: 4 g/L CHU basal salts, 1.0 mL/L 1000×Eriksson's vitamin mix, 0.5 mg/L thiamin HCL, 1.5 mg/L 2,4-D, 0.69 g/L L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 μM acetosyringone, filter-sterilized before using. [0413]2. PHI-B: PHI-A without glucose, increased 2,4-D to 2 mg/L, reduced sucrose to 30 g/L and supplemented with 0.85 mg/L silver nitrate (filter-sterilized), 3.0 g/L gelrite, 100 μM acetosyringone (filter-sterilized), 5.8. [0414]3. PHI-C: PHI-B without gelrite and acetosyringonee, reduced 2,4-D to 1.5 mg/L and supplemented with 8.0 g/L agar, 0.5 g/L Ms-morpholino ethane sulfonic acid (MES) buffer, 100 mg/L carbenicillin (filter-sterilized). [0415]4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos (filter-sterilized). [0416]5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL 11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5 mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5 mg/L zeatin (Sigma, cat. no. Z-0164), 1 mg/L indole acetic acid (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L bialaphos (filter-sterilized), 100 mg/L carbenicillin (fileter-sterilized), 8 g/L agar, pH 5.6. [0417]6. PHI-F: PHI-E without zeatin, IAA, ABA; sucrose reduced to 40 g/L; replacing agar with 1.5 g/L gelrite; pH 5.6.
[0418]Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).
Phenotypic analysis of transgenic T0 plants and T1 plants can be performed.
[0419]T1 plants can be analyzed for phenotypic changes. Using image analysis T1 plants can be analyzed for phenotypical changes in plant area, volume, growth rate and color analysis can be taken at multiple times during growth of the plants. Alteration in root architecture can be assayed as described in Example 20.
[0420]Subsequent analysis of alterations in agronomic characteristics can be done to determine whether plants containing the validated Arabidopsis lead gene have an improvement of at least one agronomic characteristic, when compared to the control (or reference) plants that do not contain the validated Arabidopsis lead gene. The alterations may also be studied under various environmental conditions.
[0421]Expression constructs that result in a significant alteration in root architecture will be considered evidence that the Arabidopsis gene functions in maize to alter root architecture.
Example 14A
Construction of Maize Expression Vectors with the Arabidopsis Lead Gene (AT3G03650) Using Agrobacterium Mediated Transformation
[0422]Maize expression vectors were prepared with the Arabidopsis EXST gene (AT3G03650) under the control of the NAS2 (SEQ ID NO:44) and GOS2 (SEQ ID NO:45) promoter. PINII was the terminator (SEQ ID NO:48)
Using Invitrogen® Gateway® technology the entry clone, created as described in Example 5A, PHP 28739, containing the Arabidopsis EXST gene (AT3G03650) was used in separate Gateway® LR reactions with:
[0423]1) the constitutive maize GOS2 promoter entry clone (PHP28408, FIG. 11, SEQ ID NO:11) and the PinII Terminator entry clone (PHP20234, FIG. 9, SEQ ID NO:9) into the destination vector PHP28529 (FIG. 10, SEQ ID NO:10). The resulting vector was named PHP28976.
[0424]2) the root maize NAS2 promoter entry clone (PHP22020, FIG. 12, SEQ ID NO:12) and the PinII Terminator entry clone (PHP20234, FIG. 9, SEQ ID NO:9) into the destination vector PHP28529 (FIG. 10, SEQ ID NO:10). The resulting vector was named PHP28913.
The destination vector PHP28529 added to each of the final vectors (PHP28983 and PHP28984) also an: [0425]1) RD29A promoter::yellow fluorescent protein::PinII terminator cassette for Arabidopsis seed sorting [0426]2) a Ubiquitin promoter::moPAT/red fluorescent protein fusion::PinII terminator cassette for transformation selection and Z. mays seed sorting.
Example 14B
Preparation of Maize Expression Constructs Containing the Arabidopsis EXST Gene and Homologs Thereof
[0427]The Arabidopsis EXST gene and the corresponding homologs from maize and other species (Table 1) can be transformed into maize lines using the procedures outlined in Examples 5A and 14A. Maize expression vectors with Arabidopsis EXST gene and the corresponding homologs from maize and other species (Table 1) can be prepared as outlined in Examples 5A and 14A. In addition to the GOS2 or NAS2 promoter, other promoters such as, but not limited to the ubiquitin promoter, the S2A and S2B promoter, the maize ROOTMET2 promoter, the maize Cyclo, the CR1BIO, the CRWAQ81 and the maize ZRP2.4447 are useful for directing expression of EXST and EXST-like genes in maize. Furthermore, a variety of terminators, such as, but not limited to the PINII terminator, could be used to achieve expression of the gene of interest in maize.
Example 14C
Transformation of Maize Lines with the Arabidopsis Lead Gene (AT3G03650) and Corresponding Homologs from Other Species Using Agrobacterium Mediated Transformation
[0428]The final vectors (vectors for expression in Maize, Example 14A and B) can be then electroporated separately into LBA4404 Agrobacterium containing PHP10523 (FIG. 7; SEQ ID NO:7, Komari et al. Plant J 10:165-174 (1996), NCBI GI: 59797027) to create the co-integrate vectors for maize transformation. The co-integrate vectors are formed by recombination of the final vectors (maize expression vectors) with PHP10523, through the COS recombination sites contained on each vector. The co-integrate vectors contain in addition to the expression cassettes described in Examples 14A-B, also genes needed for the Agrobacterium strain and the Agrobacterium mediated transformation, (TET, TET, TRFA, ORI terminator, CTL, ORI V, VIR C1, VIR C2, VIR G, VIR B). Transformation into a maize line can be performed as described in Example 13.
Example 15
Preparation of the Destination Vectors PHP23236 and PHP29635 for Transformation of Gaspe Flint Derived Maize Lines
[0429]Destination vector PHP23236 (FIG. 6, SEQ ID NO:6) was obtained by transformation of Agrobacterium strain LBA4404 containing plasmid PHP10523 (FIG. 7, SEQ ID NO:7) with plasmid PHP23235 (FIG. 8, SEQ ID NO:8) and isolation of the resulting co-integration product. Destination vector PHP23236, can be used in a recombination reaction with an entry clone as described in Example 16 to create a maize expression vector for transformation of Gaspe Flint derived maize lines. Expression of the gene of interest is under control of the ubiquitin promoter (SEQ ID NO:46).
[0430]PHP29635 (FIG. 13, SEQ ID NO:13) was obtained by transformation of Agrobacterium strain LBA4404 containing plasmid PHP10523 with plasmid PIIOXS2a-FRT87(ni)m (FIG. 14, SEQ ID NO:43) and isolation of the resulting co-integration product. Destination vector PHP29635 can be used in a recombination reaction with an entry clone as described in Example 16 to create a maize expression vector for transformation of Gaspe Flint derived maize lines. Expression of the gene of interest is under control of the S2A promoter (SEQ ID NO:47).
Example 16
Preparation of Plasmids for Transformation of Gaspe Flint Derived Maize Lines
[0431]Using Invitrogen® Gateway® Recombination technology, entry clones containing the Arabidopsis EXST gene (AT3G03650) or a maize EXST-like homolog can be created, as described in Examples 5A and 9 and used to directionally clone each gene into destination vector PHP23236 (Example 15) for expression under the ubiquitin promoter or into destination vector PHP29635 (Example 15) for expression under the S2A promoter. Each of the expression vectors are T-DNA binary vectors for Agrobacterium-mediated transformation into corn.
Gaspe Flint Derived Maize Lines can be transformed with the expression constructs as described in Example 17.
Example 17
Transformation of Gaspe Flint Derived Maize Lines with Validated Arabidopsis Lead Genes and Corresponding Homologs from Other Species
[0432]Maize plants can be transformed as described in Example 16 to overexpress the Arabidopsis AT3G03650 gene and the corresponding homologs from other species, such as the ones listed in Table 1, in order to examine the resulting phenotype. In addition to the promoters described in Example 16 other promoters such the S2B promoter, the maize ROOTMET2 promoter, the maize Cyclo, the CR1BIO, the CRWAQ81 and the maize ZRP2.4447 are useful for directing expression of exst and exst-like genes in maize. Furthermore, a variety of terminators, such as, but not limited to the PINII terminator, can be used to achieve expression of the gene of interest in Gaspe Flint Derived Maize Lines.
[0433]Recipient Plants
[0434]Recipient plant cells can be from a uniform maize line having a short life cycle ("fast cycling"), a reduced size, and high transformation potential. Typical of these plant cells for maize are plant cells from any of the publicly available Gaspe Flint (GF) line varieties. One possible candidate plant line variety is the F1 hybrid of GF×QTM (Quick Turnaround Maize, a publicly available form of Gaspe Flint selected for growth under greenhouse conditions) disclosed in Tomes et al. U.S. Patent Application Publication No. 2003/0221212. Transgenic plants obtained from this line are of such a reduced size that they can be grown in four inch pots (1/4 the space needed for a normal sized maize plant) and mature in less than 2.5 months. (Traditionally 3.5 months is required to obtain transgenic T0 seed once the transgenic plants are acclimated to the greenhouse.) Another suitable line is a double haploid line of GS3 (a highly transformable line) X Gaspe Flint. Yet another suitable line is a transformable elite inbred line carrying a transgene which causes early flowering, reduced stature, or both.
[0435]Transformation Protocol
[0436]Any suitable method may be used to introduce the transgenes into the maize cells, including but not limited to inoculation type procedures using Agrobacterium based vectors as described in Example 9. Transformation may be performed on immature embryos of the recipient (target) plant.
[0437]Precision Growth and Plant Tracking
[0438]The event population of transgenic (T0) plants resulting from the transformed maize embryos is grown in a controlled greenhouse environment using a modified randomized block design to reduce or eliminate environmental error. A randomized block design is a plant layout in which the experimental plants are divided into groups (e.g., thirty plants per group), referred to as blocks, and each plant is randomly assigned a location with the block.
[0439]For a group of thirty plants, twenty-four transformed, experimental plants and six control plants (plants with a set phenotype) (collectively, a "replicate group") are placed in pots which are arranged in an array (a.k.a. a replicate group or block) on a table located inside a greenhouse. Each plant, control or experimental, is randomly assigned to a location with the block which is mapped to a unique, physical greenhouse location as well as to the replicate group. Multiple replicate groups of thirty plants each may be grown in the same greenhouse in a single experiment. The layout (arrangement) of the replicate groups should be determined to minimize space requirements as well as environmental effects within the greenhouse. Such a layout may be referred to as a compressed greenhouse layout.
[0440]An alternative to the addition of a specific control group is to identify those transgenic plants that do not express the gene of interest. A variety of techniques such as RT-PCR can be applied to quantitatively assess the expression level of the introduced gene. T0 plants that do not express the transgene can be compared to those which do.
[0441]Each plant in the event population is identified and tracked throughout the evaluation process, and the data gathered from that plant is automatically associated with that plant so that the gathered data can be associated with the transgene carried by the plant. For example, each plant container can have a machine readable label (such as a Universal Product Code (UPC) bar code) which includes information about the plant identity, which in turn is correlated to a greenhouse location so that data obtained from the plant can be automatically associated with that plant.
[0442]Alternatively any efficient, machine readable, plant identification system can be used, such as two-dimensional matrix codes or even radio frequency identification tags (RFID) in which the data is received and interpreted by a radio frequency receiver/processor. See U.S. Published Patent Application No. 2004/0122592, incorporated herein by reference.
[0443]Phenotypic Analysis Using Three-Dimensional Imaging
[0444]Each greenhouse plant in the T0 event population, including any control plants, is analyzed for agronomic characteristics of interest, and the agronomic data for each plant is recorded or stored in a manner so that it is associated with the identifying data (see above) for that plant. Confirmation of a phenotype (gene effect) can be accomplished in the T1 generation with a similar experimental design to that described above.
[0445]The T0 plants are analyzed at the phenotypic level using quantitative, non-destructive imaging technology throughout the plant's entire greenhouse life cycle to assess the traits of interest. Preferably, a digital imaging analyzer is used for automatic multi-dimensional analyzing of total plants. The imaging may be done inside the greenhouse. Two camera systems, located at the top and side, and an apparatus to rotate the plant, are used to view and image plants from all sides. Images are acquired from the top, front and side of each plant. All three images together provide sufficient information to evaluate the biomass, size and morphology of each plant.
[0446]Due to the change in size of the plants from the time the first leaf appears from the soil to the time the plants are at the end of their development, the early stages of plant development are best documented with a higher magnification from the top. This may be accomplished by using a motorized zoom lens system that is fully controlled by the imaging software.
[0447]In a single imaging analysis operation, the following events occur: (1) the plant is conveyed inside the analyzer area, rotated 360 degrees so its machine readable label can be read, and left at rest until its leaves stop moving; (2) the side image is taken and entered into a database; (3) the plant is rotated 90 degrees and again left at rest until its leaves stop moving, and (4) the plant is transported out of the analyzer.
[0448]Plants are allowed at least six hours of darkness per twenty four hour period in order to have a normal day/night cycle.
[0449]Imaging Instrumentation
[0450]Any suitable imaging instrumentation may be used, including but not limited to light spectrum digital imaging instrumentation commercially available from LemnaTec GmbH of Wurselen, Germany. The images are taken and analyzed with a LemnaTec Scanalyzer HTS LT-0001-2 having a 1/2'' IT Progressive Scan IEE CCD imaging device. The imaging cameras may be equipped with a motor zoom, motor aperture and motor focus. All camera settings may be made using LemnaTec software. Preferably, the instrumental variance of the imaging analyzer is less than about 5% for major components and less than about 10% for minor components.
[0451]Software
[0452]The imaging analysis system comprises a LemnaTec HTS Bonit software program for color and architecture analysis and a server database for storing data from about 500,000 analyses, including the analysis dates. The original images and the analyzed images are stored together to allow the user to do as much reanalyzing as desired. The database can be connected to the imaging hardware for automatic data collection and storage. A variety of commercially available software systems (e.g. Matlab, others) can be used for quantitative interpretation of the imaging data, and any of these software systems can be applied to the image data set.
[0453]Conveyor System
[0454]A conveyor system with a plant rotating device may be used to transport the plants to the imaging area and rotate them during imaging. For example, up to four plants, each with a maximum height of 1.5 m, are loaded onto cars that travel over the circulating conveyor system and through the imaging measurement area. In this case the total footprint of the unit (imaging analyzer and conveyor loop) is about 5 m×5 m.
[0455]The conveyor system can be enlarged to accommodate more plants at a time. The plants are transported along the conveyor loop to the imaging area and are analyzed for up to 50 seconds per plant. Three views of the plant are taken. The conveyor system, as well as the imaging equipment, should be capable of being used in greenhouse environmental conditions.
[0456]Illumination
[0457]Any suitable mode of illumination may be used for the image acquisition. For example, a top light above a black background can be used. Alternatively, a combination of top- and backlight using a white background can be used. The illuminated area should be housed to ensure constant illumination conditions. The housing should be longer than the measurement area so that constant light conditions prevail without requiring the opening and closing or doors. Alternatively, the illumination can be varied to cause excitation of either transgene (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP)) or endogenous (e.g. Chlorophyll) fluorophores.
[0458]Biomass Estimation Based on Three-Dimensional Imaging
[0459]For best estimation of biomass the plant images should be taken from at least three axes, preferably the top and two side (sides 1 and 2) views. These images are then analyzed to separate the plant from the background, pot and pollen control bag (if applicable). The volume of the plant can be estimated by the calculation:
Volume(voxels)= {square root over (TopArea(pixels))}× {square root over (Side1Area(pixels))}× {square root over (Side2Area(pixels))}
[0460]In the equation above the units of volume and area are "arbitrary units". Arbitrary units are entirely sufficient to detect gene effects on plant size and growth in this system because what is desired is to detect differences (both positive-larger and negative-smaller) from the experimental mean, or control mean. The arbitrary units of size (e.g. area) may be trivially converted to physical measurements by the addition of a physical reference to the imaging process. For instance, a physical reference of known area can be included in both top and side imaging processes. Based on the area of these physical references a conversion factor can be determined to allow conversion from pixels to a unit of area such as square centimeters (cm2). The physical reference may or may not be an independent sample. For instance, the pot, with a known diameter and height, could serve as an adequate physical reference.
[0461]Color Classification
[0462]The imaging technology may also be used to determine plant color and to assign plant colors to various color classes. The assignment of image colors to color classes is an inherent feature of the LemnaTec software. With other image analysis software systems color classification may be determined by a variety of computational approaches.
[0463]For the determination of plant size and growth parameters, a useful classification scheme is to define a simple color scheme including two or three shades of green and, in addition, a color class for chlorosis, necrosis and bleaching, should these conditions occur. A background color class which includes non plant colors in the image (for example pot and soil colors) is also used and these pixels are specifically excluded from the determination of size. The plants are analyzed under controlled constant illumination so that any change within one plant over time, or between plants or different batches of plants (e.g. seasonal differences) can be quantified.
[0464]In addition to its usefulness in determining plant size growth, color classification can be used to assess other yield component traits. For these other yield component traits additional color classification schemes may be used. For instance, the trait known as "staygreen", which has been associated with improvements in yield, may be assessed by a color classification that separates shades of green from shades of yellow and brown (which are indicative of senescing tissues). By applying this color classification to images taken toward the end of the T0 or T1 plants' life cycle, plants that have increased amounts of green colors relative to yellow and brown colors (expressed, for instance, as Green/Yellow Ratio) may be identified. Plants with a significant difference in this Green/Yellow ratio can be identified as carrying transgenes which impact this important agronomic trait.
[0465]The skilled plant biologist will recognize that other plant colors arise which can indicate plant health or stress response (for instance anthocyanins), and that other color classification schemes can provide further measures of gene action in traits related to these responses.
[0466]Plant Architecture Analysis
[0467]Transgenes which modify plant architecture parameters may also be identified using the present invention, including such parameters as maximum height and width, internodal distances, angle between leaves and stem, number of leaves starting at nodes and leaf length. The LemnaTec system software may be used to determine plant architecture as follows. The plant is reduced to its main geometric architecture in a first imaging step and then, based on this image, parameterized identification of the different architecture parameters can be performed. Transgenes that modify any of these architecture parameters either singly or in combination can be identified by applying the statistical approaches previously described.
[0468]Pollen Shed Date
[0469]Pollen shed date is an important parameter to be analyzed in a transformed plant, and may be determined by the first appearance on the plant of an active male flower. To find the male flower object, the upper end of the stem is classified by color to detect yellow or violet anthers. This color classification analysis is then used to define an active flower, which in turn can be used to calculate pollen shed date.
[0470]Alternatively, pollen shed date and other easily visually detected plant attributes (e.g. pollination date, first silk date) can be recorded by the personnel responsible for performing plant care. To maximize data integrity and process efficiency this data is tracked by utilizing the same barcodes utilized by the LemnaTec light spectrum digital analyzing device. A computer with a barcode reader, a palm device, or a notebook PC may be used for ease of data capture recording time of observation, plant identifier, and the operator who captured the data.
[0471]Orientation of the Plants
[0472]Mature maize plants grown at densities approximating commercial planting often have a planar architecture. That is, the plant has a clearly discernable broad side, and a narrow side. The image of the plant from the broadside is determined. To each plant a well defined basic orientation is assigned to obtain the maximum difference between the broadside and edgewise images. The top image is used to determine the main axis of the plant, and an additional rotating device is used to turn the plant to the appropriate orientation prior to starting the main image acquisition.
Example 18
Screening of Gaspe Flint Derived Maize Lines Under Nitrogen Limiting Conditions
[0473]Transgenic plants will contain two or three doses of Gaspe Flint-3 with one dose of GS3 (GS3/(Gaspe-3)2× or GS3/(Gaspe-3)3×) and will segregate 1:1 for a dominant transgene. Plants will be planted in Turface, a commercial potting medium, and watered four times each day with 1 mM KNO3 growth medium and with 2 mM KNO3, or higher, growth medium (see FIG. 23). Control plants grown in 1 mM KNO3 medium will be less green, produce less biomass and have a smaller ear at anthesis (see FIG. 24 for an illustration of sample data).
[0474]Statistics are used to decide if differences seen between treatments are really different. FIG. 18 illustrates one method which places letters after the values. Those values in the same column that have the same letter (not group of letters) following them are not significantly different. Using this method, if there are no letters following the values in a column, then there are no significant differences between any of the values in that column or, in other words, all the values in that column are equal.
[0475]Expression of a transgene will result in plants with improved plant growth in 1 mM KNO3 when compared to a transgenic null. Thus biomass and greenness (as described in Example 17) will be monitored during growth and compared to a transgenic null. Improvements in growth, greenness and ear size at anthesis will be indications of increased nitrogen tolerance.
Example 19
Yield Analysis of Maize Lines with Validated Arabidopsis Lead Gene (AT3G03650)
[0476]A recombinant DNA construct containing a validated Arabidopsis gene can be introduced into a maize line either by direct transformation or introgression from a separately transformed line.
[0477]Transgenic plants, either inbred or hybrid, can undergo more vigorous field-based experiments to study yield enhancement and/or stability under various environmental conditions, such as variations in water and nutrient availability.
[0478]Subsequent yield analysis can be done to determine whether plants that contain the validated Arabidopsis lead gene have an improvement in yield performance under various environmental conditions, when compared to the control plants that do not contain the validated Arabidopsis lead gene. Reduction in yield can be measured for both. Plants containing the validated Arabidopsis lead gene have less yield loss relative to the control plants, preferably 50% less yield loss.
Example 20
Assays to Determine Alterations of Root Architecture in Maize
[0479]Transgenic maize plants are assayed for changes in root architecture at seedling stage, flowering time or maturity. Assays to measure alterations of root architecture of maize plants include, but are not limited to the methods outlined below. To facilitate manual or automated assays of root architecture alterations, corn plants can be grown in clear pots. [0480]1) Root mass (dry weights). Plants are grown in Turface, a growth media that allows easy separation of roots. Oven-dried shoot and root tissues are weighed and a root/shoot ratio calculated. [0481]2) Levels of lateral root branching. The extent of lateral root branching (e.g. lateral root number, lateral root length) is determined by sub-sampling a complete root system, imaging with a flat-bed scanner or a digital camera and analyzing with WinRHIZO® software (Regent Instruments Inc.). [0482]3) Root band width measurements. The root band is the band or mass of roots that forms at the bottom of greenhouse pots as the plants mature. The thickness of the root band is measured in mm at maturity as a rough estimate of root mass. [0483]4) Nodal root count. The number of crown roots coming off the upper nodes can be determined after separating the root from the support medium (e.g. potting mix). In addition the angle of crown roots and/or brace roots can be measured. Digital analysis of the nodal roots and amount of branching of nodal roots form another extension to the aforementioned manual method.All data taken on root phenotype are subjected to statistical analysis, normally a t-test to compare the transgenic roots with that of non-transgenic sibling plants. One-way ANOVA may also be used in cases where multiple events and/or constructs are involved in the analysis.
Example 21
Nitrogen Utilization Efficiency Seedling Assay
[0484]Seed of transgenic events are separated into transgene (heterozygous) and null seed using a seed color marker. Two different random assignments of treatments are made to each block of 54 pots arranged 6 rows by 9 columns using 9 replicates of all treatments.
[0485]Two seed of each treatment are planted in 4 inch, square pots containing Turface on 8 inch, staggered centers and watered four times each day with a solution containing the following nutrients:
TABLE-US-00005 1 mM CaCl2 2 mM MgSO4 0.5 mM KH2PO4 83 ppm Sprint330 3 mM KCl 1 mM KNO3 1 μM ZnSO4 1 μM MnCl2 3 μM H3BO4 1 μM MnCl2 0.1 μM CuSO4 0.1 μM NaMoO4
[0486]After emergence the plants are thinned to one seed per pot. At harvest, plants are removed from the pots and the Turface is washed from the roots. The roots are separated from the shoot, placed in a paper bag and dried at 70° C. for 70 hr. The dried plant parts (roots and shoots) are weighed and placed in a 50 ml conical tube with approximately 20 5/32 inch steel balls and ground by shaking in a paint shaker. Approximately, 30 mg of the ground tissue (weight recorded for later adjustment) is hydrolyzed in 2 ml of 20% H2O2 and 6M H2SO4 for 30 min at 170° C. After cooling, water is added to 20 ml, mixed thoroughly, and a 50 μl aliquot is removed and added to 950 μl 1M Na2CO3. The ammonia in this solution is used to estimate total reduced plant nitrogen by placing 100 μl of this solution into individual wells of a 96 well plate followed by adding 50 μl of OPA solution. Fluorescence, excitation=360 nM/emission=530 nM, is determined and compared to NH4Cl standards dissolved in a similar solution and treated with OPA solution.
OPA solution--5 μl Mercaptoethanol+1 ml OPA stock solutionOPA stock--50 mg o-phthadialdehyde (OPA--Sigma #P0657) dissolved in 1.5 ml methanol+4.4 ml 1M Borate buffer pH9.5 (3.09 g H3BO4+1 g NaOH in 50 ml water)+0.55 ml 20% SDS
[0487]Using these data the following parameters are measured and means compared to null mean parameters using a Student's t test:
Total Plant Biomass
Root Biomass
Shoot Biomass
Root/Shoot Ratio
[0488]Plant N concentration
Total Plant N
[0489]Variance is calculated within each block using a nearest neighbor calculation as well as by Analysis of Variance (ANOVA) using a completely random design (CRD) model. An overall treatment effect for each block is calculated using an F statistic by dividing overall block treatment mean square by the overall block error mean square. The probability of a greater Student's t test is calculated for each transgenic mean compared to the appropriate null (either construct bulked or individual event null mean) mean. A minimum (P<t) of 0.1 is used as a cut off.
Example 22
Analysis of Roots of Maize Seedlings Containing the Arabidopsis Exostosin Gene Compared to Roots from Seedlings not Containing the Exostosin Gene
[0490]A maize expression vector, containing the maize NAS2 promoter (SEQ ID NO:44) and the Arabidopsis exostosin gene (SEQ ID NO:33) was prepared as described in Example 14A.
[0491]Transformation of maize was achieved via Agrobacterium mediated transformation as described in Example 14C by creating a cointegrate vector (PHP29009) and roots were assayed as described in Example 20 using a seedling assay.
[0492]All 10 events from construct PHP29009 (ZM-NAS2::AT-EXST) were assayed in a greenhouse experiment, where 9 plants per each event were grown in Turface media to V4 stage. Seeds were from T1 generation (from ears collected off T0 plants). The control experiment included 15 plants of bulked nulls (non-transgenic segregates) grown to the same stage. Seeds were planted using a complete random block design. Plants were harvested 18 days after planting, when they reached V4 stage. Roots were washed and collected separately from shoots. All samples were oven-dried before dry weights were taken on an analytical balance.
[0493]A total of 4 events were found to have significant changes in root dry weights, 5 events in shoot dry weights, and 7 events in root to shoot ratios, when compared to the bulked null control, ata P-value less than 0.1. Six events, #4, #5, #6. #7, #9, and #10, had significant increases in Root/Shoot ratios, and 1 event, #1, had a significant decrease.
[0494]T-test analysis was performed to show significant differences between each transgenic event and the control. The p-values are shown for each trait: root dry weights, shoot dry weights, and root-to-shoot ratios. Bold face fonts indicate the transgenic had a higher value that the control. Those that had a p-value of less than 0.1 are indicated with an asterix (*).
TABLE-US-00006 TABLE 5 Comparison of transgenic and control seedlings EVENT Root Dry Weight Shoot Dry Weight Root/Shoot Ratio 1 0.407 1.000 0.057* 2 0.737 0.909 0.501 3 0.931 0.893 0.576 4 0.012* 0.093* 0.000* 5 0.000* 0.431 0.000* 6 0.022* 0.859 0.005* 7 0.431 0.001* 0.061* 8 0.949 0.017* 0.163 9 0.003* 0.001* 0.000* 10 0.404 0.000* 0.027*
Example 23
Yield Testing of Transgenic Hybrids Under Normal and Under Nitrogen Depleted Conditions in the Field
[0495]A field experiment was carried out on a farm in Johnston, Iowa for the 2007 season.
[0496]Seven (7) transgenic events expressing the exostosin gene driven by the maize NAS2 promoter, and two controls were included in the experiment. One control was a non-transgenic null with nulls bulked across 7 events. The other control was the wild type used for transformation. All the events were hybrids generated from a common inbred tester.
[0497]Two treatments were applied, consisting of conditions wherein the plants were either grown under "normal" nitrogen or under nitrogen "depleted" (stress) conditions. The "normal" treatment included application of a nitrogen fertilizer at a rate of 250 lb per acre. Nitrogen "depleted" conditions were achieved by growing the transgenic and non-transgenic control maize lines in a field wherein soil nitrogen levels had been withdrawn by crops grown in previous years in the absence of fertilizer.
[0498]Nitrogen depletion was controlled at the level that caused 30% yield reduction, compared to the normal nitrogen treatment, and required a 100 lb per acre nitrogen fertilization rate. The experiments were set up as 2-row plots with a density of 32000 plants per acre. Four (4) and six (6) replications were included in the normal and the depleted nitrogen treatment, respectively. Plants were planted May 21, 2007 and combine harvested on Sep. 26 and 27, 2007. Yield was measured as bushels per acre.
[0499]The combine yield data in bushels per acre from the experiments are summarized as percent increases over the null controls, in Table 6. Event # 7 was not tested under normal nitrogen due to seed shortage. Overall, there were 3 events under low nitrogen and 4 events under normal nitrogen that had significant increase in yield over the bulked null control. All events tested showed a positive trend in yield increase over nulls.
TABLE-US-00007 TABLE 6 Yield Tests of Transgenic versus Control Plants under Low and Normal Nitrogen Conditions Event Yield increase over null Significance Treatment 1 16.15% P = 0.1 Low nitrogen 2 6.15% 1 stand error Low nitrogen 3 6.15% 1 stand error Low nitrogen 4 3.08% Low nitrogen 5 7.69% P = 0.1 Low nitrogen 6 2.31% Low nitrogen 7 7.69% P = 0.1 Low nitrogen 1 7.53% P = 0.1 Normal nitrogen 2 7.65% P = 0.1 Normal nitrogen 3 8.82% P = 0.1 Normal nitrogen 4 12.35% P = 0.1 Normal nitrogen 5 4.71% Normal nitrogen 6 7.53% 1 stand error Normal nitrogen
Example 24
Genome-Wide Association Mapping Analysis
[0500]An association mapping strategy can be undertaken to identify markers associated with alterations in root architecture in maize. In this association analysis, a collection of maize lines can be analyzed by DNA sequencing at several thousand genes (genetic loci). The lines can encompass elite germplasm, commercially released cultivars, and other public varieties.
[0501]Phenotypic scores for an alteration in root architecture or in at least one agronomic characteristic will be obtained. Lines with extreme phenotypes will be tested against genotypes in a whole genome association test (using 2×2 contingency tables with Fisher's exact test). A structure-based association analysis will be used, where the population structure is controlled using marker data. The model-based cluster analysis software, Structure, developed by Pritchard et al., (Genetics 155:945-959 (2000)) will be used with haplotype data for hundreds of elite maize inbreds at several hundred markers to estimate admixture coefficients and assign the inbreds to a number of subpopulations. This reduces the occurrence of false positives that can arise due to the effect of population structure on association mapping statistics. Kuiper's statistic for testing whether two distributions are the same was used to test a given marker for association between haplotype and phenotype in a given subpopulation (Press et al., Numerical Recipes in C, second edition, Cambridge University Press, NY (2002)).
[0502]At least one strong peak in at least one subpopulation is indicative of significant marker-trait associations (e.g. p<0.001). Marker positions are given in cM, with position zero being the first (most distal from the centromere) marker known at the beginning of a chromosome. These map positions are not absolute, and represent an estimate of map position based on the internally derived genetic map.
Example 25
Candidate Gene Association Mapping
[0503]Primers are designed to amplify a portion of the candidate gene locus from individual inbred lines. Genotypes are obtained for each of the inbred lines at this locus and the polymorphic loci are tested for statistically significant associations with altered root architecture or at least one alteration in one agronomic characteristic.
Sequence CWU
1
48118491DNAartificial sequencevector 1catgaatcaa acaaacatac acagcgactt
attcacacga gctcaaatta caacggtata 60tatcctgccg tcgacaacca tggtctagac
aggatccccg ggtaccgagc tcgaatttgc 120aggtcgactg cgtcatccct tacgtcagtg
gagatatcac atcaatccac ttgctttgaa 180gacgtggttg gaacgtcttc tttttccacg
atgctcctcg tgggtggggg tccatctttg 240ggaccactgt cggcagaggc atcttgaacg
atagcctttc ctttatcgca atgatggcat 300ttgtaggtgc caccttcctt ttctactgtc
cttttgatga agtgacagat agctgggcaa 360tggaatccga ggaggtttcc cgatattacc
ctttgttgaa aagtctcaat tgccctttgg 420tcttctgaga ctgttgcgtc atcccttacg
tcagtggaga tatcacatca atccacttgc 480tttgaagacg tggttggaac gtcttctttt
tccacgatgc tcctcgtggg tgggggtcca 540tctttgggac cactgtcggc agaggcatct
tgaacgatag cctttccttt atcgcaatga 600tggcatttgt aggtgccacc ttccttttct
actgtccttt tgatgaagtg acagatagct 660gggcaatgga atccgaggag gtttcccgat
attacccttt gttgaaaagt ctcagttaac 720ccgcgatcct gcgtcatccc ttacgtcagt
ggagatatca catcaatcca cttgctttga 780agacgtggtt ggaacgtctt ctttttccac
gatgctcctc gtgggtgggg gtccatcttt 840gggaccactg tcggcagagg catcttgaac
gatagccttt cctttatcgc aatgatggca 900tttgtaggtg ccaccttcct tttctactgt
ccttttgatg aagtgacaga tagctgggca 960atggaatccg aggaggtttc ccgatattac
cctttgttga aaagtctcaa ttgccctttg 1020gtcttctgag actgttgcgt catcccttac
gtcagtggag atatcacatc aatccacttg 1080ctttgaagac gtggttggaa cgtcttcttt
ttccacgatg ctcctcgtgg gtgggggtcc 1140atctttggga ccactgtcgg cagaggcatc
ttgaacgata gcctttcctt tatcgcaatg 1200atggcatttg taggtgccac cttccttttc
tactgtcctt ttgatgaagt gacagatagc 1260tgggcaatgg aatccgagga ggtttcccga
tattaccctt tgttgaaaag tctcagttaa 1320cccgcaattc actggccgtc gttttacaac
gtcgtgactg ggaaaaccct ggcgttaccc 1380aacttaatcg ccttgcagca catccccctt
tcgccagctg gcgtaatagc gaagaggccc 1440gcaccgatcg cccttcccaa cagttgcgca
gcctgaatgg cgaatggatc gatccgtcga 1500tcgaccaaag cggccatcgt gcctccccac
tcctgcagtt cgggggcatg gatgcgcgga 1560tagccgctgc tggtttcctg gatgccgacg
gatttgcact gccggtagaa ctccgcgagg 1620tcgtccagcc tcaggcagca gctgaaccaa
ctcgcgaggg gatcgagccc ctgctgagcc 1680tcgacatgtt gtcgcaaaat tcgccctgga
cccgcccaac gatttgtcgt cactgtcaag 1740gtttgacctg cacttcattt ggggcccaca
tacaccaaaa aaatgctgca taattctcgg 1800ggcagcaagt cggttacccg gccgccgtgc
tggaccgggt tgaatggtgc ccgtaacttt 1860cggtagagcg gacggccaat actcaacttc
aaggaatctc acccatgcgc gccggcgggg 1920aaccggagtt cccttcagtg aacgttatta
gttcgccgct cggtgtgtcg tagatactag 1980cccctggggc cttttgaaat ttgaataaga
tttatgtaat cagtctttta ggtttgaccg 2040gttctgccgc tttttttaaa attggatttg
taataataaa acgcaattgt ttgttattgt 2100ggcgctctat catagatgtc gctataaacc
tattcagcac aatatattgt tttcatttta 2160atattgtaca tataagtagt agggtacaat
cagtaaattg aacggagaat attattcata 2220aaaatacgat agtaacgggt gatatattca
ttagaatgaa ccgaaaccgg cggtaaggat 2280ctgagctaca catgctcagg ttttttacaa
cgtgcacaac agaattgaaa gcaaatatca 2340tgcgatcata ggcgtctcgc atatctcatt
aaagcagggg gtgggcgaag aactccagca 2400tgagatcccc gcgctggagg atcatccagc
cggcgtcccg gaaaacgatt ccgaagccca 2460acctttcata gaaggcggcg gtggaatcga
aatctcgtga tggcaggttg ggcgtcgctt 2520ggtcggtcat ttcgaacccc agagtcccgc
tcagaagaac tcgtcaagaa ggcgatagaa 2580ggcgatgcgc tgcgaatcgg gagcggcgat
accgtaaagc acgaggaagc ggtcagccca 2640ttcgccgcca agctcttcag caatatcacg
ggtagccaac gctatgtcct gatagcggtc 2700cgccacaccc agccggccac agtcgatgaa
tccagaaaag cggccatttt ccaccatgat 2760attcggcaag caggcatcgc catgggtcac
gacgagatcc tcgccgtcgg gcatgccccc 2820caattcactg gccgtcgttt tacaacgtcg
tgactgggaa aaccctggcg ttacccaact 2880taatcgcctt gcagcacatc cccctttcgc
cagctggcgt aatagcgaag aggcccgcac 2940cgatcgccct tcccaacagt tgcgcagcct
gaatggcgaa tggcgcctga tgcggtattt 3000tctccttacg catctgtgcg gtatttcaca
ccgcatatgg tgcactctca gtacaatctg 3060ctctgatgcc gcatagttaa gccagccccg
acacccgcca acacccgctg acgcgccctg 3120acgggcttgt ctgctcccgg catccgctta
cagacaagct gtgaccgtct ccgggagctg 3180catgtgtcag aggttttcac cgtcatcacc
gaaacgcgcg agacgaaagg gcctcgtgat 3240acgcctattt ttataggtta atgtcatgat
aataatggtt tcttagacgt caggtggcac 3300ttttcgggga aatgtgcgcg gaacccctat
ttgtttattt ttctaaatac attcaaatat 3360gtatccgctc atgagacaat aaccctgata
aatgcttcaa taatattgaa aaaggaagag 3420tatgagtatt caacatttcc gtgtcgccct
tattcccttt tttgcggcat tttgccttcc 3480tgtttttgct cacccagaaa cgctggtgaa
agtaaaagat gctgaagatc agttgggtgc 3540acgagtgggt tacatcgaac tggatctcaa
cagcggtaag atccttgaga gttttcgccc 3600cgaagaacgt tttccaatga tgagcacttt
taaagttctg ctatgtggcg cggtattatc 3660ccgtattgac gccgggcaag agcaactcgg
tcgccgcata cactattctc agaatgactt 3720ggttgagtac tcaccagtca cagaaaagca
tcttacggat ggcatgacag taagagaatt 3780atgcagtgct gccataacca tgagtgataa
cactgcggcc aacttacttc tgacaacgat 3840cggaggaccg aaggagctaa ccgctttttt
gcacaacatg ggggatcatg taactcgcct 3900tgatcgttgg gaaccggagc tgaatgaagc
cataccaaac gacgagcgtg acaccacgat 3960gcctgtagca atggcaacaa cgttgcgcaa
actattaact ggcgaactac ttactctagc 4020ttcccggcaa caattaatag actggatgga
ggcggataaa gttgcaggac cacttctgcg 4080ctcggccctt ccggctggct ggtttattgc
tgataaatct ggagccggtg agcgtgggtc 4140tcgcggtatc attgcagcac tggggccaga
tggtaagccc tcccgtatcg tagttatcta 4200cacgacgggg agtcaggcaa ctatggatga
acgaaataga cagatcgctg agataggtgc 4260ctcactgatt aagcattggt aactgtcaga
ccaagtttac tcatatatac tttagattga 4320tttaaaactt catttttaat ttaaaaggat
ctaggtgaag atcctttttg ataatctcat 4380gaccaaaatc ccttaacgtg agttttcgtt
ccactgagcg tcagaccccg tagaaaagat 4440caaaggatct tcttgagatc ctttttttct
gcgcgtaatc tgctgcttgc aaacaaaaaa 4500accaccgcta ccagcggtgg tttgtttgcc
ggatcaagag ctaccaactc tttttccgaa 4560ggtaactggc ttcagcagag cgcagatacc
aaatactgtc cttctagtgt agccgtagtt 4620aggccaccac ttcaagaact ctgtagcacc
gcctacatac ctcgctctgc taatcctgtt 4680accagtggct gctgccagtg gcgataagtc
gtgtcttacc gggttggact caagacgata 4740gttaccggat aaggcgcagc ggtcgggctg
aacggggggt tcgtgcacac agcccagctt 4800ggagcgaacg acctacaccg aactgagata
cctacagcgt gagcattgag aaagcgccac 4860gcttcccgaa gggagaaagg cggacaggta
tccggtaagc ggcagggtcg gaacaggaga 4920gcgcacgagg gagcttccag ggggaaacgc
ctggtatctt tatagtcctg tcgggtttcg 4980ccacctctga cttgagcgtc gatttttgtg
atgctcgtca ggggggcgga gcctatggaa 5040aaacgccagc aacgcggcct ttttacggtt
cctggccttt tgctggcctt ttgctcacat 5100gttctttcct gcgttatccc ctgattctgt
ggataaccgt attaccgcct ttgagtgagc 5160tgataccgct cgccgcagcc gaacgaccga
gcgcagcgag tcagtgagcg aggaagcgga 5220agagcgccca atacgcaaac cgcctctccc
cgcgcgttgg ccgattcatt aatgcagctg 5280gcacgacagg tttcccgact ggaaagcggg
cagtgagcgc aacgcaatta atgtgagtta 5340gctcactcat taggcacccc aggctttaca
ctttatgctt ccggctcgta tgttgtgtgg 5400aattgtgagc ggataacaat ttcacacagg
aaacagctat gaccatgatt acgccaagct 5460ttctaggggg ggggtaccga tctgagatcg
gtaacgaaaa cgaacgggta gggatgaaaa 5520cggtcggtaa cggtcggtaa aatacctcta
ccgttttcat tttcatattt aacttgcggg 5580acggaaacga aaacgggata taccggtaac
gaaaacgaac gggataaata cggtaatcga 5640aaaccgatac gatccggtcg ggttaaagtc
gaaatcggac gggaaccggt atttttgttc 5700ggtaaaatca cacatgaaaa catatattca
aaacttaaaa acaaatataa aaaattgtaa 5760acacaagtct taatgatcac tagtggcgcg
cctaggagat ctcgagtagg gataacaggg 5820taatacatag ataaaatcca tataaatctg
gagcacacat agtttaatgt agcacataag 5880tgataagtct tgggctcttg gctaacataa
gaagccatat aagtctacta gcacacatga 5940cacaatataa agtttaaaac acatattcat
aatcacttgc tcacatctgg atcacttagc 6000atgctacagc tagtgcaata ttagacactt
tccaatattt ctcaaacttt tcactcattg 6060caacggccat tctcctaatg acaaattttt
catgaacaca ccattggtca atcaaatcct 6120ttatctcaca gaaacctttg taaaataaat
ttgcagtgga atattgagta ccagatagga 6180gttcagtgag atcaaaaaac ttcttcaaac
acttaaaaag agttaatgcc atcttccact 6240cctcggcttt aggacaaatt gcatcgtacc
tacaataatt gacatttgat taattgagaa 6300tttataatga tgacatgtac aacaattgag
acaaacatac ctgcgaggat cacttgtttt 6360aagccgtgtt agtgcaggct tataatataa
ggcatccctc aacatcaaat aggttgaatt 6420ccatctagtt gagacatcat atgagatccc
tttagattta tccaagtcac attcactagc 6480acacttcatt agttcttccc actgcaaagg
agaagatttt acagcaagaa caatcgcttt 6540gattttctca attgttcctg caattacagc
caagccatcc tttgcaacca agttcagtat 6600gtgacaagca cacctcacat gaaagaaagc
accatcacaa actagatttg aatcagtgtc 6660ctgcaaatcc tcaattatat cgtgcacagc
tacttcattt gcactagcat tatccaaaga 6720caaggcaaac aattttttct caatgttcca
cttaaccatg attgcagtga aggtttgtga 6780taacctttgg ccagtgtggc gcccttcaac
atgaaaaaag ccaacaattc ttttttggag 6840acaccaatca tcatcaatcc aatggatggt
gacacacatg tatgacttat tttgacaaga 6900tgtccacata tccatagttg tactgaagcg
agactgaaca tcttttagtt ttccatacaa 6960cttttctttt tcttccaaat acaaatccat
gatatatttt ctagcagtga cacgggactt 7020tattggaaag tgagggcgca gagacttaac
aaactcaaca aagtactcat gttctacaat 7080attgaaagga tattcatgca tgattattgc
caaatgaagc ttctttaggc taaccacttc 7140atcgtactta taaggctcaa tgagatttat
gtctttgcca tgatcctttt cactttttag 7200acacaactga cctttaacta aactatgtga
tgttctcaag tgatttcgaa atccgcttgt 7260tccatgatga ccctcagccc tatacttagc
cttgcaatta ggaaagttgc aatgtcccca 7320tacctgaacg tatttctttc catcgacctc
cacttcaatt tccttcttgg tgaaatgctg 7380ccatacatcc gatgtgcact tctttgccct
cttctgtggt gcttcttctt cgggttcagg 7440ttgtggctgt ggttgtggtt ctggttgtgg
ttgtggttgt ggttgtggtt catgaacaat 7500agccatatca tcttgactcg gatctgtagc
tgtaccattt gcattactac tgcttacact 7560ctgaataaaa tgcctctcgg cctcagctgt
tgatgatgat ggtgatgtgc ggccacatcc 7620atgcccacgc gcacgtgcac gtacattctg
aatccgacta gaagaggctt cagcttttct 7680tttcaaccct gttataaaca gatttttcgt
attattctac agtcaatatg atgcttccca 7740atctacaacc aattagtaat gctaatgcta
ttgctactgt ttttctaata tataccttga 7800gcatatgcag agaatacgga atttgttttg
cgagtagaag gcgctcttgt ggtagacatc 7860aacttggcca atcttatggc tgagcctgag
ggaggattat ttccaaccgg aggcgtcatc 7920tgaggaatgg agtcgtagcc ggctagccga
agtggagagc agagccctgg acagcaggtg 7980ttcagcaatc agcttggtgc tgtactgctg
tgacttgtga gcacctggac ggctggacag 8040caatcagcag gtgttgcaga gcccctggac
agcacacaaa tgacacaaca gcttggtgca 8100atggtgctga cgtgctgtac tgctaagtgc
tgtgagcctg tgagcagccg tggagacagg 8160gagaccgcgg atggccggat gggcgagcgc
cgagcagtgg aggtctggag gaccgctgac 8220cgcagatggc ggatggcgga tgggcggacc
gcggatgggc gagcagtgga gtggaggtct 8280gggcggatgg gcggaccgcg gcgcggatgg
gcgagtcgcg agcagtggag tggagggcgg 8340accgtggatg gcggcgtctg cgtccggcgt
gccgcgtcac ggccgtcacc gcgtgtggtg 8400cctggtgcag cccagcggcc ggccggctgg
gagacaggga gagtcggaga gagcaggcga 8460gagcgagacg cgtcgccggc gtcggcgtgc
ggctggcggc gtccggactc cggcgtgggc 8520gcgtggcggc gtgtgaatgt gtgatgctgt
tactcgtgtg gtgcctggcc gcctgggaga 8580gaggcagagc agcgttcgct aggtatttct
tacatgggct gggcctcagt ggttatggat 8640gggagttgga gctggccata ttgcagtcat
cccgaattag aaaatacggt aacgaaacgg 8700gatcatcccg attaaaaacg ggatcccggt
gaaacggtcg ggaaactagc tctaccgttt 8760ccgtttccgt ttaccgtttt gtatatcccg
tttccgttcc gttttcgttt tttacctcgg 8820gttcgaaatc gatcgggata aaactaacaa
aatcggttat acgataacgg tcggtacggg 8880attttcccat cctactttca tccctgagat
tattgtcgtt tctttcgcag atcggtaccc 8940cccccctaga gtcgacatcg atctagtaac
atagatgaca ccgcgcgcga taatttatcc 9000tagtttgcgc gctatatttt gttttctatc
gcgtattaaa tgtataattg cgggactcta 9060atcataaaaa cccatctcat aaataacgtc
atgcattaca tgttaattat tacatgctta 9120acgtaattca acagaaatta tatgataatc
atcgcaagac cggcaacagg attcaatctt 9180aagaaacttt attgccaaat gtttgaacga
tctgcttcga cgcactcctt ctttaggtac 9240ggactagatc tcggtgacgg gcaggaccgg
acggggcggt accggcaggc tgaagtccag 9300ctgccagaaa cccacgtcat gccagttccc
gtgcttgaag ccggccgccc gcagcatgcc 9360gcggggggca tatccgagcg cctcgtgcat
gcgcacgctc gggtcgttgg gcagcccgat 9420gacagcgacc acgctcttga agccctgtgc
ctccagggac ttcagcaggt gggtgtagag 9480cgtggagccc agtcccgtcc gctggtggcg
gggggagacg tacacggtcg actcggccgt 9540ccagtcgtag gcgttgcgtg ccttccaggg
gcccgcgtag gcgatgccgg cgacctcgcc 9600gtccacctcg gcgacgagcc agggatagcg
ctcccgcaga cggacgaggt cgtccgtcca 9660ctcctgcggt tcctgcggct cggtacggaa
gttgaccgtg cttgtctcga tgtagtggtt 9720gacgatggtg cagaccgccg gcatgtccgc
ctcggtggca cggcggatgt cggccgggcg 9780tcgttctggg ctcatggatc tggattgaga
gtgaatatga gactctaatt ggataccgag 9840gggaatttat ggaacgtcag tggagcattt
ttgacaagaa atatttgcta gctgatagtg 9900accttaggcg acttttgaac gcgcaataat
ggtttctgac gtatgtgctt agctcattaa 9960actccagaaa cccgcggctg agtggctcct
tcaatcgttg cggttctgtc agttccaaac 10020gtaaaacggc ttgtcccgcg tcatcggcgg
gggtcataac gtgactccct taattctccg 10080ctcatgatcc ccgggtaccg agctcgaatt
gcggctgagt ggctccttca atcgttgcgg 10140ttctgtcagt tccaaacgta aaacggcttg
tcccgcgtca tcggcggggg tcataacgtg 10200actcccttaa ttctccgctc atgatcttga
tcccctgcgc catcagatcc ttggcggcaa 10260gaaagccatc cagtttactt tgcagggctt
cccaacctta ccagagggcg ccccagctgg 10320caattccggt tcgcttgctg tatcgatatg
gtggatttat cacaaatggg acccgccgcc 10380gacagaggtg tgatgttagg ccaggacttt
gaaaatttgc gcaactatcg tatagtggcc 10440gacaaattga cgccgagttg acagactgcc
tagcatttga gtgaattatg tgaggtaatg 10500ggctacactg aattggtagc tcaaactgtc
agtatttatg tatatgagtg tatattttcg 10560cataatctca gaccaatctg aagatgaaat
gggtatctgg gaatggcgaa atcaaggcat 10620cgatcgtgaa gtttctcatc taagccccca
tttggacgtg aatgtagaca cgtcgaaata 10680aagatttccg aattagaata atttgtttat
tgctttcgcc tataaatacg acggatcgta 10740atttgtcgtt ttatcaaaat gtactttcat
tttataataa cgctgcggac atctacattt 10800ttgaattgaa aaaaaattgg taattactct
ttctttttct ccatattgac catcatactc 10860attgctgatc catgtagatt tcccggacat
gaagccattt acaattgaat atatcctgcc 10920gccgctgccg ctttgcaccc ggtggagctt
gcatgttggt ttctacgcag aactgagccg 10980gttaggcaga taatttccat tgagaactga
gccatgtgca ccttcccccc aacacggtga 11040gcgacggggc aacggagtga tccacatggg
acttttaaac atcatccgtc ggatggcgtt 11100gcgagagaag cagtcgatcc gtgagatcag
ccgacgcacc gggcaggcgc gcaacacgat 11160cgcaaagtat ttgaacgcag gtacaatcga
gccgacgttc accgtcaccc tggatgctgt 11220aggcataggc ttggttatgc cggtactgcc
gggcctcttg cgggatatcg tccattccga 11280cagcatcgcc agtcactatg gcgtgctgct
agcgctatat gcgttgatgc aatttctatg 11340cgcacccgtt ctcggagcac tgtccgaccg
ctttggccgc cgcccagtcc tgctcgcttc 11400gctacttgga gccactatcg actacgcgat
catggcgacc acacccgtcc tgtggtccaa 11460cccctccgct gctatagtgc agtcggcttc
tgacgttcag tgcagccgtc ttctgaaaac 11520gacatgtcgc acaagtccta agttacgcga
caggctgccg ccctgccctt ttcctggcgt 11580tttcttgtcg cgtgttttag tcgcataaag
tagaatactt gcgactagaa ccggagacat 11640tacgccatga acaagagcgc cgccgctggc
ctgctgggct atgcccgcgt cagcaccgac 11700gaccaggact tgaccaacca acgggccgaa
ctgcacgcgg ccggctgcac caagctgttt 11760tccgagaaga tcaccggcac caggcgcgac
cgcccggagc tggccaggat gcttgaccac 11820ctacgccctg gcgacgttgt gacagtgacc
aggctagacc gcctggcccg cagcacccgc 11880gacctactgg acattgccga gcgcatccag
gaggccggcg cgggcctgcg tagcctggca 11940gagccgtggg ccgacaccac cacgccggcc
ggccgcatgg tgttgaccgt gttcgccggc 12000attgccgagt tcgagcgttc cctaatcatc
gaccgcaccc ggagcgggcg cgaggccgcc 12060aaggcccgag gcgtgaagtt tggcccccgc
cctaccctca ccccggcaca gatcgcgcac 12120gcccgcgagc tgatcgacca ggaaggccgc
accgtgaaag aggcggctgc actgcttggc 12180gtgcatcgct cgaccctgta ccgcgcactt
gagcgcagcg aggaagtgac gcccaccgag 12240gccaggcggc gcggtgcctt ccgtgaggac
gcattgaccg aggccgacgc cctggcggcc 12300gccgagaatg aacgccaaga ggaacaagca
tgaaaccgca ccaggacggc caggacgaac 12360cgtttttcat taccgaagag atcgaggcgg
agatgatcgc ggccgggtac gtgttcgagc 12420cgcccgcgca cgtctcaacc gtgcggctgc
atgaaatcct ggccggtttg tctgatgcca 12480agctggcggc ctggccggcc agcttggccg
ctgaagaaac cgagcgccgc cgtctaaaaa 12540ggtgatgtgt atttgagtaa aacagcttgc
gtcatgcggt cgctgcgtat atgatgcgat 12600gagtaaataa acaaatacgc aagggaacgc
atgaagttat cgctgtactt aaccagaaag 12660gcgggtcagg caagacgacc atcgcaaccc
atctagcccg cgccctgcaa ctcgccgggg 12720ccgatgttct gttagtcgat tccgatcccc
agggcagtgc ccgcgattgg gcggccgtgc 12780gggaagatca accgctaacc gttgtcggca
tcgaccgccc gacgattgac cgcgacgtga 12840aggccatcgg ccggcgcgac ttcgtagtga
tcgacggagc gccccaggcg gcggacttgg 12900ctgtgtccgc gatcaaggca gccgacttcg
tgctgattcc ggtgcagcca agcccttacg 12960acatatgggc caccgccgac ctggtggagc
tggttaagca gcgcattgag gtcacggatg 13020gaaggctaca agcggccttt gtcgtgtcgc
gggcgatcaa aggcacgcgc atcggcggtg 13080aggttgccga ggcgctggcc gggtacgagc
tgcccattct tgagtcccgt atcacgcagc 13140gcgtgagcta cccaggcact gccgccgccg
gcacaaccgt tcttgaatca gaacccgagg 13200gcgacgctgc ccgcgaggtc caggcgctgg
ccgctgaaat taaatcaaaa ctcatttgag 13260ttaatgaggt aaagagaaaa tgagcaaaag
cacaaacacg ctaagtgccg gccgtccgag 13320cgcacgcagc agcaaggctg caacgttggc
cagcctggca gacacgccag ccatgaagcg 13380ggtcaacttt cagttgccgg cggaggatca
caccaagctg aagatgtacg cggtacgcca 13440aggcaagacc attaccgagc tgctatctga
atacatcgcg cagctaccag agtaaatgag 13500caaatgaata aatgagtaga tgaattttag
cggctaaagg aggcggcatg gaaaatcaag 13560aacaaccagg caccgacgcc gtggaatgcc
ccatgtgtgg aggaacgggc ggttggccag 13620gcgtaagcgg ctgggttgtc tgccggccct
gcaatggcac tggaaccccc aagcccgagg 13680aatcggcgtg agcggtcgca aaccatccgg
cccggtacaa atcggcgcgg cgctgggtga 13740tgacctggtg gagaagttga aggccgcgca
ggccgcccag cggcaacgca tcgaggcaga 13800agcacgcccc ggtgaatcgt ggcaagcggc
cgctgatcga atccgcaaag aatcccggca 13860accgccggca gccggtgcgc cgtcgattag
gaagccgccc aagggcgacg agcaaccaga 13920ttttttcgtt ccgatgctct atgacgtggg
cacccgcgat agtcgcagca tcatggacgt 13980ggccgttttc cgtctgtcga agcgtgaccg
acgagctggc gaggtgatcc gctacgagct 14040tccagacggg cacgtagagg tttccgcagg
gccggccggc atggccagtg tgtgggatta 14100cgacctggta ctgatggcgg tttcccatct
aaccgaatcc atgaaccgat accgggaagg 14160gaagggagac aagcccggcc gcgtgttccg
tccacacgtt gcggacgtac tcaagttctg 14220ccggcgagcc gatggcggaa agcagaaaga
cgacctggta gaaacctgca ttcggttaaa 14280caccacgcac gttgccatgc agcgtacgaa
gaaggccaag aacggccgcc tggtgacggt 14340atccgagggt gaagccttga ttagccgcta
caagatcgta aagagcgaaa ccgggcggcc 14400ggagtacatc gagatcgagc tagctgattg
gatgtaccgc gagatcacag aaggcaagaa 14460cccggacgtg ctgacggttc accccgatta
ctttttgatc gatcccggca tcggccgttt 14520tctctaccgc ctggcacgcc gcgccgcagg
caaggcagaa gccagatggt tgttcaagac 14580gatctacgaa cgcagtggca gcgccggaga
gttcaagaag ttctgtttca ccgtgcgcaa 14640gctgatcggg tcaaatgacc tgccggagta
cgatttgaag gaggaggcgg ggcaggctgg 14700cccgatccta gtcatgcgct accgcaacct
gatcgagggc gaagcatccg ccggttccta 14760atgtacggag cagatgctag ggcaaattgc
cctagcaggg gaaaaaggtc gaaaaggtct 14820ctttcctgtg gatagcacgt acattgggaa
cccaaagccg tacattggga accggaaccc 14880gtacattggg aacccaaagc cgtacattgg
gaaccggtca cacatgtaag tgactgatat 14940aaaagagaaa aaaggcgatt tttccgccta
aaactcttta aaacttatta aaactcttaa 15000aacccgcctg gcctgtgcat aactgtctgg
ccagcgcaca gccgaagagc tgcaaaaagc 15060gcctaccctt cggtcgctgc gctccctacg
ccccgccgct tcgcgtcggc ctatcgcggc 15120cgctggccgc tcaaaaatgg ctggcctacg
gccaggcaat ctaccagggc gcggacaagc 15180cgcgccgtcg ccactcgacc gccggcgccc
acatcaaggc accctgcctc gcgcgtttcg 15240gtgatgacgg tgaaaacctc tgacacatgc
agctcccgga gacggtcaca gcttgtctgt 15300aagcggatgc cgggagcaga caagcccgtc
agggcgcgtc agcgggtgtt ggcgggtgtc 15360ggggcgcagc catgacccag tcacgtagcg
atagcggagt gtatactggc ttaactatgc 15420ggcatcagag cagattgtac tgagagtgca
ccatatgcgg tgtgaaatac cgcacagatg 15480cgtaaggaga aaataccgca tcaggcgctc
ttccgcttcc tcgctcactg actcgctgcg 15540ctcggtcgtt cggctgcggc gagcggtatc
agctcactca aaggcggtaa tacggttatc 15600cacagaatca ggggataacg caggaaagaa
catgtgagca aaaggccagc aaaaggccag 15660gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg ctccgccccc ctgacgagca 15720tcacaaaaat cgacgctcaa gtcagaggtg
gcgaaacccg acaggactat aaagatacca 15780ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt ccgaccctgc cgcttaccgg 15840atacctgtcc gcctttctcc cttcgggaag
cgtggcgctt tctcatagct cacgctgtag 15900gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc tgtgtgcacg aaccccccgt 15960tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt gagtccaacc cggtaagaca 16020cgacttatcg ccactggcag cagccactgg
taacaggatt agcagagcga ggtatgtagg 16080cggtgctaca gagttcttga agtggtggcc
taactacggc tacactagaa ggacagtatt 16140tggtatctgc gctctgctga agccagttac
cttcggaaaa agagttggta gctcttgatc 16200cggcaaacaa accaccgctg gtagcggtgg
tttttttgtt tgcaagcagc agattacgcg 16260cagaaaaaaa ggatctcaag aagatccttt
gatcttttct acggggtctg acgctcagtg 16320gaacgaaaac tcacgttaag ggattttggt
catgagatta tcaaaaagga tcttcaccta 16380gatcctttta aattaaaaat gaagttttaa
atcaatctaa agtatatatg agtaaacttg 16440gtctgacagt taccaatgct taatcagtga
ggcacctatc tcagcgatct gtctatttcg 16500ttcatccata gttgcctgac tccccgtcgt
gtagataact acgatacggg agggcttacc 16560atctggcccc agtgctgcaa tgataccgcg
agacccacgc tcaccggctc cagatttatc 16620agcaataaac cagccagccg gaagggccga
gcgcagaagt ggtcctgcaa ctttatccgc 16680ctccatccag tctattaatt gttgccggga
agctagagta agtagttcgc cagttaatag 16740tttgcgcaac gttgttgcca ttgctacagg
catcgtggtg tcacgctcgt cgtttggtat 16800ggcttcattc agctccggtt cccaacgatc
aaggcgagtt acatgatccc ccatgttgtg 16860caaaaaagcg gttagctcct tcggtcctcc
gatcgttgtc agaagtaagt tggccgcagt 16920gttatcactc atggttatgg cagcactgca
taattctctt actgtcatgc catccgtaag 16980atgcttttct gtgactggtg agtactcaac
caagtcattc tgagaatagt gtatgcggcg 17040accgagttgc tcttgcccgg cgtcaacacg
ggataatacc gcgccacata gcagaacttt 17100aaaagtgctc atcattggaa aagacctgca
gggggggggg ggaaagccac gttgtgtctc 17160aaaatctctg atgttacatt gcacaagata
aaaatatatc atcatgaaca ataaaactgt 17220ctgcttacat aaacagtaat acaaggggtg
ttatgagcca tattcaacgg gaaacgtctt 17280gctcgaggcc gcgattaaat tccaacatgg
atgctgattt atatgggtat aaatgggctc 17340gcgataatgt cgggcaatca ggtgcgacaa
tctatcgatt gtatgggaag cccgatgcgc 17400cagagttgtt tctgaaacat ggcaaaggta
gcgttgccaa tgatgttaca gatgagatgg 17460tcagactaaa ctggctgacg gaatttatgc
ctcttccgac catcaagcat tttatccgta 17520ctcctgatga tgcatggtta ctcaccactg
cgatccccgg gaaaacagca ttccaggtat 17580tagaagaata tcctgattca ggtgaaaata
ttgttgatgc gctggcagtg ttcctgcgcc 17640ggttgcattc gattcctgtt tgtaattgtc
cttttaacag cgatcgcgta tttcgtctcg 17700ctcaggcgca atcacgaatg aataacggtt
tggttgatgc gagtgatttt gatgacgagc 17760gtaatggctg gcctgttgaa caagtctgga
aagaaatgca taagcttttg ccattctcac 17820cggattcagt cgtcactcat ggtgatttct
cacttgataa ccttattttt gacgagggga 17880aattaatagg ttgtattgat gttggacgag
tcggaatcgc agaccgatac caggatcttg 17940ccatcctatg gaactgcctc ggtgagtttt
ctccttcatt acagaaacgg ctttttcaaa 18000aatatggtat tgataatcct gatatgaata
aattgcagtt tcatttgatg ctcgatgagt 18060ttttctaatc agaattggtt aattggttgt
aacactggca gagcattacg ctgacttgac 18120gggacggcgg ctttgttgaa taaatcgaac
ttttgctgag ttgaaggatc agatcacgca 18180tcttcccgac aacgcagacc gttccgtggc
aaagcaaaag ttcaaaatca ccaactggtc 18240cacctacaac aaagctctca tcaaccgtgg
ctccctcact ttctggctgg atgatggggc 18300gattcaggcc tggtatgagt cagcaacacc
ttcttcacga ggcagacctc agcgcccccc 18360cccccctgca ggtcaattcg gtcgatatgg
ctattacgaa gaaggctcgt gcgcggagtc 18420ccgtgaactt tcccacgcaa caagtgaacc
gcaccgggtt tgccggaggc catttcgtta 18480aaatgcgcag c
1849124291DNAartificial sequencevector
2ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
60taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
120gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
180cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
240tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
300gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
360acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
420caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
480gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
540aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
600ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
660agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa
720aaaacagact acataatact gtaaaacaca acatatccag tcactatgaa tcaactactt
780agatggtatt agtgacctgt agtcgaccga cagccttcca aatgttcttc gggtgatgct
840gccaacttag tcgaccgaca gccttccaaa tgttcttctc aaacggaatc gtcgtatcca
900gcctactcgc tattgtcctc aatgccgtat taaatcataa aaagaaataa gaaaaagagg
960tgcgagcctc ttttttgtgt gacaaaataa aaacatctac ctattcatat acgctagtgt
1020catagtcctg aaaatcatct gcatcaagaa caatttcaca actcttatac ttttctctta
1080caagtcgttc ggcttcatct ggattttcag cctctatact tactaaacgt gataaagttt
1140ctgtaatttc tactgtatcg acctgcagac tggctgtgta taagggagcc tgacatttat
1200attccccaga acatcaggtt aatggcgttt ttgatgtcat tttcgcggtg gctgagatca
1260gccacttctt ccccgataac ggagaccggc acactggcca tatcggtggt catcatgcgc
1320cagctttcat ccccgatatg caccaccggg taaagttcac gggagacttt atctgacagc
1380agacgtgcac tggccagggg gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc
1440tgtacatcca caaacagacg ataacggctc tctcttttat aggtgtaaac cttaaactgc
1500atttcaccag cccctgttct cgtcagcaaa agagccgttc atttcaataa accgggcgac
1560ctcagccatc ccttcctgat tttccgcttt ccagcgttcg gcacgcagac gacgggcttc
1620attctgcatg gttgtgctta ccagaccgga gatattgaca tcatatatgc cttgagcaac
1680tgatagctgt cgctgtcaac tgtcactgta atacgctgct tcatagcata cctctttttg
1740acatacttcg ggtatacata tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat
1800acgcatactg ttatctggct tttagtaagc cggatccacg cggcgtttac gccccgccct
1860gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac
1920agacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat
1980atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa
2040aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt
2100tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa
2160actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat
2220ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg
2280ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat
2340aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg
2400tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc
2460attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag
2520ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt
2580gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccagg
2640gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca
2700caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttagtc gactacaggt
2760cactaatacc atctaagtag ttgattcata gtgactggat atgttgtgtt ttacagtatt
2820atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
2880tttctcgttc agctttcttg tacaaagttg gcattataag aaagcattgc ttatcaattt
2940gttgcaacga acaggtcact atcagtcaaa ataaaatcat tatttgccat ccagctgata
3000tcccctatag tgagtcgtat tacatggtca tagctgtttc ctggcagctc tggcccgtgt
3060ctcaaaatct ctgatgttac attgcacaag ataaaataat atcatcatga tcagtcctgc
3120tcctcggcca cgaagtgcac gcagttgccg gccgggtcgc gcagggcgaa ctcccgcccc
3180cacggctgct cgccgatctc ggtcatggcc ggcccggagg cgtcccggaa gttcgtggac
3240acgacctccg accactcggc gtacagctcg tccaggccgc gcacccacac ccaggccagg
3300gtgttgtccg gcaccacctg gtcctggacc gcgctgatga acagggtcac gtcgtcccgg
3360accacaccgg cgaagtcgtc ctccacgaag tcccgggaga acccgagccg gtcggtccag
3420aactcgaccg ctccggcgac gtcgcgcgcg gtgagcaccg gaacggcact ggtcaacttg
3480gccatggttt agttcctcac cttgtcgtat tatactatgc cgatatacta tgccgatgat
3540taattgtcaa cacgtgctga tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc
3600actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
3660gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
3720atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
3780atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
3840ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
3900gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
3960cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
4020tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
4080cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
4140ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
4200gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
4260tggccttttg ctggcctttt gctcacatgt t
429134762DNAartificial sequencevector 3ctttcctgcg ttatcccctg attctgtgga
taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg
cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc
gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag
tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa
aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt
cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt
gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc
gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc
atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggcccca
aataatgatt ttattttgac tgatagtgac 600ctgttcgttg caacacattg atgagcaatg
cttttttata atgccaactt tgtacaaaaa 660agctgaacga gaaacgtaaa atgatataaa
tatcaatata ttaaattaga ttttgcataa 720aaaacagact acataatact gtaaaacaca
acatatccag tcactatgaa tcaactactt 780agatggtatt agtgacctgt agtcgaccga
cagccttcca aatgttcttc gggtgatgct 840gccaacttag tcgaccgaca gccttccaaa
tgttcttctc aaacggaatc gtcgtatcca 900gcctactcgc tattgtcctc aatgccgtat
taaatcataa aaagaaataa gaaaaagagg 960tgcgagcctc ttttttgtgt gacaaaataa
aaacatctac ctattcatat acgctagtgt 1020catagtcctg aaaatcatct gcatcaagaa
caatttcaca actcttatac ttttctctta 1080caagtcgttc ggcttcatct ggattttcag
cctctatact tactaaacgt gataaagttt 1140ctgtaatttc tactgtatcg acctgcagac
tggctgtgta taagggagcc tgacatttat 1200attccccaga acatcaggtt aatggcgttt
ttgatgtcat tttcgcggtg gctgagatca 1260gccacttctt ccccgataac ggagaccggc
acactggcca tatcggtggt catcatgcgc 1320cagctttcat ccccgatatg caccaccggg
taaagttcac gggagacttt atctgacagc 1380agacgtgcac tggccagggg gatcaccatc
cgtcgcccgg gcgtgtcaat aatatcactc 1440tgtacatcca caaacagacg ataacggctc
tctcttttat aggtgtaaac cttaaactgc 1500atttcaccag cccctgttct cgtcagcaaa
agagccgttc atttcaataa accgggcgac 1560ctcagccatc ccttcctgat tttccgcttt
ccagcgttcg gcacgcagac gacgggcttc 1620attctgcatg gttgtgctta ccagaccgga
gatattgaca tcatatatgc cttgagcaac 1680tgatagctgt cgctgtcaac tgtcactgta
atacgctgct tcatagcata cctctttttg 1740acatacttcg ggtatacata tcagtatata
ttcttatacc gcaaaaatca gcgcgcaaat 1800acgcatactg ttatctggct tttagtaagc
cggatccacg cggcgtttac gccccgccct 1860gccactcatc gcagtactgt tgtaattcat
taagcattct gccgacatgg aagccatcac 1920agacggcatg atgaacctga atcgccagcg
gcatcagcac cttgtcgcct tgcgtataat 1980atttgcccat ggtgaaaacg ggggcgaaga
agttgtccat attggccacg tttaaatcaa 2040aactggtgaa actcacccag ggattggctg
agacgaaaaa catattctca ataaaccctt 2100tagggaaata ggccaggttt tcaccgtaac
acgccacatc ttgcgaatat atgtgtagaa 2160actgccggaa atcgtcgtgg tattcactcc
agagcgatga aaacgtttca gtttgctcat 2220ggaaaacggt gtaacaaggg tgaacactat
cccatatcac cagctcaccg tctttcattg 2280ccatacggaa ttccggatga gcattcatca
ggcgggcaag aatgtgaata aaggccggat 2340aaaacttgtg cttatttttc tttacggtct
ttaaaaaggc cgtaatatcc agctgaacgg 2400tctggttata ggtacattga gcaactgact
gaaatgcctc aaaatgttct ttacgatgcc 2460attgggatat atcaacggtg gtatatccag
tgattttttt ctccatttta gcttccttag 2520ctcctgaaaa tctcgataac tcaaaaaata
cgcccggtag tgatcttatt tcattatggt 2580gaaagttgga acctcttacg tgccgatcaa
cgtctcattt tcgccaaaag ttggcccagg 2640gcttcccggt atcaacaggg acaccaggat
ttatttattc tgcgaagtga tcttccgtca 2700caggtattta ttcggcgcaa agtgcgtcgg
gtgatgctgc caacttagtc gactacaggt 2760cactaatacc atctaagtag ttgattcata
gtgactggat atgttgtgtt ttacagtatt 2820atgtagtctg ttttttatgc aaaatctaat
ttaatatatt gatatttata tcattttacg 2880tttctcgttc agctttcttg tacaaagttg
gcattataag aaagcattgc ttatcaattt 2940gttgcaacga acaggtcact atcagtcaaa
ataaaatcat tatttgccat ccagctgata 3000tcccctatag tgagtcgtat tacatggtca
tagctgtttc ctggcagctc tggcccgtgt 3060ctcaaaatct ctgatgttac attgcacaag
ataaaataat atcatcatga acaataaaac 3120tgtctgctta cataaacagt aatacaaggg
gtgttatgag ccatattcaa cgggaaacgt 3180cgaggccgcg attaaattcc aacatggatg
ctgatttata tgggtataaa tgggctcgcg 3240ataatgtcgg gcaatcaggt gcgacaatct
atcgcttgta tgggaagccc gatgcgccag 3300agttgtttct gaaacatggc aaaggtagcg
ttgccaatga tgttacagat gagatggtca 3360gactaaactg gctgacggaa tttatgcctc
ttccgaccat caagcatttt atccgtactc 3420ctgatgatgc atggttactc accactgcga
tccccggaaa aacagcattc caggtattag 3480aagaatatcc tgattcaggt gaaaatattg
ttgatgcgct ggcagtgttc ctgcgccggt 3540tgcattcgat tcctgtttgt aattgtcctt
ttaacagcga tcgcgtattt cgtctcgctc 3600aggcgcaatc acgaatgaat aacggtttgg
ttgatgcgag tgattttgat gacgagcgta 3660atggctggcc tgttgaacaa gtctggaaag
aaatgcataa acttttgcca ttctcaccgg 3720attcagtcgt cactcatggt gatttctcac
ttgataacct tatttttgac gaggggaaat 3780taataggttg tattgatgtt ggacgagtcg
gaatcgcaga ccgataccag gatcttgcca 3840tcctatggaa ctgcctcggt gagttttctc
cttcattaca gaaacggctt tttcaaaaat 3900atggtattga taatcctgat atgaataaat
tgcagtttca tttgatgctc gatgagtttt 3960tctaatcaga attggttaat tggttgtaac
actggcagag cattacgctg acttgacggg 4020acggcgcaag ctcatgacca aaatccctta
acgtgagtta cgcgtcgttc cactgagcgt 4080cagaccccgt agaaaagatc aaaggatctt
cttgagatcc tttttttctg cgcgtaatct 4140gctgcttgca aacaaaaaaa ccaccgctac
cagcggtggt ttgtttgccg gatcaagagc 4200taccaactct ttttccgaag gtaactggct
tcagcagagc gcagatacca aatactgttc 4260ttctagtgta gccgtagtta ggccaccact
tcaagaactc tgtagcaccg cctacatacc 4320tcgctctgct aatcctgtta ccagtggctg
ctgccagtgg cgataagtcg tgtcttaccg 4380ggttggactc aagacgatag ttaccggata
aggcgcagcg gtcgggctga acggggggtt 4440cgtgcacaca gcccagcttg gagcgaacga
cctacaccga actgagatac ctacagcgtg 4500agctatgaga aagcgccacg cttcccgaag
ggagaaaggc ggacaggtat ccggtaagcg 4560gcagggtcgg aacaggagag cgcacgaggg
agcttccagg gggaaacgcc tggtatcttt 4620atagtcctgt cgggtttcgc cacctctgac
ttgagcgtcg atttttgtga tgctcgtcag 4680gggggcggag cctatggaaa aacgccagca
acgcggcctt tttacggttc ctggcctttt 4740gctggccttt tgctcacatg tt
4762416843DNAartificial sequencevector
4ccgggctggt tgccctcgcc gctgggctgg cggccgtcta tggccctgca aacgcgccag
60aaacgccgtc gaagccgtgt gcgagacacc gcggccgccg gcgttgtgga tacctcgcgg
120aaaacttggc cctcactgac agatgagggg cggacgttga cacttgaggg gccgactcac
180ccggcgcggc gttgacagat gaggggcagg ctcgatttcg gccggcgacg tggagctggc
240cagcctcgca aatcggcgaa aacgcctgat tttacgcgag tttcccacag atgatgtgga
300caagcctggg gataagtgcc ctgcggtatt gacacttgag gggcgcgact actgacagat
360gaggggcgcg atccttgaca cttgaggggc agagtgctga cagatgaggg gcgcacctat
420tgacatttga ggggctgtcc acaggcagaa aatccagcat ttgcaagggt ttccgcccgt
480ttttcggcca ccgctaacct gtcttttaac ctgcttttaa accaatattt ataaaccttg
540tttttaacca gggctgcgcc ctgtgcgcgt gaccgcgcac gccgaagggg ggtgcccccc
600cttctcgaac cctcccggcc cgctaacgcg ggcctcccat ccccccaggg gctgcgcccc
660tcggccgcga acggcctcac cccaaaaatg gcagcgctgg cagtccttgc cattgccggg
720atcggggcag taacgggatg ggcgatcagc ccgagcgcga cgcccggaag cattgacgtg
780ccgcaggtgc tggcatcgac attcagcgac caggtgccgg gcagtgaggg cggcggcctg
840ggtggcggcc tgcccttcac ttcggccgtc ggggcattca cggacttcat ggcggggccg
900gcaattttta ccttgggcat tcttggcata gtggtcgcgg gtgccgtgct cgtgttcggg
960ggtgcgataa acccagcgaa ccatttgagg tgataggtaa gattataccg aggtatgaaa
1020acgagaattg gacctttaca gaattactct atgaagcgcc atatttaaaa agctaccaag
1080acgaagagga tgaagaggat gaggaggcag attgccttga atatattgac aatactgata
1140agataatata tcttttatat agaagatatc gccgtatgta aggatttcag ggggcaaggc
1200ataggcagcg cgcttatcaa tatatctata gaatgggcaa agcataaaaa cttgcatgga
1260ctaatgcttg aaacccagga caataacctt atagcttgta aattctatca taattgggta
1320atgactccaa cttattgata gtgttttatg ttcagataat gcccgatgac tttgtcatgc
1380agctccaccg attttgagaa cgacagcgac ttccgtccca gccgtgccag gtgctgcctc
1440agattcaggt tatgccgctc aattcgctgc gtatatcgct tgctgattac gtgcagcttt
1500cccttcaggc gggattcata cagcggccag ccatccgtca tccatatcac cacgtcaaag
1560ggtgacagca ggctcataag acgccccagc gtcgccatag tgcgttcacc gaatacgtgc
1620gcaacaaccg tcttccggag actgtcatac gcgtaaaaca gccagcgctg gcgcgattta
1680gccccgacat agccccactg ttcgtccatt tccgcgcaga cgatgacgtc actgcccggc
1740tgtatgcgcg aggttaccga ctgcggcctg agttttttaa gtgacgtaaa atcgtgttga
1800ggccaacgcc cataatgcgg gctgttgccc ggcatccaac gccattcatg gccatatcaa
1860tgattttctg gtgcgtaccg ggttgagaag cggtgtaagt gaactgcagt tgccatgttt
1920tacggcagtg agagcagaga tagcgctgat gtccggcggt gcttttgccg ttacgcacca
1980ccccgtcagt agctgaacag gagggacagc tgatagacac agaagccact ggagcacctc
2040aaaaacacca tcatacacta aatcagtaag ttggcagcat cacccataat tgtggtttca
2100aaatcggctc cgtcgatact atgttatacg ccaactttga aaacaacttt gaaaaagctg
2160ttttctggta tttaaggttt tagaatgcaa ggaacagtga attggagttc gtcttgttat
2220aattagcttc ttggggtatc tttaaatact gtagaaaaga ggaaggaaat aataaatggc
2280taaaatgaga atatcaccgg aattgaaaaa actgatcgaa aaataccgct gcgtaaaaga
2340tacggaagga atgtctcctg ctaaggtata taagctggtg ggagaaaatg aaaacctata
2400tttaaaaatg acggacagcc ggtataaagg gaccacctat gatgtggaac gggaaaagga
2460catgatgcta tggctggaag gaaagctgcc tgttccaaag gtcctgcact ttgaacggca
2520tgatggctgg agcaatctgc tcatgagtga ggccgatggc gtcctttgct cggaagagta
2580tgaagatgaa caaagccctg aaaagattat cgagctgtat gcggagtgca tcaggctctt
2640tcactccatc gacatatcgg attgtcccta tacgaatagc ttagacagcc gcttagccga
2700attggattac ttactgaata acgatctggc cgatgtggat tgcgaaaact gggaagaaga
2760cactccattt aaagatccgc gcgagctgta tgatttttta aagacggaaa agcccgaaga
2820ggaacttgtc ttttcccacg gcgacctggg agacagcaac atctttgtga aagatggcaa
2880agtaagtggc tttattgatc ttgggagaag cggcagggcg gacaagtggt atgacattgc
2940cttctgcgtc cggtcgatca gggaggatat cggggaagaa cagtatgtcg agctattttt
3000tgacttactg gggatcaagc ctgattggga gaaaataaaa tattatattt tactggatga
3060attgttttag tacctagatg tggcgcaacg atgccggcga caagcaggag cgcaccgact
3120tcttccgcat caagtgtttt ggctctcagg ccgaggccca cggcaagtat ttgggcaagg
3180ggtcgctggt attcgtgcag ggcaagattc ggaataccaa gtacgagaag gacggccaga
3240cggtctacgg gaccgacttc attgccgata aggtggatta tctggacacc aaggcaccag
3300gcgggtcaaa tcaggaataa gggcacattg ccccggcgtg agtcggggca atcccgcaag
3360gagggtgaat gaatcggacg tttgaccgga aggcatacag gcaagaactg atcgacgcgg
3420ggttttccgc cgaggatgcc gaaaccatcg caagccgcac cgtcatgcgt gcgccccgcg
3480aaaccttcca gtccgtcggc tcgatggtcc agcaagctac ggccaagatc gagcgcgaca
3540gcgtgcaact ggctccccct gccctgcccg cgccatcggc cgccgtggag cgttcgcgtc
3600gtctcgaaca ggaggcggca ggtttggcga agtcgatgac catcgacacg cgaggaacta
3660tgacgaccaa gaagcgaaaa accgccggcg aggacctggc aaaacaggtc agcgaggcca
3720agcaggccgc gttgctgaaa cacacgaagc agcagatcaa ggaaatgcag ctttccttgt
3780tcgatattgc gccgtggccg gacacgatgc gagcgatgcc aaacgacacg gcccgctctg
3840ccctgttcac cacgcgcaac aagaaaatcc cgcgcgaggc gctgcaaaac aaggtcattt
3900tccacgtcaa caaggacgtg aagatcacct acaccggcgt cgagctgcgg gccgacgatg
3960acgaactggt gtggcagcag gtgttggagt acgcgaagcg cacccctatc ggcgagccga
4020tcaccttcac gttctacgag ctttgccagg acctgggctg gtcgatcaat ggccggtatt
4080acacgaaggc cgaggaatgc ctgtcgcgcc tacaggcgac ggcgatgggc ttcacgtccg
4140accgcgttgg gcacctggaa tcggtgtcgc tgctgcaccg cttccgcgtc ctggaccgtg
4200gcaagaaaac gtcccgttgc caggtcctga tcgacgagga aatcgtcgtg ctgtttgctg
4260gcgaccacta cacgaaattc atatgggaga agtaccgcaa gctgtcgccg acggcccgac
4320ggatgttcga ctatttcagc tcgcaccggg agccgtaccc gctcaagctg gaaaccttcc
4380gcctcatgtg cggatcggat tccacccgcg tgaagaagtg gcgcgagcag gtcggcgaag
4440cctgcgaaga gttgcgaggc agcggcctgg tggaacacgc ctgggtcaat gatgacctgg
4500tgcattgcaa acgctagggc cttgtggggt cagttccggc tgggggttca gcagccagcg
4560ctttactggc atttcaggaa caagcgggca ctgctcgacg cacttgcttc gctcagtatc
4620gctcgggacg cacggcgcgc tctacgaact gccgataaac agaggattaa aattgacaat
4680tgtgattaag gctcagattc gacggcttgg agcggccgac gtgcaggatt tccgcgagat
4740ccgattgtcg gccctgaaga aagctccaga gatgttcggg tccgtttacg agcacgagga
4800gaaaaagccc atggaggcgt tcgctgaacg gttgcgagat gccgtggcat tcggcgccta
4860catcgacggc gagatcattg ggctgtcggt cttcaaacag gaggacggcc ccaaggacgc
4920tcacaaggcg catctgtccg gcgttttcgt ggagcccgaa cagcgaggcc gaggggtcgc
4980cggtatgctg ctgcgggcgt tgccggcggg tttattgctc gtgatgatcg tccgacagat
5040tccaacggga atctggtgga tgcgcatctt catcctcggc gcacttaata tttcgctatt
5100ctggagcttg ttgtttattt cggtctaccg cctgccgggc ggggtcgcgg cgacggtagg
5160cgctgtgcag ccgctgatgg tcgtgttcat ctctgccgct ctgctaggta gcccgatacg
5220attgatggcg gtcctggggg ctatttgcgg aactgcgggc gtggcgctgt tggtgttgac
5280accaaacgca gcgctagatc ctgtcggcgt cgcagcgggc ctggcggggg cggtttccat
5340ggcgttcgga accgtgctga cccgcaagtg gcaacctccc gtgcctctgc tcacctttac
5400cgcctggcaa ctggcggccg gaggacttct gctcgttcca gtagctttag tgtttgatcc
5460gccaatcccg atgcctacag gaaccaatgt tctcggcctg gcgtggctcg gcctgatcgg
5520agcgggttta acctacttcc tttggttccg ggggatctcg cgactcgaac ctacagttgt
5580ttccttactg ggctttctca gccccagatc tggggtcgat cagccgggga tgcatcaggc
5640cgacagtcgg aacttcgggt ccccgacctg taccattcgg tgagcaatgg ataggggagt
5700tgatatcgtc aacgttcact tctaaagaaa tagcgccact cagcttcctc agcggcttta
5760tccagcgatt tcctattatg tcggcatagt tctcaagatc gacagcctgt cacggttaag
5820cgagaaatga ataagaaggc tgataattcg gatctctgcg agggagatga tatttgatca
5880caggcagcaa cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt
5940gtttcaaacc cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag
6000tctgccgcct tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat
6060cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga
6120tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt
6180taatgtactg gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
6240accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
6300aaatcctgtt tgatggtggt tccgaaatcg gcaaaatccc ttataaatca aaagaatagc
6360ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
6420actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cctgtatggc
6480cgcattcgca aaacacacct agactagatt tgttttgcta acccaattga tattaattat
6540atatgattaa tatttatatg tatatggatt tggttaatga aatgcatctg gttcatcaaa
6600gaattataaa gacacgtgac attcatttag gataagaaat atggatgatc tctttctctt
6660ttattcagat aactagtaat tacacataac acacaacttt gatgcccaca ttatagtgat
6720tagcatgtca ctatgtgtgc atccttttat ttcatacatt aattaagttg gccaatccag
6780aagatggaca agtctaggtt aaccatgtgg tacctacgcg ttcgaatatc catgggccgc
6840ttcaggccag ggcgctgggg aaggcgatgg cgtgctcggt cagctgccac ttctggttct
6900tggcgtcgct ccggtcctcc cgcagcagct tgtgctggat gaagtgccac tcgggcatct
6960tgctgggcac gctcttggcc ttgtacacgg tgtcgaactg gcaccggtac cggccgccgt
7020ccttcagcag caggtacatg ctcacgtcgc ccttcaggat gccctgctta ggcacgggca
7080tgatcttctc gcagctggcc tcccagttgg tggtcatctt cttcatcacg gggccgtcgg
7140cggggaagtt cacgccgttg aagatgctct tgtggtagat gcagttctcc ttcacgctca
7200cggtgatgtc cacgttacag atgcacacgg cgccgtcctc gaacaggaag ctccggcccc
7260aggtgtagcc ggcggggcag ctgttcttga agtagtccac gatgtcctgg gggtactcgg
7320tgaagatccg gtcgccgtac ttgaagccgg cgctcaggat gtcctcgctg aagggcaggg
7380ggccgccctc gatcacgcac aggttgatgg tctgcttgcc cttgaagggg tagccgatgc
7440cctcgccggt gatcacgaac ttgtggccgt tcacgcagcc ctccatgtgg tacttcatgg
7500tcatctcctc cttcaggccg tgcttgctgt gggccatggt ggcgaccggt gaattcgagc
7560tcggtacccg gggatcctga gtaaaacaga ggagggtctc actaagttta tagagagact
7620gagagagata aagggacacg tatgaagcgt ctgttttcgt ggtgtgacgt caaagtcatt
7680ttgctctcta cgcgtgtctg tgtcggcttg atcttttttt ttgctttttg gaactcatgt
7740cggtagtata tcttttattt attttttctt tttttccctt ttctttcaaa ctgatgtcgg
7800tatgatattt attccatcct aaaatgtaac ttactattat tagtagtcgg tccatgtcta
7860ttggcccatc atgtggtcat tttacgttta cgtcgtgtgg ctgtttatta taacaaacgg
7920cacatccttc tcattcgaat tgtatttctc cttaatcgtt ctaataggta tgatctttta
7980ttttatacgt aaaattaaaa ttgaatgatg tcaagaacga aaattaattt gtatttacaa
8040aggagctaaa tattgtttat tcctctactg gtagaagata aaagaagtag atgaaataat
8100gatcttacta gagaatattc ctcatttaca ctagtcaaat ggaaatcttg taaactttta
8160caataattta tcctgaaaat atgaaaaaat agaagaaaat gtttacctcc tctctcctct
8220taattcacct acgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
8280gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
8340cgacggccag tgaattcgag ctcggtaccc ggggatcctc tagagtcgac ctgcaggcat
8400gcaagcttgt tgaaacatcc ctgaagtgtc tcattttatt ttatttattc tttgctgata
8460aaaaaataaa ataaaagaag ctaagcacac ggtcaaccat tgctctactg ctaaaagggt
8520tatgtgtagt gttttactgc ataaattatg cagcaaacaa gacaactcaa attaaaaaat
8580ttcctttgct tgtttttttg ttgtctctga cttgactttc ttgtggaagt tggttgtata
8640aggattggga cacaccattg tccttcttaa tttaatttta tttctttgct gataaaaaaa
8700aaaaatttca tatagtgtta aataataatt tgttaaataa ccaaaaagtc aaatatgttt
8760actctcgttt aaataattga gagtcgtcca gcaaggctaa acgattgtat agatttatga
8820caatatttac ttttttatag ataaatgtta tattataata aatttatata catatattat
8880atgttattta ttatttatta ttattttaaa tccttcaata ttttatcaaa ccaactcata
8940attttttttt tatctgtaag aagcaataaa attaaataga cccactttaa ggatgatcca
9000acctttatac agagtaagag agttcaaata gtaccctttc atatacatat caactaaaat
9060attagaaata tcatggatca aaccttataa agacattaaa taagtggata agtataatat
9120ataaatgggt agtatataat atataaatgg atacaaactt ctctctttat aattgttatg
9180tctccttaac atcctaatat aatacataag tgggtaatat ataatatata aatggagaca
9240aacttcttcc attataattg ttatgtcttc ttaacactta tgtctcgttc acaatgctaa
9300agttagaatt gtttagaaag tcttatagta cacatttgtt tttgtactat ttgaagcatt
9360ccataagccg tcacgattca gatgatttat aataataaga ggaaatttat catagaacaa
9420taaggtgcat agatagagtg ttaatatatc ataacatcct ttgtttattc atagaagaag
9480tgagatggag ctcagttatt atactgttac atggtcggat acaatattcc atgctctcca
9540tgagctctta cacctacatg cattttagtt catacttcat gcacgtggcc atcacagcta
9600gctgcagcta catatttaca ttttacaaca ccaggagaac tgccctgtta gtgcataaca
9660atcagaagat ggccgtggct actcgagtta tcgaaccact ttgtacaaga aagctgaacg
9720agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata aaaaacagac
9780tacataatac tgtaaaacac aacatatcca gtcactatgg tcgacctgca gactggctgt
9840gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
9900cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg
9960ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
10020cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc
10080cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
10140tataggtgta aaccttaaac tgcatttcac cagtccctgt tctcgtcagc aaaagagccg
10200ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
10260tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
10320acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
10380gcttcatagc acacctcttt ttgacatact tcgggtatac atatcagtat atattcttat
10440accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc
10500tctagattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct
10560gccgacatgg aagccatcac agacggcatg atgaacctga atcgccagcg gcatcagcac
10620cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
10680attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
10740catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
10800ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
10860aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
10920cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
10980aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
11040cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
11100aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
11160ctccatttta gcttccttag ctcctgaaaa tctcgccgga tcctaactca aaatccacac
11220attatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgcgg ccgccatagt
11280gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt
11340aatatattga tatttatatc attttacgtt tctcgttcag cttttttgta caaacttgtt
11400tgataaccgg tactagtgtg cacgtcgagc gtgtcctctc caaatgaaat gaacttcctt
11460atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg
11520agatgtcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga
11580tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaatga
11640tagcctttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt tctactgtcc
11700tttcgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gaaattatcc
11760tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg acatttttgg
11820agtagaccag agtgtcgtgc tccaccatgt tgacgaagat tttcttcttg tcattgagtc
11880gtaaaagact ctgtatgaac tgttcgccag tcttcacggc gagttctgtt agatcctcga
11940tttgaatctt agactccatg catggcctta gattcagtag gaactacctt tttagagact
12000ccaatctcta ttacttgcct tggtttatga agcaagcctt gaatcgtcca tactggaata
12060gtacttctga tcttgagaaa tatgtctttc tctgtgttct tgatgcaatt agtcctgaat
12120cttttgactg catctttaac cttcttggga aggtatttga tctcctggag attgttactc
12180gggtagatcg tcttgatgag acctgctgcg taggcctctc taaccatctg tgggtcagca
12240ttctttctga aattgaagag gctaaccttc tcattatcag tggtgaacat agtgtcgtca
12300ccttcacctt cgaacttcct tcctagatcg taaagataga ggaaatcgtc cattgtaatc
12360tccggggcaa aggagatctc ttttggggct ggatcactgc tgggcctttt ggttcctagc
12420gtgagccagt gggctttttg ctttggtggg cttgttaggg ccttagcaaa gctcttgggc
12480ttgagttgag cttctccttt ggggatgaag ttcaacctgt ctgtttgctg acttgttgtg
12540tacgcgtcag ctgctgctct tgcctctgta atagtggcaa atttcttgtg tgcaactccg
12600ggaacgccgt ttgttgccgc ctttgtacaa ccccagtcat cgtatatacc ggcatgtgga
12660ccgttataca caacgtagta gttgatatga gggtgttgaa tacccgattc tgctctgaga
12720ggagcaactg tgctgttaag ctcagatttt tgtgggattg gaattggatc ctctagagca
12780aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
12840tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
12900ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
12960ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggccaaa
13020gacaaaaggg cgacattcaa ccgattgagg gagggaaggt aaatattgac ggaaattatt
13080cattaaaggt gaattatcac cgtcaccgac ttgagccatt tgggaattag agccagcaaa
13140atcaccagta gcaccattac cattagcaag gccggaaacg tcaccaatga aaccatcatc
13200tagtaacata gatgacaccg cgcgcgataa tttatcctag tttgcgcgct atattttgtt
13260ttctatcgcg tattaaatgt ataattgcgg gactctaatc ataaaaaccc atctcataaa
13320taacgtcatg cattacatgt taattattac atgcttaacg taattcaaca gaaattatat
13380gataatcatc gcaagaccgg caacaggatt caatcttaag aaactttatt gccaaatgtt
13440tgaacgatct gcttcgacgc actccttctt taggtacgga ctagatctcg gtgacgggca
13500ggaccggacg gggcggtacc ggcaggctga agtccagctg ccagaaaccc acgtcatgcc
13560agttcccgtg cttgaagccg gccgcccgca gcatgccgcg gggggcatat ccgagcgcct
13620cgtgcatgcg cacgctcggg tcgttgggca gcccgatgac agcgaccacg ctcttgaagc
13680cctgtgcctc cagggacttc agcaggtggg tgtagagcgt ggagcccagt cccgtccgct
13740ggtggcgggg ggagacgtac acggtcgact cggccgtcca gtcgtaggcg ttgcgtgcct
13800tccaggggcc cgcgtaggcg atgccggcga cctcgccgtc cacctcggcg acgagccagg
13860gatagcgctc ccgcagacgg acgaggtcgt ccgtccactc ctgcggttcc tgcggctcgg
13920tacggaagtt gaccgtgctt gtctcgatgt agtggttgac gatggtgcag accgccggca
13980tgtccgcctc ggtggcacgg cggatgtcgg ccgggcgtcg ttctgggctc atggatctgg
14040attgagagtg aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg
14100agcatttttg acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg
14160caataatggt ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctgagt
14220ggctccttca acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat
14280cggcgggggt cataacgtga ctcccttaat tctccgctca tgatcagatt gtcgtttccc
14340gccttcagtt taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa
14400agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc
14460catttgtatg tgcatgccaa ccacagggtt ccccagatct ggcgccggcc agcgagacga
14520gcaagattgg ccgccgcccg aaacgatccg acagcgcgcc cagcacaggt gcgcaggcaa
14580attgcaccaa cgcatacagc gccagcagaa tgccatagtg ggcggtgacg tcgttcgagt
14640gaaccagatc gcgcaggagg cccggcagca ccggcataat caggccgatg ccgacagcgt
14700cgagcgcgac agtgctcaga attacgatca ggggtatgtt gggtttcacg tctggcctcc
14760ggaccagcct ccgctggtcc gattgaacgc gcggattctt tatcactgat aagttggtgg
14820acatattatg tttatcagtg ataaagtgtc aagcatgaca aagttgcagc cgaatacagt
14880gatccgtgcc gccctggacc tgttgaacga ggtcggcgta gacggtctga cgacacgcaa
14940actggcggaa cggttggggg ttcagcagcc ggcgctttac tggcacttca ggaacaagcg
15000ggcgctgctc gacgcactgg ccgaagccat gctggcggag aatcatacgc attcggtgcc
15060gagagccgac gacgactggc gctcatttct gatcgggaat gcccgcagct tcaggcaggc
15120gctgctcgcc taccgcgatg gcgcgcgcat ccatgccggc acgcgaccgg gcgcaccgca
15180gatggaaacg gccgacgcgc agcttcgctt cctctgcgag gcgggttttt cggccgggga
15240cgccgtcaat gcgctgatga caatcagcta cttcactgtt ggggccgtgc ttgaggagca
15300ggccggcgac agcgatgccg gcgagcgcgg cggcaccgtt gaacaggctc cgctctcgcc
15360gctgttgcgg gccgcgatag acgccttcga cgaagccggt ccggacgcag cgttcgagca
15420gggactcgcg gtgattgtcg atggattggc gaaaaggagg ctcgttgtca ggaacgttga
15480aggaccgaga aagggtgacg attgatcagg accgctgccg gagcgcaacc cactcactac
15540agcagagcca tgtagacaac atcccctccc cctttccacc gcgtcagacg cccgtagcag
15600cccgctacgg gctttttcat gccctgccct agcgtccaag cctcacggcc gcgctcggcc
15660tctctggcgg ccttctggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
15720gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
15780tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
15840aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
15900aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
15960ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
16020tccgcctttc tcccttcggg aagcgtggcg cttttccgct gcataaccct gcttcggggt
16080cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca
16140aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga
16200agtaggccca cccgcgagcg ggtgttcctt cttcactgtc ccttattcgc acctggcggt
16260gctcaacggg aatcctgctc tgcgaggctg gccggctacc gccggcgtaa cagatgaggg
16320caagcggatg gctgatgaaa ccaagccaac caggaagggc agcccaccta tcaaggtgta
16380ctgccttcca gacgaacgaa gagcgattga ggaaaaggcg gcggcggccg gcatgagcct
16440gtcggcctac ctgctggccg tcggccaggg ctacaaaatc acgggcgtcg tggactatga
16500gcacgtccgc gagctggccc gcatcaatgg cgacctgggc cgcctgggcg gcctgctgaa
16560actctggctc accgacgacc cgcgcacggc gcggttcggt gatgccacga tcctcgccct
16620gctggcgaag atcgaagaga agcaggacga gcttggcaag gtcatgatgg gcgtggtccg
16680cccgagggca gagccatgac ttttttagcc gctaaaacgg ccggggggtg cgcgtgattg
16740ccaagcacgt ccccatgcgc tccatcaaga agagcgactt cgcggagctg gtgaagtaca
16800tcaccgacga gcaaggcaag accgagcgcc tttgcgacgc tca
1684359142DNAartificial sequencevector 5ctagttatct gaataaaaga gaaagagatc
atccatattt cttatcctaa atgaatgtca 60cgtgtcttta taattctttg atgaaccaga
tgcatttcat taaccaaatc catatacata 120taaatattaa tcatatataa ttaatatcaa
ttgggttagc aaaacaaatc tagtctaggt 180gtgttttgcg aattcgatat caagcttgat
gggtaccggc gcgcccgatc atccggatat 240agttcctcct ttcagcaaaa aacccctcaa
gacccgttta gaggccccaa ggggttatgc 300tagttattgc tcagcggtgg cagcagccaa
ctcagcttcc tttcgggctt tgttagcagc 360cggatcgatc caagctgtac ctcactattc
ctttgccctc ggacgagtgc tggggcgtcg 420gtttccacta tcggcgagta cttctacaca
gccatcggtc cagacggccg cgcttctgcg 480ggcgatttgt gtacgcccga cagtcccggc
tccggatcgg acgattgcgt cgcatcgacc 540ctgcgcccaa gctgcatcat cgaaattgcc
gtcaaccaag ctctgataga gttggtcaag 600accaatgcgg agcatatacg cccggagccg
cggcgatcct gcaagctccg gatgcctccg 660ctcgaagtag cgcgtctgct gctccataca
agccaaccac ggcctccaga agaagatgtt 720ggcgacctcg tattgggaat ccccgaacat
cgcctcgctc cagtcaatga ccgctgttat 780gcggccattg tccgtcagga cattgttgga
gccgaaatcc gcgtgcacga ggtgccggac 840ttcggggcag tcctcggccc aaagcatcag
ctcatcgaga gcctgcgcga cggacgcact 900gacggtgtcg tccatcacag tttgccagtg
atacacatgg ggatcagcaa tcgcgcatat 960gaaatcacgc catgtagtgt attgaccgat
tccttgcggt ccgaatgggc cgaacccgct 1020cgtctggcta agatcggccg cagcgatcgc
atccatagcc tccgcgaccg gctgcagaac 1080agcgggcagt tcggtttcag gcaggtcttg
caacgtgaca ccctgtgcac ggcgggagat 1140gcaataggtc aggctctcgc tgaattcccc
aatgtcaagc acttccggaa tcgggagcgc 1200ggccgatgca aagtgccgat aaacataacg
atctttgtag aaaccatcgg cgcagctatt 1260tacccgcagg acatatccac gccctcctac
atcgaagctg aaagcacgag attcttcgcc 1320ctccgagagc tgcatcaggt cggagacgct
gtcgaacttt tcgatcagaa acttctcgac 1380agacgtcgcg gtgagttcag gcttttccat
gggtatatct ccttcttaaa gttaaacaaa 1440attatttcta gagggaaacc gttgtggtct
ccctatagtg agtcgtatta atttcgcggg 1500atcgagatct gatcaacctg cattaatgaa
tcggccaacg cgcggggaga ggcggtttgc 1560gtattgggcg ctcttccgct tcctcgctca
ctgactcgct gcgctcggtc gttcggctgc 1620ggcgagcggt atcagctcac tcaaaggcgg
taatacggtt atccacagaa tcaggggata 1680acgcaggaaa gaacatgtga gcaaaaggcc
agcaaaaggc caggaaccgt aaaaaggccg 1740cgttgctggc gtttttccat aggctccgcc
cccctgacga gcatcacaaa aatcgacgct 1800caagtcagag gtggcgaaac ccgacaggac
tataaagata ccaggcgttt ccccctggaa 1860gctccctcgt gcgctctcct gttccgaccc
tgccgcttac cggatacctg tccgcctttc 1920tcccttcggg aagcgtggcg ctttctcaat
gctcacgctg taggtatctc agttcggtgt 1980aggtcgttcg ctccaagctg ggctgtgtgc
acgaaccccc cgttcagccc gaccgctgcg 2040ccttatccgg taactatcgt cttgagtcca
acccggtaag acacgactta tcgccactgg 2100cagcagccac tggtaacagg attagcagag
cgaggtatgt aggcggtgct acagagttct 2160tgaagtggtg gcctaactac ggctacacta
gaaggacagt atttggtatc tgcgctctgc 2220tgaagccagt taccttcgga aaaagagttg
gtagctcttg atccggcaaa caaaccaccg 2280ctggtagcgg tggttttttt gtttgcaagc
agcagattac gcgcagaaaa aaaggatctc 2340aagaagatcc tttgatcttt tctacggggt
ctgacgctca gtggaacgaa aactcacgtt 2400aagggatttt ggtcatgaca ttaacctata
aaaataggcg tatcacgagg ccctttcgtc 2460tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 2520cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg tcagcgggtg 2580ttggcgggtg tcggggctgg cttaactatg
cggcatcaga gcagattgta ctgagagtgc 2640accatatgga catattgtcg ttagaacgcg
gctacaatta atacataacc ttatgtatca 2700tacacatacg atttaggtga cactatagaa
cggcgcgcca agctgggtct agaactagaa 2760acgtgatgcc acttgttatt gaagtcgatt
acagcatcta ttctgtttta ctatttataa 2820ctttgccatt tctgactttt gaaaactatc
tctggatttc ggtatcgctt tgtgaagatc 2880gagcaaaaga gacgttttgt ggacgcaatg
gtccaaatcc gttctacatg aacaaattgg 2940tcacaatttc cactaaaagt aaataaatgg
caagttaaaa aaggaatatg cattttactg 3000attgcctagg tgagctccaa gagaagttga
atctacacgt ctaccaaccg ctaaaaaaag 3060aaaaacattg aatatgtaac ctgattccat
tagcttttga cttcttcaac agattctcta 3120cttagatttc taacagaaat attattacta
gcacatcatt ttcagtctca ctacagcaaa 3180aaatccaacg gcacaataca gacaacagga
gatatcagac tacagagata gatagatgct 3240actgcatgta gtaagttaaa taaaaggaaa
ataaaatgtc ttgctaccaa aactactaca 3300gactatgatg ctcaccacag gccaaatcct
gcaactagga cagcattatc ttatatatat 3360tgtacaaaac aagcatcaag gaacatttgg
tctaggcaat cagtacctcg ttctaccatc 3420accctcagtt atcacatcct tgaaggatcc
attactggga atcatcggca acacatgctc 3480ctgatggggc acaatgacat caagaaggta
ggggccaggg gtgtccaaca ttctctgaat 3540tgccgctcta agctcttcct tcttcgtcac
tcgcgctgcc ggtatcccac aagcatcagc 3600aaacttgagc atgtttggga atatctcgct
ctcgctagac ggatctccaa gataggtgtg 3660agctctattg gacttgtaga acctatcctc
caactgaacc accataccca aatgctgatt 3720gttcaacaac aatatcttaa ctgggagatt
ctccactctt atagtggcca actcctgaac 3780attcatgatg aaactaccat ccccatcaat
gtcaaccaca acagccccag ggttagcaac 3840agcagcacca atagccgcag gcaatccaaa
acccatggct ccaagacccc ctgaggtcaa 3900ccactgcctc ggtctcttgt acttgtaaaa
ctgcgcagcc cacatttgat gctgcccaac 3960cccagtacta acaatagcat ctccattagt
caactcatca agaacctcga tagcatgctg 4020cggagaaatc gcgtcctgga atgtcttgta
acccaatgga aacttgtgtt tctgcacatt 4080aatctcttct ctccaacctc caagatcaaa
cttaccctcc actcctttct cctccaaaat 4140catattaatt cccttcaagg ccaacttcaa
atccgcgcaa accgacacgt gcgcctgctt 4200gttcttccca atctcggcag aatcaatatc
aatgtgaaca atcttagccc tactagcaaa 4260agcctcaagc ttcccagtaa cacggtcatc
aaaccttacc ccaaaggcaa gcaacaaatc 4320actattgtca acagcatagt tagcataaac
agtaccatgc atacccagca tctgaaggga 4380atattcatca ccaataggaa aagttccaag
acccattaaa gtgctagcaa cgggaatacc 4440agtgagttca acaaagcgcc tcaattcagc
actggaattc aaactgccac cgccgacgta 4500gagaacgggc ttttgggcct ccatgatgag
tctgacaatg tgttccaatt gggcctcggc 4560ggggggcctg ggcagcctgg cgaggtaacc
ggggaggtta acgggctcgt cccaattagg 4620cacggcgagt tgctgctgaa cgtctttggg
aatgtcgatg aggaccggac cggggcggcc 4680ggaggtggcg acgaagaaag cctcggcgac
gacgcggggg atgtcgtcga cgtcgaggat 4740gaggtagttg tgcttcgtga tggatctgct
cacctccacg atcggggttt cttggaaggc 4800gtcggtgccg atcatccggc gggcgacctg
gccggtgatg gcgacgactg ggacgctgtc 4860cattaaagcg tcggcgaggc cgctcacgag
gttggtggcg ccggggccgg aggtggcaat 4920gcagacgccg gggaggccgg aggaacgcgc
gtagccttcg gcggcgaaga cgccgccctg 4980ctcgtggcgc gggagcacgt tgcggatggc
ggcggagcgc gtgagcgcct ggtggatctc 5040catcgacgca ccgccggggt acgcgaacac
cgtcgtcacg ccctgcctct ccagcgcctc 5100cacaaggatg tccgcgccct tgcgaggttc
gccggaggcg aaccgtgaca cgaagggctc 5160cgtggtcggc gcttccttgg tgaagggcgc
cgccgtgggg ggtttggaga tggaacattt 5220gattttgaga gcgtggttgg gtttggtgag
ggtttgatga gagagaggga gggtggatct 5280agtaatgcgt ttggggaagg tggggtgtga
agaggaagaa gagaatcggg tggttctgga 5340agcggtggcc gccattgtgt tgtgtggcat
ggttatactt caaaaactgc acaacaagcc 5400tagagttagt acctaaacag taaatttaca
acagagagca aagacacatg caaaaatttc 5460agccataaaa aaagttataa tagaatttaa
agcaaaagtt tcatttttta aacatatata 5520caaacaaact ggatttgaag gaagggatta
attcccctgc tcaaagtttg aattcctatt 5580gtgacctata ctcgaataaa attgaagcct
aaggaatgta tgagaaacaa gaaaacaaaa 5640caaaactaca gacaaacaag tacaattaca
aaattcgcta aaattctgta atcaccaaac 5700cccatctcag tcagcacaag gcccaaggtt
tattttgaaa taaaaaaaaa gtgattttat 5760ttctcataag ctaaaagaaa gaaaggcaat
tatgaaatga tttcgactag atctgaaagt 5820caaacgcgta ttccgcagat attaaagaaa
gagtagagtt tcacatggat cctagatgga 5880cccagttgag gaaaaagcaa ggcaaagcaa
accagaagtg caagatccga aattgaacca 5940cggaatctag gatttggtag agggagaaga
aaagtacctt gagaggtaga agagaagaga 6000agagcagaga gatatatgaa cgagtgtgtc
ttggtctcaa ctctgaagcg atacgagttt 6060agaggggagc attgagttcc aatttatagg
gaaaccgggt ggcaggggtg agttaatgac 6120ggaaaagccc ctaagtaacg agattggatt
gtgggttaga ttcaaccgtt tgcatccgcg 6180gcttagattg gggaagtcag agtgaatctc
aaccgttgac tgagttgaaa attgaatgta 6240gcaaccaatt gagccaaccc cagcctttgc
cctttgattt tgatttgttt gttgcatact 6300ttttatttgt cttctggttc tgactctctt
tctctcgttt caatgccagg ttgcctactc 6360ccacaccact cacaagaaga ttctactgtt
agtattaaat attttttaat gtattaaatg 6420atgaatgctt ttgtaaacag aacaagacta
tgtctaataa gtgtcttgca acatttttta 6480agaaattaaa aaaaatatat ttattatcaa
aatcaaatgt atgaaaaatc atgaataata 6540taattttata cattttttta aaaaatcttt
taatttctta attaatatct taaaaataat 6600gattaatatt taacccaaaa taattagtat
gattggtaag gaagatatcc atgttatgtt 6660tggatgtgag tttgatctag agcaaagctt
actagagtcg acctgcagcc cctccaccgc 6720ggtggcggcc gctctagaga tccgtcaaca
tggtggagca cgacactctc gtctactcca 6780agaatatcaa agatacagtc tcagaagacc
aaagggctat tgagactttt caacaaaggg 6840taatatcggg aaacctcctc ggattccatt
gcccagctat ctgtcacttc atcaaaagga 6900cagtagaaaa ggaaggtggc acctacaaat
gccatcattg cgataaagga aaggctatcg 6960ttcaagatgc ctctgccgac agtggtccca
aagatggacc cccacccacg aggagcatcg 7020tggaaaaaga agacgttcca accacgtctt
caaagcaagt ggattgatgt gatgatccta 7080tgcgtatggt atgacgtgtg ttcaagatga
tgacttcaaa cctacctatg acgtatggta 7140tgacgtgtgt cgactgatga cttagatcca
ctcgagcggc tataaatacg tacctacgca 7200ccctgcgcta ccatccctag agctgcagct
tatttttaca acaattacca acaacaacaa 7260acaacaaaca acattacaat tactatttac
aattacagtc gacccatcaa caagtttgta 7320caaaaaagct gaacgagaaa cgtaaaatga
tataaatatc aatatattaa attagatttt 7380gcataaaaaa cagactacat aatactgtaa
aacacaacat atccagtcat attggcggcc 7440gcattaggca ccccaggctt tacactttat
gcttccggct cgtataatgt gtggattttg 7500agttaggatc cgtcgagatt ttcaggagct
aaggaagcta aaatggagaa aaaaatcact 7560ggatatacca ccgttgatat atcccaatgg
catcgtaaag aacattttga ggcatttcag 7620tcagttgctc aatgtaccta taaccagacc
gttcagctgg atattacggc ctttttaaag 7680accgtaaaga aaaataagca caagttttat
ccggccttta ttcacattct tgcccgcctg 7740atgaatgctc atccggaatt ccgtatggca
atgaaagacg gtgagctggt gatatgggat 7800agtgttcacc cttgttacac cgttttccat
gagcaaactg aaacgttttc atcgctctgg 7860agtgaatacc acgacgattt ccggcagttt
ctacacatat attcgcaaga tgtggcgtgt 7920tacggtgaaa acctggccta tttccctaaa
gggtttattg agaatatgtt tttcgtctca 7980gccaatccct gggtgagttt caccagtttt
gatttaaacg tggccaatat ggacaacttc 8040ttcgcccccg ttttcaccat gggcaaatat
tatacgcaag gcgacaaggt gctgatgccg 8100ctggcgattc aggttcatca tgccgtttgt
gatggcttcc atgtcggcag aatgcttaat 8160gaattacaac agtactgcga tgagtggcag
ggcggggcgt aaagatctgg atccggctta 8220ctaaaagcca gataacagta tgcgtatttg
cgcgctgatt tttgcggtat aagaatatat 8280actgatatgt atacccgaag tatgtcaaaa
agaggtatgc tatgaagcag cgtattacag 8340tgacagttga cagcgacagc tatcagttgc
tcaaggcata tatgatgtca atatctccgg 8400tctggtaagc acaaccatgc agaatgaagc
ccgtcgtctg cgtgccgaac gctggaaagc 8460ggaaaatcag gaagggatgg ctgaggtcgc
ccggtttatt gaaatgaacg gctcttttgc 8520tgacgagaac aggggctggt gaaatgcagt
ttaaggttta cacctataaa agagagagcc 8580gttatcgtct gtttgtggat gtacagagtg
atattattga cacgcccggg cgacggatgg 8640tgatccccct ggccagtgca cgtctgctgt
cagataaagt ctcccgtgaa ctttacccgg 8700tggtgcatat cggggatgaa agctggcgca
tgatgaccac cgatatggcc agtgtgccgg 8760tctccgttat cggggaagaa gtggctgatc
tcagccaccg cgaaaatgac atcaaaaacg 8820ccattaacct gatgttctgg ggaatataaa
tgtcaggctc ccttatacac agccagtctg 8880caggtcgacc atagtgactg gatatgttgt
gttttacagt attatgtagt ctgtttttta 8940tgcaaaatct aatttaatat attgatattt
atatcatttt acgtttctcg ttcagctttc 9000ttgtacaaag tggttgataa cctagacttg
tccatcttct ggattggcca acttaattaa 9060tgtatgaaat aaaaggatgc acacatagtg
acatgctaat cactataatg tgggcatcaa 9120agttgtgtgt tatgtgtaat ta
9142649911DNAartificial sequencevector
6gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta
60taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt
120atacatatat ttaaacttta ctctacgaat aatataatct atagtactac aataatatca
180gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt
240ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg
300caaatagctt cacctatata atacttcatc cattttatta gtacatccat ttagggttta
360gggttaatgg tttttataga ctaatttttt tagtacatct attttattct attttagcct
420ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa
480tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta
540aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt
600ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca
660cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg
720ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag
780gcggcctcct cctcctctca cggcacggca gctacggggg attcctttcc caccgctcct
840tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc
900aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc
960ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc taccttctct
1020agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc atgtttgtgt
1080tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg cgacctgtac
1140gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc
1200tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt
1260tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt
1320tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc
1380ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg
1440tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata
1500ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg
1560gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac
1620tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct
1680tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat
1740gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac
1800cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat
1860acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg
1920ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact
1980tctgcaggtc gactctagag gatccacaag tttgtacaaa aaagctgaac gagaaacgta
2040aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
2100ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca
2160ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatttaaa tacgcgttga
2220tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata
2280agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc
2340gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa
2400tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg
2460ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg
2520ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa
2580gagagagccg ttatcgtctg tttgtggatg tacagagtga tatcattgac acgcccggtc
2640gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
2700tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
2760gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
2820tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca
2880gccagtctgc aggtcgacca tagtgactgg atatgttgtg ttttacagta ttatgtagtc
2940tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt
3000tcagctttct tgtacaaagt ggtgttaacc tagacttgtc catcttctgg attggccaac
3060ttaattaatg tatgaaataa aaggatgcac acatagtgac atgctaatca ctataatgtg
3120ggcatcaaag ttgtgtgtta tgtgtaatta ctagttatct gaataaaaga gaaagagatc
3180atccatattt cttatcctaa atgaatgtca cgtgtcttta taattctttg atgaaccaga
3240tgcatttcat taaccaaatc catatacata taaatattaa tcatatataa ttaatatcaa
3300ttgggttagc aaaacaaatc tagtctaggt gtgttttgcg aattgcggcc gccaccgcgg
3360tggagctcga attccggtcc gggtcacctt tgtccaccaa gatggaactg cggccgctca
3420ttaattaagt caggcgcgcc tctagttgaa gacacgttca tgtcttcatc gtaagaagac
3480actcagtagt cttcggccag aatggccatc tggattcagc aggcctagaa ggccatttaa
3540atcctgagga tctggtcttc ctaaggaccc gggatatcgg accgattaaa ctttaattcg
3600gtccgaagct tgcatgcctg cagtgcagcg tgacccggtc gtgcccctct ctagagataa
3660tgagcattgc atgtctaagt tataaaaaat taccacatat tttttttgtc acacttgttt
3720gaagtgcagt ttatctatct ttatacatat atttaaactt tactctacga ataatataat
3780ctatagtact acaataatat cagtgtttta gagaatcata taaatgaaca gttagacatg
3840gtctaaagga caattgagta ttttgacaac aggactctac agttttatct ttttagtgtg
3900catgtgttct cctttttttt tgcaaatagc ttcacctata taatacttca tccattttat
3960tagtacatcc atttagggtt tagggttaat ggtttttata gactaatttt tttagtacat
4020ctattttatt ctattttagc ctctaaatta agaaaactaa aactctattt tagttttttt
4080atttaataat ttagatataa aatagaataa aataaagtga ctaaaaatta aacaaatacc
4140ctttaagaaa ttaaaaaaac taaggaaaca tttttcttgt ttcgagtaga taatgccagc
4200ctgttaaacg ccgtcgacga gtctaacgga caccaaccag cgaaccagca gcgtcgcgtc
4260gggccaagcg aagcagacgg cacggcatct ctgtcgctgc ctctggaccc ctctcgagag
4320ttccgctcca ccgttggact tgctccgctg tcggcatcca gaaattgcgt ggcggagcgg
4380cagacgtgag ccggcacggc aggcggcctc ctcctcctct cacggcaccg gcagctacgg
4440gggattcctt tcccaccgct ccttcgcttt cccttcctcg cccgccgtaa taaatagaca
4500ccccctccac accctctttc cccaacctcg tgttgttcgg agcgcacaca cacacaacca
4560gatctccccc aaatccaccc gtcggcacct ccgcttcaag gtacgccgct cgtcctcccc
4620cccccccctc tctaccttct ctagatcggc gttccggtcc atgcatggtt agggcccggt
4680agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag
4740cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac ttgccagtgt
4800ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc gatttcatga
4860ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc aatatatgcc
4920gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg tgatgatgtg
4980gtctggttgg gcggtcgttc tagatcggag tagaattctg tttcaaacta cctggtggat
5040ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga attgaagatg
5100atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt actgatgcat
5160atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc
5220attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt attaattttg
5280gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga tggaaatatc
5340gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca tgatggcata
5400tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata aacaagtatg
5460ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca gctatatgtg
5520gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt cttttgtcga
5580tgctcaccct gttgtttggt gttacttctg caggtcgact ttaacttagc ctaggatcca
5640cacgacacca tgtcccccga gcgccgcccc gtcgagatcc gcccggccac cgccgccgac
5700atggccgccg tgtgcgacat cgtgaaccac tacatcgaga cctccaccgt gaacttccgc
5760accgagccgc agaccccgca ggagtggatc gacgacctgg agcgcctcca ggaccgctac
5820ccgtggctcg tggccgaggt ggagggcgtg gtggccggca tcgcctacgc cggcccgtgg
5880aaggcccgca acgcctacga ctggaccgtg gagtccaccg tgtacgtgtc ccaccgccac
5940cagcgcctcg gcctcggctc caccctctac acccacctcc tcaagagcat ggaggcccag
6000ggcttcaagt ccgtggtggc cgtgatcggc ctcccgaacg acccgtccgt gcgcctccac
6060gaggccctcg gctacaccgc ccgcggcacc ctccgcgccg ccggctacaa gcacggcggc
6120tggcacgacg tcggcttctg gcagcgcgac ttcgagctgc cggccccgcc gcgcccggtg
6180cgcccggtga cgcagatctg agtcgaaacc tagacttgtc catcttctgg attggccaac
6240ttaattaatg tatgaaataa aaggatgcac acatagtgac atgctaatca ctataatgtg
6300ggcatcaaag ttgtgtgtta tgtgtaatta ctagttatct gaataaaaga gaaagagatc
6360atccatattt cttatcctaa atgaatgtca cgtgtcttta taattctttg atgaaccaga
6420tgcatttcat taaccaaatc catatacata taaatattaa tcatatataa ttaatatcaa
6480ttgggttagc aaaacaaatc tagtctaggt gtgttttgcg aattgcggcc gccaccgcgg
6540tggagctcga attcattccg attaatcgtg gcctcttgct cttcaggatg aagagctatg
6600tttaaacgtg caagcgctac tagacaattc agtacattaa aaacgtccgc aatgtgttat
6660taagttgtct aagcgtcaat ttggtttaca ccacaatata tcctgccacc agccagccaa
6720cagctccccg accggcagct cggcacaaaa tcaccactcg atacaggcag cccatcagtc
6780cgggacggcg tcagcgggag agccgttgta aggcggcaga ctttgctcat gttaccgatg
6840ctattcggaa gaacggcaac taagctgccg ggtttgaaac acggatgatc tcgcggaggg
6900tagcatgttg attgtaacga tgacagagcg ttgctgcctg tgatcaaata tcatctccct
6960cgcagagatc cgaattatca gccttcttat tcatttctcg cttaaccgtg acaggctgtc
7020gatcttgaga actatgccga cataatagga aatcgctgga taaagccgct gaggaagctg
7080agtggcgcta tttctttaga agtgaacgtt gacgatcgtc gaccgtaccc cgatgaatta
7140attcggacgt acgttctgaa cacagctgga tacttacttg ggcgattgtc atacatgaca
7200tcaacaatgt acccgtttgt gtaaccgtct cttggaggtt cgtatgacac tagtggttcc
7260cctcagcttg cgactagatg ttgaggccta acattttatt agagagcagg ctagttgctt
7320agatacatga tcttcaggcc gttatctgtc agggcaagcg aaaattggcc atttatgacg
7380accaatgccc cgcagaagct cccatctttg ccgccataga cgccgcgccc cccttttggg
7440gtgtagaaca tccttttgcc agatgtggaa aagaagttcg ttgtcccatt gttggcaatg
7500acgtagtagc cggcgaaagt gcgagaccca tttgcgctat atataagcct acgatttccg
7560ttgcgactat tgtcgtaatt ggatgaacta ttatcgtagt tgctctcaga gttgtcgtaa
7620tttgatggac tattgtcgta attgcttatg gagttgtcgt agttgcttgg agaaatgtcg
7680tagttggatg gggagtagtc atagggaaga cgagcttcat ccactaaaac aattggcagg
7740tcagcaagtg cctgccccga tgccatcgca agtacgaggc ttagaaccac cttcaacaga
7800tcgcgcatag tcttccccag ctctctaacg cttgagttaa gccgcgccgc gaagcggcgt
7860cggcttgaac gaattgttag acattatttg ccgactacct tggtgatctc gcctttcacg
7920tagtgaacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttgtccaaga
7980taagcctgcc tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc
8040cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg
8100gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc
8160gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc
8220tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc
8280agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt gcgctgccat
8340tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca
8400acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc
8460aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacagt caccgtaacc
8520agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt
8580acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga
8640gtcgatactt cggcgatcac cgcttccctc atgatgttta actcctgaat taagccgcgc
8700cgcgaagcgg tgtcggcttg aatgaattgt taggcgtcat cctgtgctcc cgagaaccag
8760taccagtaca tcgctgtttc gttcgagact tgaggtctag ttttatacgt gaacaggtca
8820atgccgccga gagtaaagcc acattttgcg tacaaattgc aggcaggtac attgttcgtt
8880tgtgtctcta atcgtatgcc aaggagctgt ctgcttagtg cccacttttt cgcaaattcg
8940atgagactgt gcgcgactcc tttgcctcgg tgcgtgtgcg acacaacaat gtgttcgata
9000gaggctagat cgttccatgt tgagttgagt tcaatcttcc cgacaagctc ttggtcgatg
9060aatgcgccat agcaagcaga gtcttcatca gagtcatcat ccgagatgta atccttccgg
9120taggggctca cacttctggt agatagttca aagccttggt cggataggtg cacatcgaac
9180acttcacgaa caatgaaatg gttctcagca tccaatgttt ccgccacctg ctcagggatc
9240accgaaatct tcatatgacg cctaacgcct ggcacagcgg atcgcaaacc tggcgcggct
9300tttggcacaa aaggcgtgac aggtttgcga atccgttgct gccacttgtt aacccttttg
9360ccagatttgg taactataat ttatgttaga ggcgaagtct tgggtaaaaa ctggcctaaa
9420attgctgggg atttcaggaa agtaaacatc accttccggc tcgatgtcta ttgtagatat
9480atgtagtgta tctacttgat cgggggatct gctgcctcgc gcgtttcggt gatgacggtg
9540aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
9600ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
9660tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
9720gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
9780ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
9840gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
9900ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
9960ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
10020acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
10080tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
10140ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
10200ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
10260ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
10320actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
10380gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
10440tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
10500caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
10560atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
10620acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
10680ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
10740ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
10800tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
10860tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
10920gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
10980tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
11040tgttgccatt gctgcagggg gggggggggg gggggacttc cattgttcat tccacggaca
11100aaaacagaga aaggaaacga cagaggccaa aaagcctcgc tttcagcacc tgtcgtttcc
11160tttcttttca gagggtattt taaataaaaa cattaagtta tgacgaagaa gaacggaaac
11220gccttaaacc ggaaaatttt cataaatagc gaaaacccgc gaggtcgccg ccccgtaacc
11280tacctgtcgg atcaccggaa aggacccgta aagtgataat gattatcatc tacatatcac
11340aacgtgcgtg gaggccatca aaccacgtca aataatcaat tatgacgcag gtatcgtatt
11400aattgatctg catcaactta acgtaaaaac aacttcagac aatacaaatc agcgacactg
11460aatacggggc aacctcatgt cccccccccc cccccccctg caggcatcgt ggtgtcacgc
11520tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
11580tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
11640aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
11700atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
11760tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca
11820catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
11880aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
11940tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
12000gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
12060tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
12120tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
12180taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
12240cgtcttcaag aattcggagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga
12300tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg
12360acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga
12420gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat
12480gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
12540gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcggctttg ttgaataaat
12600cgaacttttg ctgagttgaa ggatcagatc acgcatcttc ccgacaacgc agaccgttcc
12660gtggcaaagc aaaagttcaa aatcaccaac tggtccacct acaacaaagc tctcatcaac
12720cgtggctccc tcactttctg gctggatgat ggggcgattc aggcctggta tgagtcagca
12780acaccttctt cacgaggcag acctcagcgc cagaaggccg ccagagaggc cgagcgcggc
12840cgtgaggctt ggacgctagg gcagggcatg aaaaagcccg tagcgggctg ctacgggcgt
12900ctgacgcggt ggaaaggggg aggggatgtt gtctacatgg ctctgctgta gtgagtgggt
12960tgcgctccgg cagcggtcct gatcaatcgt caccctttct cggtccttca acgttcctga
13020caacgagcct ccttttcgcc aatccatcga caatcaccgc gagtccctgc tcgaacgctg
13080cgtccggacc ggcttcgtcg aaggcgtcta tcgcggcccg caacagcggc gagagcggag
13140cctgttcaac ggtgccgccg cgctcgccgg catcgctgtc gccggcctgc tcctcaagca
13200cggccccaac agtgaagtag ctgattgtca tcagcgcatt gacggcgtcc ccggccgaaa
13260aacccgcctc gcagaggaag cgaagctgcg cgtcggccgt ttccatctgc ggtgcgcccg
13320gtcgcgtgcc ggcatggatg cgcgcgccat cgcggtaggc gagcagcgcc tgcctgaagc
13380tgcgggcatt cccgatcaga aatgagcgcc agtcgtcgtc ggctctcggc accgaatgcg
13440tatgattctc cgccagcatg gcttcggcca gtgcgtcgag cagcgcccgc ttgttcctga
13500agtgccagta aagcgccggc tgctgaaccc ccaaccgttc cgccagtttg cgtgtcgtca
13560gaccgtctac gccgacctcg ttcaacaggt ccagggcggc acggatcact gtattcggct
13620gcaactttgt catgcttgac actttatcac tgataaacat aatatgtcca ccaacttatc
13680agtgataaag aatccgcgcg ttcaatcgga ccagcggagg ctggtccgga ggccagacgt
13740gaaacccaac atacccctga tcgtaattct gagcactgtc gcgctcgacg ctgtcggcat
13800cggcctgatt atgccggtgc tgccgggcct cctgcgcgat ctggttcact cgaacgacgt
13860caccgcccac tatggcattc tgctggcgct gtatgcgttg gtgcaatttg cctgcgcacc
13920tgtgctgggc gcgctgtcgg atcgtttcgg gcggcggcca atcttgctcg tctcgctggc
13980cggcgccact gtcgactacg ccatcatggc gacagcgcct ttcctttggg ttctctatat
14040cgggcggatc gtggccggca tcaccggggc gactggggcg gtagccggcg cttatattgc
14100cgatatcact gatggcgatg agcgcgcgcg gcacttcggc ttcatgagcg cctgtttcgg
14160gttcgggatg gtcgcgggac ctgtgctcgg tgggctgatg ggcggtttct ccccccacgc
14220tccgttcttc gccgcggcag ccttgaacgg cctcaatttc ctgacgggct gtttcctttt
14280gccggagtcg cacaaaggcg aacgccggcc gttacgccgg gaggctctca acccgctcgc
14340ttcgttccgg tgggcccggg gcatgaccgt cgtcgccgcc ctgatggcgg tcttcttcat
14400catgcaactt gtcggacagg tgccggccgc gctttgggtc attttcggcg aggatcgctt
14460tcactgggac gcgaccacga tcggcatttc gcttgccgca tttggcattc tgcattcact
14520cgcccaggca atgatcaccg gccctgtagc cgcccggctc ggcgaaaggc gggcactcat
14580gctcggaatg attgccgacg gcacaggcta catcctgctt gccttcgcga cacggggatg
14640gatggcgttc ccgatcatgg tcctgcttgc ttcgggtggc atcggaatgc cggcgctgca
14700agcaatgttg tccaggcagg tggatgagga acgtcagggg cagctgcaag gctcactggc
14760ggcgctcacc agcctgacct cgatcgtcgg acccctcctc ttcacggcga tctatgcggc
14820ttctataaca acgtggaacg ggtgggcatg gattgcaggc gctgccctct acttgctctg
14880cctgccggcg ctgcgtcgcg ggctttggag cggcgcaggg caacgagccg atcgctgatc
14940gtggaaacga taggcctatg ccatgcgggt caaggcgact tccggcaagc tatacgcgcc
15000ctaggagtgc ggttggaacg ttggcccagc cagatactcc cgatcacgag caggacgccg
15060atgatttgaa gcgcactcag cgtctgatcc aagaacaacc atcctagcaa cacggcggtc
15120cccgggctga gaaagcccag taaggaaaca actgtaggtt cgagtcgcga gatcccccgg
15180aaccaaagga agtaggttaa acccgctccg atcaggccga gccacgccag gccgagaaca
15240ttggttcctg taggcatcgg gattggcgga tcaaacacta aagctactgg aacgagcaga
15300agtcctccgg ccgccagttg ccaggcggta aaggtgagca gaggcacggg aggttgccac
15360ttgcgggtca gcacggttcc gaacgccatg gaaaccgccc ccgccaggcc cgctgcgacg
15420ccgacaggat ctagcgctgc gtttggtgtc aacaccaaca gcgccacgcc cgcagttccg
15480caaatagccc ccaggaccgc catcaatcgt atcgggctac ctagcagagc ggcagagatg
15540aacacgacca tcagcggctg cacagcgcct accgtcgccg cgaccccgcc cggcaggcgg
15600tagaccgaaa taaacaacaa gctccagaat agcgaaatat taagtgcgcc gaggatgaag
15660atgcgcatcc accagattcc cgttggaatc tgtcggacga tcatcacgag caataaaccc
15720gccggcaacg cccgcagcag cataccggcg acccctcggc ctcgctgttc gggctccacg
15780aaaacgccgg acagatgcgc cttgtgagcg tccttggggc cgtcctcctg tttgaagacc
15840gacagcccaa tgatctcgcc gtcgatgtag gcgccgaatg ccacggcatc tcgcaaccgt
15900tcagcgaacg cctccatggg ctttttctcc tcgtgctcgt aaacggaccc gaacatctct
15960ggagctttct tcagggccga caatcggatc tcgcggaaat cctgcacgtc ggccgctcca
16020agccgtcgaa tctgagcctt aatcacaatt gtcaatttta atcctctgtt tatcggcagt
16080tcgtagagcg cgccgtgcgt cccgagcgat actgagcgaa gcaagtgcgt cgagcagtgc
16140ccgcttgttc ctgaaatgcc agtaaagcgc tggctgctga acccccagcc ggaactgacc
16200ccacaaggcc ctagcgtttg caatgcacca ggtcatcatt gacccaggcg tgttccacca
16260ggccgctgcc tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc
16320gggtggaatc cgatccgcac atgaggcgga aggtttccag cttgagcggg tacggctccc
16380ggtgcgagct gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg cggtacttct
16440cccatatgaa tttcgtgtag tggtcgccag caaacagcac gacgatttcc tcgtcgatca
16500ggacctggca acgggacgtt ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg
16560acaccgattc caggtgccca acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc
16620gcgacaggca ttcctcggcc ttcgtgtaat accggccatt gatcgaccag cccaggtcct
16680ggcaaagctc gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact
16740ccaacacctg ctgccacacc agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg
16800tgatcttcac gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga
16860ttttcttgtt gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc atcgctcgca
16920tcgtgtccgg ccacggcgca atatcgaaca aggaaagctg catttccttg atctgctgct
16980tcgtgtgttt cagcaacgcg gcctgcttgg cctcgctgac ctgttttgcc aggtcctcgc
17040cggcggtttt tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg
17100ccaaacctgc cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg
17160gcagggcagg gggagccagt tgcacgctgt cgcgctcgat cttggccgta gcttgctgga
17220ccatcgagcc gacggactgg aaggtttcgc ggggcgcacg catgacggtg cggcttgcga
17280tggtttcggc atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc
17340ggtcaaacgt ccgattcatt caccctcctt gcgggattgc cccgactcac gccggggcaa
17400tgtgccctta ttcctgattt gacccgcctg gtgccttggt gtccagataa tccaccttat
17460cggcaatgaa gtcggtcccg tagaccgtct ggccgtcctt ctcgtacttg gtattccgaa
17520tcttgccctg cacgaatacc agcgacccct tgcccaaata cttgccgtgg gcctcggcct
17580gagagccaaa acacttgatg cggaagaagt cggtgcgctc ctgcttgtcg ccggcatcgt
17640tgcgccactc ttcattaacc gctatatcga aaattgcttg cggcttgtta gaattgccat
17700gacgtacctc ggtgtcacgg gtaagattac cgataaactg gaactgatta tggctcatat
17760cgaaagtctc cttgagaaag gagactctag tttagctaaa cattggttcc gctgtcaaga
17820actttagcgg ctaaaatttt gcgggccgcg accaaaggtg cgaggggcgg cttccgctgt
17880gtacaaccag atatttttca ccaacatcct tcgtctgctc gatgagcggg gcatgacgaa
17940acatgagctg tcggagaggg caggggtttc aatttcgttt ttatcagact taaccaacgg
18000taaggccaac ccctcgttga aggtgatgga ggccattgcc gacgccctgg aaactcccct
18060acctcttctc ctggagtcca ccgaccttga ccgcgaggca ctcgcggaga ttgcgggtca
18120tcctttcaag agcagcgtgc cgcccggata cgaacgcatc agtgtggttt tgccgtcaca
18180taaggcgttt atcgtaaaga aatggggcga cgacacccga aaaaagctgc gtggaaggct
18240ctgacgccaa gggttagggc ttgcacttcc ttctttagcc gctaaaacgg ccccttctct
18300gcgggccgtc ggctcgcgca tcatatcgac atcctcaacg gaagccgtgc cgcgaatggc
18360atcgggcggg tgcgctttga cagttgtttt ctatcagaac ccctacgtcg tgcggttcga
18420ttagctgttt gtcttgcagg ctaaacactt tcggtatatc gtttgcctgt gcgataatgt
18480tgctaatgat ttgttgcgta ggggttactg aaaagtgagc gggaaagaag agtttcagac
18540catcaaggag cgggccaagc gcaagctgga acgcgacatg ggtgcggacc tgttggccgc
18600gctcaacgac ccgaaaaccg ttgaagtcat gctcaacgcg gacggcaagg tgtggcacga
18660acgccttggc gagccgatgc ggtacatctg cgacatgcgg cccagccagt cgcaggcgat
18720tatagaaacg gtggccggat tccacggcaa agaggtcacg cggcattcgc ccatcctgga
18780aggcgagttc cccttggatg gcagccgctt tgccggccaa ttgccgccgg tcgtggccgc
18840gccaaccttt gcgatccgca agcgcgcggt cgccatcttc acgctggaac agtacgtcga
18900ggcgggcatc atgacccgcg agcaatacga ggtcattaaa agcgccgtcg cggcgcatcg
18960aaacatcctc gtcattggcg gtactggctc gggcaagacc acgctcgtca acgcgatcat
19020caatgaaatg gtcgccttca acccgtctga gcgcgtcgtc atcatcgagg acaccggcga
19080aatccagtgc gccgcagaga acgccgtcca ataccacacc agcatcgacg tctcgatgac
19140gctgctgctc aagacaacgc tgcgtatgcg ccccgaccgc atcctggtcg gtgaggtacg
19200tggccccgaa gcccttgatc tgttgatggc ctggaacacc gggcatgaag gaggtgccgc
19260caccctgcac gcaaacaacc ccaaagcggg cctgagccgg ctcgccatgc ttatcagcat
19320gcacccggat tcaccgaaac ccattgagcc gctgattggc gaggcggttc atgtggtcgt
19380ccatatcgcc aggaccccta gcggccgtcg agtgcaagaa attctcgaag ttcttggtta
19440cgagaacggc cagtacatca ccaaaaccct gtaaggagta tttccaatga caacggctgt
19500tccgttccgt ctgaccatga atcgcggcat tttgttctac cttgccgtgt tcttcgttct
19560cgctctcgcg ttatccgcgc atccggcgat ggcctcggaa ggcaccggcg gcagcttgcc
19620atatgagagc tggctgacga acctgcgcaa ctccgtaacc ggcccggtgg ccttcgcgct
19680gtccatcatc ggcatcgtcg tcgccggcgg cgtgctgatc ttcggcggcg aactcaacgc
19740cttcttccga accctgatct tcctggttct ggtgatggcg ctgctggtcg gcgcgcagaa
19800cgtgatgagc accttcttcg gtcgtggtgc cgaaatcgcg gccctcggca acggggcgct
19860gcaccaggtg caagtcgcgg cggcggatgc cgtgcgtgcg gtagcggctg gacggctcgc
19920ctaatcatgg ctctgcgcac gatccccatc cgtcgcgcag gcaaccgaga aaacctgttc
19980atgggtggtg atcgtgaact ggtgatgttc tcgggcctga tggcgtttgc gctgattttc
20040agcgcccaag agctgcgggc caccgtggtc ggtctgatcc tgtggttcgg ggcgctctat
20100gcgttccgaa tcatggcgaa ggccgatccg aagatgcggt tcgtgtacct gcgtcaccgc
20160cggtacaagc cgtattaccc ggcccgctcg accccgttcc gcgagaacac caatagccaa
20220gggaagcaat accgatgatc caagcaattg cgattgcaat cgcgggcctc ggcgcgcttc
20280tgttgttcat cctctttgcc cgcatccgcg cggtcgatgc cgaactgaaa ctgaaaaagc
20340atcgttccaa ggacgccggc ctggccgatc tgctcaacta cgccgctgtc gtcgatgacg
20400gcgtaatcgt gggcaagaac ggcagcttta tggctgcctg gctgtacaag ggcgatgaca
20460acgcaagcag caccgaccag cagcgcgaag tagtgtccgc ccgcatcaac caggccctcg
20520cgggcctggg aagtgggtgg atgatccatg tggacgccgt gcggcgtcct gctccgaact
20580acgcggagcg gggcctgtcg gcgttccctg accgtctgac ggcagcgatt gaagaagagc
20640gctcggtctt gccttgctcg tcggtgatgt acttcaccag ctccgcgaag tcgctcttct
20700tgatggagcg catggggacg tgcttggcaa tcacgcgcac cccccggccg ttttagcggc
20760taaaaaagtc atggctctgc cctcgggcgg accacgccca tcatgacctt gccaagctcg
20820tcctgcttct cttcgatctt cgccagcagg gcgaggatcg tggcatcacc gaaccgcgcc
20880gtgcgcgggt cgtcggtgag ccagagtttc agcaggccgc ccaggcggcc caggtcgcca
20940ttgatgcggg ccagctcgcg gacgtgctca tagtccacga cgcccgtgat tttgtagccc
21000tggccgacgg ccagcaggta ggccgacagg ctcatgccgg ccgccgccgc cttttcctca
21060atcgctcttc gttcgtctgg aaggcagtac accttgatag gtgggctgcc cttcctggtt
21120ggcttggttt catcagccat ccgcttgccc tcatctgtta cgccggcggt agccggccag
21180cctcgcagag caggattccc gttgagcacc gccaggtgcg aataagggac agtgaagaag
21240gaacacccgc tcgcgggtgg gcctacttca cctatcctgc ccggctgacg ccgttggata
21300caccaaggaa agtctacacg aaccctttgg caaaatcctg tatatcgtgc gaaaaaggat
21360ggatataccg aaaaaatcgc tataatgacc ccgaagcagg gttatgcagc ggaaaagcgc
21420tgcttccctg ctgttttgtg gaatatctac cgactggaaa caggcaaatg caggaaatta
21480ctgaactgag gggacaggcg agagacgatg ccaaagagct acaccgacga gctggccgag
21540tgggttgaat cccgcgcggc caagaagcgc cggcgtgatg aggctgcggt tgcgttcctg
21600gcggtgaggg cggatgtcga ggcggcgtta gcgtccggct atgcgctcgt caccatttgg
21660gagcacatgc gggaaacggg gaaggtcaag ttctcctacg agacgttccg ctcgcacgcc
21720aggcggcaca tcaaggccaa gcccgccgat gtgcccgcac cgcaggccaa ggctgcggaa
21780cccgcgccgg cacccaagac gccggagcca cggcggccga agcagggggg caaggctgaa
21840aagccggccc ccgctgcggc cccgaccggc ttcaccttca acccaacacc ggacaaaaag
21900gatctactgt aatggcgaaa attcacatgg ttttgcaggg caagggcggg gtcggcaagt
21960cggccatcgc cgcgatcatt gcgcagtaca agatggacaa ggggcagaca cccttgtgca
22020tcgacaccga cccggtgaac gcgacgttcg agggctacaa ggccctgaac gtccgccggc
22080tgaacatcat ggccggcgac gaaattaact cgcgcaactt cgacaccctg gtcgagctga
22140ttgcgccgac caaggatgac gtggtgatcg acaacggtgc cagctcgttc gtgcctctgt
22200cgcattacct catcagcaac caggtgccgg ctctgctgca agaaatgggg catgagctgg
22260tcatccatac cgtcgtcacc ggcggccagg ctctcctgga cacggtgagc ggcttcgccc
22320agctcgccag ccagttcccg gccgaagcgc ttttcgtggt ctggctgaac ccgtattggg
22380ggcctatcga gcatgagggc aagagctttg agcagatgaa ggcgtacacg gccaacaagg
22440cccgcgtgtc gtccatcatc cagattccgg ccctcaagga agaaacctac ggccgcgatt
22500tcagcgacat gctgcaagag cggctgacgt tcgaccaggc gctggccgat gaatcgctca
22560cgatcatgac gcggcaacgc ctcaagatcg tgcggcgcgg cctgtttgaa cagctcgacg
22620cggcggccgt gctatgagcg accagattga agagctgatc cgggagattg cggccaagca
22680cggcatcgcc gtcggccgcg acgacccggt gctgatcctg cataccatca acgcccggct
22740catggccgac agtgcggcca agcaagagga aatccttgcc gcgttcaagg aagagctgga
22800agggatcgcc catcgttggg gcgaggacgc caaggccaaa gcggagcgga tgctgaacgc
22860ggccctggcg gccagcaagg acgcaatggc gaaggtaatg aaggacagcg ccgcgcaggc
22920ggccgaagcg atccgcaggg aaatcgacga cggccttggc cgccagctcg cggccaaggt
22980cgcggacgcg cggcgcgtgg cgatgatgaa catgatcgcc ggcggcatgg tgttgttcgc
23040ggccgccctg gtggtgtggg cctcgttatg aatcgcagag gcgcagatga aaaagcccgg
23100cgttgccggg ctttgttttt gcgttagctg ggcttgtttg acaggcccaa gctctgactg
23160cgcccgcgct cgcgctcctg ggcctgtttc ttctcctgct cctgcttgcg catcagggcc
23220tggtgccgtc gggctgcttc acgcatcgaa tcccagtcgc cggccagctc gggatgctcc
23280gcgcgcatct tgcgcgtcgc cagttcctcg atcttgggcg cgtgaatgcc catgccttcc
23340ttgatttcgc gcaccatgtc cagccgcgtg tgcagggtct gcaagcgggc ttgctgttgg
23400gcctgctgct gctgccaggc ggcctttgta cgcggcaggg acagcaagcc gggggcattg
23460gactgtagct gctgcaaacg cgcctgctga cggtctacga gctgttctag gcggtcctcg
23520atgcgctcca cctggtcatg ctttgcctgc acgtagagcg caagggtctg ctggtaggtc
23580tgctcgatgg gcgcggattc taagagggcc tgctgttccg tctcggcctc ctgggccgcc
23640tgtagcaaat cctcgccgct gttgccgctg gactgcttta ctgccgggga ctgctgttgc
23700cctgctcgcg ccgtcgtcgc agttcggctt gcccccactc gattgactgc ttcatttcga
23760gccgcagcga tgcgatctcg gattgcgtca acggacgggg cagcgcggag gtgtccggct
23820tctccttggg tgagtcggtc gatgccatag ccaaaggttt ccttccaaaa tgcgtccatt
23880gctggaccgt gtttctcatt gatgcccgca agcatcttcg gcttgaccgc caggtcaagc
23940gcgccttcat gggcggtcat gacggacgcc gccatgacct tgccgccgtt gttctcgatg
24000tagccgcgta atgaggcaat ggtgccgccc atcgtcagcg tgtcatcgac aacgatgtac
24060ttctggccgg ggatcacctc cccctcgaaa gtcgggttga acgccaggcg atgatctgaa
24120ccggctccgg ttcgggcgac cttctcccgc tgcacaatgt ccgtttcgac ctcaaggcca
24180aggcggtcgg ccagaacgac cgccatcatg gccggaatct tgttgttccc cgccgcctcg
24240acggcgagga ctggaacgat gcggggcttg tcgtcgccga tcagcgtctt gagctgggca
24300acagtgtcgt ccgaaatcag gcgctcgacc aaattaagcg ccgcttccgc gtcgccctgc
24360ttcgcagcct ggtattcagg ctcgttggtc aaagaaccaa ggtcgccgtt gcgaaccacc
24420ttcgggaagt ctccccacgg tgcgcgctcg gctctgctgt agctgctcaa gacgcctccc
24480tttttagccg ctaaaactct aacgagtgcg cccgcgactc aacttgacgc tttcggcact
24540tacctgtgcc ttgccacttg cgtcataggt gatgcttttc gcactcccga tttcaggtac
24600tttatcgaaa tctgaccggg cgtgcattac aaagttcttc cccacctgtt ggtaaatgct
24660gccgctatct gcgtggacga tgctgccgtc gtggcgctgc gacttatcgg ccttttgggc
24720catatagatg ttgtaaatgc caggtttcag ggccccggct ttatctacct tctggttcgt
24780ccatgcgcct tggttctcgg tctggacaat tctttgccca ttcatgacca ggaggcggtg
24840tttcattggg tgactcctga cggttgcctc tggtgttaaa cgtgtcctgg tcgcttgccg
24900gctaaaaaaa agccgacctc ggcagttcga ggccggcttt ccctagagcc gggcgcgtca
24960aggttgttcc atctatttta gtgaactgcg ttcgatttat cagttacttt cctcccgctt
25020tgtgtttcct cccactcgtt tccgcgtcta gccgacccct caacatagcg gcctcttctt
25080gggctgcctt tgcctcttgc cgcgcttcgt cacgctcggc ttgcaccgtc gtaaagcgct
25140cggcctgcct ggccgcctct tgcgccgcca acttcctttg ctcctggtgg gcctcggcgt
25200cggcctgcgc cttcgctttc accgctgcca actccgtgcg caaactctcc gcttcgcgcc
25260tggtggcgtc gcgctcgccg cgaagcgcct gcatttcctg gttggccgcg tccagggtct
25320tgcggctctc ttctttgaat gcgcgggcgt cctggtgagc gtagtccagc tcggcgcgca
25380gctcctgcgc tcgacgctcc acctcgtcgg cccgctgcgt cgccagcgcg gcccgctgct
25440cggctcctgc cagggcggtg cgtgcttcgg ccagggcttg ccgctggcgt gcggccagct
25500cggccgcctc ggcggcctgc tgctctagca atgtaacgcg cgcctgggct tcttccagct
25560cgcgggcctg cgcctcgaag gcgtcggcca gctccccgcg cacggcttcc aactcgttgc
25620gctcacgatc ccagccggct tgcgctgcct gcaacgattc attggcaagg gcctgggcgg
25680cttgccagag ggcggccacg gcctggttgc cggcctgctg caccgcgtcc ggcacctgga
25740ctgccagcgg ggcggcctgc gccgtgcgct ggcgtcgcca ttcgcgcatg ccggcgctgg
25800cgtcgttcat gttgacgcgg gcggccttac gcactgcatc cacggtcggg aagttctccc
25860ggtcgccttg ctcgaacagc tcgtccgcag ccgcaaaaat gcggtcgcgc gtctctttgt
25920tcagttccat gttggctccg gtaattggta agaataataa tactcttacc taccttatca
25980gcgcaagagt ttagctgaac agttctcgac ttaacggcag gttttttagc ggctgaaggg
26040caggcaaaaa aagccccgca cggtcggcgg gggcaaaggg tcagcgggaa ggggattagc
26100gggcgtcggg cttcttcatg cgtcggggcc gcgcttcttg ggatggagca cgacgaagcg
26160cgcacgcgca tcgtcctcgg ccctatcggc ccgcgtcgcg gtcaggaact tgtcgcgcgc
26220taggtcctcc ctggtgggca ccaggggcat gaactcggcc tgctcgatgt aggtccactc
26280catgaccgca tcgcagtcga ggccgcgttc cttcaccgtc tcttgcaggt cgcggtacgc
26340ccgctcgttg agcggctggt aacgggccaa ttggtcgtaa atggctgtcg gccatgagcg
26400gcctttcctg ttgagccagc agccgacgac gaagccggca atgcaggccc ctggcacaac
26460caggccgacg ccgggggcag gggatggcag cagctcgcca accaggaacc ccgccgcgat
26520gatgccgatg ccggtcaacc agcccttgaa actatccggc cccgaaacac ccctgcgcat
26580tgcctggatg ctgcgccgga tagcttgcaa catcaggagc cgtttctttt gttcgtcagt
26640catggtccgc cctcaccagt tgttcgtatc ggtgtcggac gaactgaaat cgcaagagct
26700gccggtatcg gtccagccgc tgtccgtgtc gctgctgccg aagcacggcg aggggtccgc
26760gaacgccgca gacggcgtat ccggccgcag cgcatcgccc agcatggccc cggtcagcga
26820gccgccggcc aggtagccca gcatggtgct gttggtcgcc ccggccacca gggccgacgt
26880gacgaaatcg ccgtcattcc ctctggattg ttcgctgctc ggcggggcag tgcgccgcgc
26940cggcggcgtc gtggatggct cgggttggct ggcctgcgac ggccggcgaa aggtgcgcag
27000cagctcgtta tcgaccggct gcggcgtcgg ggccgccgcc ttgcgctgcg gtcggtgttc
27060cttcttcggc tcgcgcagct tgaacagcat gatcgcggaa accagcagca acgccgcgcc
27120tacgcctccc gcgatgtaga acagcatcgg attcattctt cggtcctcct tgtagcggaa
27180ccgttgtctg tgcggcgcgg gtggcccgcg ccgctgtctt tggggatcag ccctcgatga
27240gcgcgaccag tttcacgtcg gcaaggttcg cctcgaactc ctggccgtcg tcctcgtact
27300tcaaccaggc atagccttcc gccggcggcc gacggttgag gataaggcgg gcagggcgct
27360cgtcgtgctc gacctggacg atggcctttt tcagcttgtc cgggtccggc tccttcgcgc
27420ccttttcctt ggcgtcctta ccgtcctggt cgccgtcctc gccgtcctgg ccgtcgccgg
27480cctccgcgtc acgctcggca tcagtctggc cgttgaaggc atcgacggtg ttgggatcgc
27540ggcccttctc gtccaggaac tcgcgcagca gcttgaccgt gccgcgcgtg atttcctggg
27600tgtcgtcgtc aagccacgcc tcgacttcct ccgggcgctt cttgaaggcc gtcaccagct
27660cgttcaccac ggtcacgtcg cgcacgcggc cggtgttgaa cgcatcggcg atcttctccg
27720gcaggtccag cagcgtgacg tgctgggtga tgaacgccgg cgacttgccg atttccttgg
27780cgatatcgcc tttcttcttg cccttcgcca gctcgcggcc aatgaagtcg gcaatttcgc
27840gcggggtcag ctcgttgcgt tgcaggttct cgataacctg gtcggcttcg ttgtagtcgt
27900tgtcgatgaa cgccgggatg gacttcttgc cggcccactt cgagccacgg tagcggcggg
27960cgccgtgatt gatgatatag cggcccggct gctcctggtt ctcgcgcacc gaaatgggtg
28020acttcacccc gcgctctttg atcgtggcac cgatttccgc gatgctctcc ggggaaaagc
28080cggggttgtc ggccgtccgc ggctgatgcg gatcttcgtc gatcaggtcc aggtccagct
28140cgatagggcc ggaaccgccc tgagacgccg caggagcgtc caggaggctc gacaggtcgc
28200cgatgctatc caaccccagg ccggacggct gcgccgcgcc tgcggcttcc tgagcggccg
28260cagcggtgtt tttcttggtg gtcttggctt gagccgcagt cattgggaaa tctccatctt
28320cgtgaacacg taatcagcca gggcgcgaac ctctttcgat gccttgcgcg cggccgtttt
28380cttgatcttc cagaccggca caccggatgc gagggcatcg gcgatgctgc tgcgcaggcc
28440aacggtggcc ggaatcatca tcttggggta cgcggccagc agctcggctt ggtggcgcgc
28500gtggcgcgga ttccgcgcat cgaccttgct gggcaccatg ccaaggaatt gcagcttggc
28560gttcttctgg cgcacgttcg caatggtcgt gaccatcttc ttgatgccct ggatgctgta
28620cgcctcaagc tcgatggggg acagcacata gtcggccgcg aagagggcgg ccgccaggcc
28680gacgccaagg gtcggggccg tgtcgatcag gcacacgtcg aagccttggt tcgccagggc
28740cttgatgttc gccccgaaca gctcgcgggc gtcgtccagc gacagccgtt cggcgttcgc
28800cagtaccggg ttggactcga tgagggcgag gcgcgcggcc tggccgtcgc cggctgcggg
28860tgcggtttcg gtccagccgc cggcagggac agcgccgaac agcttgcttg catgcaggcc
28920ggtagcaaag tccttgagcg tgtaggacgc attgccctgg gggtccaggt cgatcacggc
28980aacccgcaag ccgcgctcga aaaagtcgaa ggcaagatgc acaagggtcg aagtcttgcc
29040gacgccgcct ttctggttgg ccgtgaccaa agttttcatc gtttggtttc ctgttttttc
29100ttggcgtccg cttcccactt ccggacgatg tacgcctgat gttccggcag aaccgccgtt
29160acccgcgcgt acccctcggg caagttcttg tcctcgaacg cggcccacac gcgatgcacc
29220gcttgcgaca ctgcgcccct ggtcagtccc agcgacgttg cgaacgtcgc ctgtggcttc
29280ccatcgacta agacgccccg cgctatctcg atggtctgct gccccacttc cagcccctgg
29340atcgcctcct ggaactggct ttcggtaagc cgtttcttca tggataacac ccataatttg
29400ctccgcgcct tggttgaaca tagcggtgac agccgccagc acatgagaga agtttagcta
29460aacatttctc gcacgtcaac acctttagcc gctaaaactc gtccttggcg taacaaaaca
29520aaagcccgga aaccgggctt tcgtctcttg ccgcttatgg ctctgcaccc ggctccatca
29580ccaacaggtc gcgcacgcgc ttcactcggt tgcggatcga cactgccagc ccaacaaagc
29640cggttgccgc cgccgccagg atcgcgccga tgatgccggc cacaccggcc atcgcccacc
29700aggtcgccgc cttccggttc cattcctgct ggtactgctt cgcaatgctg gacctcggct
29760caccataggc tgaccgctcg atggcgtatg ccgcttctcc ccttggcgta aaacccagcg
29820ccgcaggcgg cattgccatg ctgcccgccg ctttcccgac cacgacgcgc gcaccaggct
29880tgcggtccag accttcggcc acggcgagct gcgcaaggac ataatcagcc gccgacttgg
29940ctccacgcgc ctcgatcagc tcttgcactc gcgcgaaatc cttggcctcc acggccgcca
30000tgaatcgcgc acgcggcgaa ggctccgcag ggccggcgtc gtgatcgccg ccgagaatgc
30060ccttcaccaa gttcgacgac acgaaaatca tgctgacggc tatcaccatc atgcagacgg
30120atcgcacgaa cccgctgaat tgaacacgag cacggcaccc gcgaccacta tgccaagaat
30180gcccaaggta aaaattgccg gccccgccat gaagtccgtg aatgccccga cggccgaagt
30240gaagggcagg ccgccaccca ggccgccgcc ctcactgccc ggcacctggt cgctgaatgt
30300cgatgccagc acctgcggca cgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca
30360tcccgttact gccccgatcc cggcaatggc aaggactgcc agcgctgcca tttttggggt
30420gaggccgttc gcggccgagg ggcgcagccc ctggggggat gggaggcccg cgttagcggg
30480ccgggagggt tcgagaaggg ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg
30540cgcagccctg gttaaaaaca aggtttataa atattggttt aaaagcaggt taaaagacag
30600gttagcggtg gccgaaaaac gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg
30660acagcccctc aaatgtcaat aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg
30720tcaaggatcg cgcccctcat ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg
30780cacttatccc caggcttgtc cacatcatct gtgggaaact cgcgtaaaat caggcgtttt
30840cgccgatttg cgaggctggc cagctccacg tcgccggccg aaatcgagcc tgcccctcat
30900ctgtcaacgc cgcgccgggt gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt
30960cagtgagggc caagttttcc gcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac
31020acggcttcga cggcgtttct ggcgcgtttg cagggccata gacggccgcc agcccagcgg
31080cgagggcaac cagcccggtg agcgtcggaa aggcgctgga agccccgtag cgacgcggag
31140aggggcgaga caagccaagg gcgcaggctc gatgcgcagc acgacatagc cggttctcgc
31200aaggacgaga atttccctgc ggtgcccctc aagtgtcaat gaaagtttcc aacgcgagcc
31260attcgcgaga gccttgagtc cacgctagat gagagctttg ttgtaggtgg accagttggt
31320gattttgaac ttttgctttg ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg
31380atccttcaac tcagcaaaag ttcgatttat tcaacaaagc cacgttgtgt ctcaaaatct
31440ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
31500cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctcgac
31560tctagagctc gttcctcgag gaacggtacc tgcggggaag cttacaataa tgtgtgttgt
31620taagtcttgt tgcctgtcat cgtctgactg actttcgtca taaatcccgg cctccgtaac
31680ccagctttgg gcaagctcac ggatttgatc cggcggaacg ggaatatcga gatgccgggc
31740tgaacgctgc agttccagct ttccctttcg ggacaggtac tccagctgat tgattatctg
31800ctgaagggtc ttggttccac ctcctggcac aatgcgaatg attacttgag cgcgatcggg
31860catccaattt tctcccgtca ggtgcgtggt caagtgctac aaggcacctt tcagtaacga
31920gcgaccgtcg atccgtcgcc gggatacgga caaaatggag cgcagtagtc catcgagggc
31980ggcgaaagcc tcgccaaaag caatacgttc atctcgcaca gcctccagat ccgatcgagg
32040gtcttcggcg taggcagata gaagcatgga tacattgctt gagagtattc cgatggactg
32100aagtatggct tccatctttt ctcgtgtgtc tgcatctatt tcgagaaagc ccccgatgcg
32160gcgcaccgca acgcgaattg ccatactatc cgaaagtccc agcaggcgcg cttgatagga
32220aaaggtttca tactcggccg atcgcagacg ggcactcacg accttgaacc cttcaacttt
32280cagggatcga tgctggttga tggtagtctc actcgacgtg gctctggtgt gttttgacat
32340agcttcctcc aaagaaagcg gaaggtctgg atactccagc acgaaatgtg cccgggtaga
32400cggatggaag tctagccctg ctcaatatga aatcaacagt acatttacag tcaatactga
32460atatacttgc tacatttgca attgtcttat aacgaatgtg aaataaaaat agtgtaacaa
32520cgcttttact catcgataat cacaaaaaca tttatacgaa caaaaataca aatgcactcc
32580ggtttcacag gataggcggg atcagaatat gcaacttttg acgttttgtt ctttcaaagg
32640gggtgctggc aaaaccaccg cactcatggg cctttgcgct gctttggcaa atgacggtaa
32700acgagtggcc ctctttgatg ccgacgaaaa ccggcctctg acgcgatgga gagaaaacgc
32760cttacaaagc agtactggga tcctcgctgt gaagtctatt ccgccgacga aatgcccctt
32820cttgaagcag cctatgaaaa tgccgagctc gaaggatttg attatgcgtt ggccgatacg
32880cgtggcggct cgagcgagct caacaacaca atcatcgcta gctcaaacct gcttctgatc
32940cccaccatgc taacgccgct cgacatcgat gaggcactat ctacctaccg ctacgtcatc
33000gagctgctgt tgagtgaaaa tttggcaatt cctacagctg ttttgcgcca acgcgtcccg
33060gtcggccgat tgacaacatc gcaacgcagg atgtcagaga cgctagagag ccttccagtt
33120gtaccgtctc ccatgcatga aagagatgca tttgccgcga tgaaagaacg cggcatgttg
33180catcttacat tactaaacac gggaactgat ccgacgatgc gcctcataga gaggaatctt
33240cggattgcga tggaggaagt cgtggtcatt tcgaaactga tcagcaaaat cttggaggct
33300tgaagatggc aattcgcaag cccgcattgt cggtcggcga agcacggcgg cttgctggtg
33360ctcgacccga gatccaccat cccaacccga cacttgttcc ccagaagctg gacctccagc
33420acttgcctga aaaagccgac gagaaagacc agcaacgtga gcctctcgtc gccgatcaca
33480tttacagtcc cgatcgacaa cttaagctaa ctgtggatgc ccttagtcca cctccgtccc
33540cgaaaaagct ccaggttttt ctttcagcgc gaccgcccgc gcctcaagtg tcgaaaacat
33600atgacaacct cgttcggcaa tacagtccct cgaagtcgct acaaatgatt ttaaggcgcg
33660cgttggacga tttcgaaagc atgctggcag atggatcatt tcgcgtggcc ccgaaaagtt
33720atccgatccc ttcaactaca gaaaaatccg ttctcgttca gacctcacgc atgttcccgg
33780ttgcgttgct cgaggtcgct cgaagtcatt ttgatccgtt ggggttggag accgctcgag
33840ctttcggcca caagctggct accgccgcgc tcgcgtcatt ctttgctgga gagaagccat
33900cgagcaattg gtgaagaggg acctatcgga acccctcacc aaatattgag tgtaggtttg
33960aggccgctgg ccgcgtcctc agtcaccttt tgagccagat aattaagagc caaatgcaat
34020tggctcaggc tgccatcgtc cccccgtgcg aaacctgcac gtccgcgtca aagaaataac
34080cggcacctct tgctgttttt atcagttgag ggcttgacgg atccgcctca agtttgcggc
34140gcagccgcaa aatgagaaca tctatactcc tgtcgtaaac ctcctcgtcg cgtactcgac
34200tggcaatgag aagttgctcg cgcgatagaa cgtcgcgggg tttctctaaa aacgcgagga
34260gaagattgaa ctcacctgcc gtaagtttca cctcaccgcc agcttcggac atcaagcgac
34320gttgcctgag attaagtgtc cagtcagtaa aacaaaaaga ccgtcggtct ttggagcgga
34380caacgttggg gcgcacgcgc aaggcaaccc gaatgcgtgc aagaaactct ctcgtactaa
34440acggcttagc gataaaatca cttgctccta gctcgagtgc aacaacttta tccgtctcct
34500caaggcggtc gccactgata attatgattg gaatatcaga ctttgccgcc agatttcgaa
34560cgatctcaag cccatcttca cgacctaaat ttagatcaac aaccacgaca tcgaccgtcg
34620cggaagagag tactctagtg aactgggtgc tgtcggctac cgcggtcact ttgaaggcgt
34680ggatcgtaag gtattcgata ataagatgcc gcatagcgac atcgtcatcg ataagaagaa
34740cgtgtttcaa cggctcacct ttcaatctaa aatctgaacc cttgttcaca gcgcttgaga
34800aattttcacg tgaaggatgt acaatcatct ccagctaaat gggcagttcg tcagaattgc
34860ggctgaccgc ggatgacgaa aatgcgaacc aagtatttca attttatgac aaaagttctc
34920aatcgttgtt acaagtgaaa cgcttcgagg ttacagctac tattgattaa ggagatcgcc
34980tatggtctcg ccccggcgtc gtgcgtccgc cgcgagccag atctcgccta cttcataaac
35040gtcctcatag gcacggaatg gaatgatgac atcgatcgcc gtagagagca tgtcaatcag
35100tgtgcgatct tccaagctag caccttgggc gctacttttg acaagggaaa acagtttctt
35160gaatccttgg attggattcg cgccgtgtat tgttgaaatc gatcccggat gtcccgagac
35220gacttcactc agataagccc atgctgcatc gtcgcgcatc tcgccaagca atatccggtc
35280cggccgcata cgcagacttg cttggagcaa gtgctcggcg ctcacagcac ccagcccagc
35340accgttcttg gagtagagta gtctaacatg attatcgtgt ggaatgacga gttcgagcgt
35400atcttctatg gtgattagcc tttcctgggg ggggatggcg ctgatcaagg tcttgctcat
35460tgttgtcttg ccgcttccgg tagggccaca tagcaacatc gtcagtcggc tgacgacgca
35520tgcgtgcaga aacgcttcca aatccccgtt gtcaaaatgc tgaaggatag cttcatcatc
35580ctgattttgg cgtttccttc gtgtctgcca ctggttccac ctcgaagcat cataacggga
35640ggagacttct ttaagaccag aaacacgcga gcttggccgt cgaatggtca agctgacggt
35700gcccgaggga acggtcggcg gcagacagat ttgtagtcgt tcaccaccag gaagttcagt
35760ggcgcagagg gggttacgtg gtccgacatc ctgctttctc agcgcgcccg ctaaaatagc
35820gatatcttca agatcatcat aagagacggg caaaggcatc ttggtaaaaa tgccggcttg
35880gcgcacaaat gcctctccag gtcgattgat cgcaatttct tcagtcttcg ggtcatcgag
35940ccattccaaa atcggcttca gaagaaagcg tagttgcgga tccacttcca tttacaatgt
36000atcctatctc taagcggaaa tttgaattca ttaagagcgg cggttcctcc cccgcgtggc
36060gccgccagtc aggcggagct ggtaaacacc aaagaaatcg aggtcccgtg ctacgaaaat
36120ggaaacggtg tcaccctgat tcttcttcag ggttggcggt atgttgatgg ttgccttaag
36180ggctgtctca gttgtctgct caccgttatt ttgaaagctg ttgaagctca tcccgccacc
36240cgagctgccg gcgtaggtgc tagctgcctg gaaggcgcct tgaacaacac tcaagagcat
36300agctccgcta aaacgctgcc agaagtggct gtcgaccgag cccggcaatc ctgagcgacc
36360gagttcgtcc gcgcttggcg atgttaacga gatcatcgca tggtcaggtg tctcggcgcg
36420atcccacaac acaaaaacgc gcccatctcc ctgttgcaag ccacgctgta tttcgccaac
36480aacggtggtg ccacgatcaa gaagcacgat attgttcgtt gttccacgaa tatcctgagg
36540caagacacac tttacatagc ctgccaaatt tgtgtcgatt gcggtttgca agatgcacgg
36600aattattgtc ccttgcgtta ccataaaatc ggggtgcggc aagagcgtgg cgctgctggg
36660ctgcagctcg gtgggtttca tacgtatcga caaatcgttc tcgccggaca cttcgccatt
36720cggcaaggag ttgtcgtcac gcttgccttc ttgtcttcgg cccgtgtcgc cctgaatggc
36780gcgtttgctg accccttgat cgccgctgct atatgcaaaa atcggtgttt cttccggccg
36840tggctcatgc cgctccggtt cgcccctcgg cggtagagga gcagcaggct gaacagcctc
36900ttgaaccgct ggaggatccg gcggcacctc aatcggagct ggatgaaatg gcttggtgtt
36960tgttgcgatc aaagttgacg gcgatgcgtt ctcattcacc ttcttttggc gcccacctag
37020ccaaatgagg cttaatgata acgcgagaac gacacctccg acgatcaatt tctgagaccc
37080cgaaagacgc cggcgatgtt tgtcggagac cagggatcca gatgcatcaa cctcatgtgc
37140cgcttgctga ctatcgttat tcatcccttc gcccccttca ggacgcgttt cacatcgggc
37200ctcaccgtgc ccgtttgcgg cctttggcca acgggatcgt aagcggtgtt ccagatacat
37260agtactgtgt ggccatccct cagacgccaa cctcgggaaa ccgaagaaat ctcgacatcg
37320ctccctttaa ctgaatagtt ggcaacagct tccttgccat caggattgat ggtgtagatg
37380gagggtatgc gtacattgcc cggaaagtgg aataccgtcg taaatccatt gtcgaagact
37440tcgagtggca acagcgaacg atcgccttgg gcgacgtagt gccaattact gtccgccgca
37500ccaagggctg tgacaggctg atccaataaa ttctcagctt tccgttgata ttgtgcttcc
37560gcgtgtagtc tgtccacaac agccttctgt tgtgcctccc ttcgccgagc cgccgcatcg
37620tcggcggggt aggcgaattg gacgctgtaa tagagatcgg gctgctcttt atcgaggtgg
37680gacagagtct tggaacttat actgaaaaca taacggcgca tcccggagtc gcttgcggtt
37740agcacgatta ctggctgagg cgtgaggacc tggcttgcct tgaaaaatag ataatttccc
37800cgcggtaggg ctgctagatc tttgctattt gaaacggcaa ccgctgtcac cgtttcgttc
37860gtggcgaatg ttacgaccaa agtagctcca accgccgtcg agaggcgcac cacttgatcg
37920ggattgtaag ccaaataacg catgcgcgga tctagcttgc ccgccattgg agtgtcttca
37980gcctccgcac cagtcgcagc ggcaaataaa catgctaaaa tgaaaagtgc ttttctgatc
38040atggttcgct gtggcctacg tttgaaacgg tatcttccga tgtctgatag gaggtgacaa
38100ccagacctgc cgggttggtt agtctcaatc tgccgggcaa gctggtcacc ttttcgtagc
38160gaactgtcgc ggtccacgta ctcaccacag gcattttgcc gtcaacgacg agggtccttt
38220tatagcgaat ttgctgcgtg cttggagtta catcatttga agcgatgtgc tcgacctcca
38280ccctgccgcg tttgccaaga atgacttgag gcgaactggg attgggatag ttgaagaatt
38340gctggtaatc ctggcgcact gttggggcac tgaagttcga taccaggtcg taggcgtact
38400gagcggtgtc ggcatcataa ctctcgcgca ggcgaacgta ctcccacaat gaggcgttaa
38460cgacggcctc ctcttgagtt gcaggcaatc gcgagacaga cacctcgctg tcaacggtgc
38520cgtccggccg tatccataga tatacgggca caagcctgct caacggcacc attgtggcta
38580tagcgaacgc ttgagcaaca tttcccaaaa tcgcgatagc tgcgacagct gcaatgagtt
38640tggagagacg tcgcgccgat ttcgctcgcg cggtttgaaa ggcttctact tccttatagt
38700gctcggcaag gctttcgcgc gccactagca tggcatattc aggccccgtc atagcgtcca
38760cccgaattgc cgagctgaag atctgacgga gtaggctgcc atcgccccac attcagcggg
38820aagatcgggc ctttgcagct cgctaatgtg tcgtttgtct ggcagccgct caaagcgaca
38880actaggcaca gcaggcaata cttcatagaa ttctccattg aggcgaattt ttgcgcgacc
38940tagcctcgct caacctgagc gaagcgacgg tacaagctgc tggcagattg ggttgcgccg
39000ctccagtaac tgcctccaat gttgccggcg atcgccggca aagcgacaat gagcgcatcc
39060cctgtcagaa aaaacatatc gagttcgtaa agaccaatga tcttggccgc ggtcgtaccg
39120gcgaaggtga ttacaccaag cataagggtg agcgcagtcg cttcggttag gatgacgatc
39180gttgccacga ggtttaagag gagaagcaag agaccgtagg tgataagttg cccgatccac
39240ttagctgcga tgtcccgcgt gcgatcaaaa atatatccga cgaggatcag aggcccgatc
39300gcgagaagca ctttcgtgag aattccaacg gcgtcgtaaa ctccgaaggc agaccagagc
39360gtgccgtaaa ggacccactg tgccccttgg aaagcaagga tgtcctggtc gttcatcgga
39420ccgatttcgg atgcgatttt ctgaaaaacg gcctgggtca cggcgaacat tgtatccaac
39480tgtgccggaa cagtctgcag aggcaagccg gttacactaa actgctgaac aaagtttggg
39540accgtctttt cgaagatgga aaccacatag tcttggtagt tagcctgccc aacaattaga
39600gcaacaacga tggtgaccgt gatcacccga gtgataccgc tacgggtatc gacttcgccg
39660cgtatgacta aaataccctg aacaataatc caaagagtga cacaggcgat caatggcgca
39720ctcaccgcct cctggatagt ctcaagcatc gagtccaagc ctgtcgtgaa ggctacatcg
39780aagatcgtat gaatggccgt aaacggcgcc ggaatcgtga aattcatcga ttggacctga
39840acttgactgg tttgtcgcat aatgttggat aaaatgagct cgcattcggc gaggatgcgg
39900gcggatgaac aaatcgccca gccttagggg agggcaccaa agatgacagc ggtcttttga
39960tgctccttgc gttgagcggc cgcctcttcc gcctcgtgaa ggccggcctg cgcggtagtc
40020atcgttaata ggcttgtcgc ctgtacattt tgaatcattg cgtcatggat ctgcttgaga
40080agcaaaccat tggtcacggt tgcctgcatg atattgcgag atcgggaaag ctgagcagac
40140gtatcagcat tcgccgtcaa gcgtttgtcc atcgtttcca gattgtcagc cgcaatgcca
40200gcgctgtttg cggaaccggt gatctgcgat cgcaacaggt ccgcttcagc atcactaccc
40260acgactgcac gatctgtatc gctggtgatc gcacgtgccg tggtcgacat tggcattcgc
40320ggcgaaaaca tttcattgtc taggtccttc gtcgaaggat actgattttt ctggttgagc
40380gaagtcagta gtccagtaac gccgtaggcc gacgtcaaca tcgtaaccat cgctatagtc
40440tgagtgagat tctccgcagt cgcgagcgca gtcgcgagcg tctcagcctc cgttgccggg
40500tcgctaacaa caaactgcgc ccgcgcgggc tgaatatata gaaagctgca ggtcaaaact
40560gttgcaataa gttgcgtcgt cttcatcgtt tcctacctta tcaatcttct gcctcgtggt
40620gacgggccat gaattcgctg agccagccag atgagttgcc ttcttgtgcc tcgcgtagtc
40680gagttgcaaa gcgcaccgtg ttggcacgcc ccgaaagcac ggcgacatat tcacgcatat
40740cccgcagatc aaattcgcag atgacgcttc cactttctcg tttaagaaga aacttacggc
40800tgccgaccgt catgtcttca cggatcgcct gaaattcctt ttcggtacat ttcagtccat
40860cgacataagc cgatcgatct gcggttggtg atggatagaa aatcttcgtc atacattgcg
40920caaccaagct ggctcctagc ggcgattcca gaacatgctc tggttgctgc gttgccagta
40980ttagcatccc gttgtttttt cgaacggtca ggaggaattt gtcgacgaca gtcgaaaatt
41040tagggtttaa caaataggcg cgaaactcat cgcagctcat cacaaaacgg cggccgtcga
41100tcatggctcc aatccgatgc aggagatatg ctgcagcggg agcgcatact tcctcgtatt
41160cgagaagatg cgtcatgtcg aagccggtaa tcgacggatc taactttact tcgtcaactt
41220cgccgtcaaa tgcccagcca agcgcatggc cccggcacca gcgttggagc cgcgctcctg
41280cgccttcggc gggcccatgc aacaaaaatt cacgtaaccc cgcgattgaa cgcatttgtg
41340gatcaaacga gagctgacga tggataccac ggaccagacg gcggttctct tccggagaaa
41400tcccaccccg accatcactc tcgatgagag ccacgatcca ttcgcgcaga aaatcgtgtg
41460aggctgctgt gttttctagg ccacgcaacg gcgccaaccc gctgggtgtg cctctgtgaa
41520gtgccaaata tgttcctcct gtggcgcgaa ccagcaattc gccaccccgg tccttgtcaa
41580agaacacgac cgtacctgca cggtcgacca tgctctgttc gagcatggct agaacaaaca
41640tcatgagcgt cgtcttaccc ctcccgatag gcccgaatat tgccgtcatg ccaacatcgt
41700gctcatgcgg gatatagtcg aaaggcgttc cgccattggt acgaaatcgg gcaatcgcgt
41760tgccccagtg gcctgagctg gcgccctctg gaaagttttc gaaagagaca aaccctgcga
41820aattgcgtga agtgattgcg ccagggcgtg tgcgccactt aaaattcccc ggcaattggg
41880accaataggc cgcttccata ccaatacctt cttggacaac cacggcacct gcatccgcca
41940ttcgtgtccg agcccgcgcg cccctgtccc caagactatt gagatcgtct gcatagacgc
42000aaaggctcaa atgatgtgag cccataacga attcgttgct cgcaagtgcg tcctcagcct
42060cggataattt gccgatttga gtcacggctt tatcgccgga actcagcatc tggctcgatt
42120tgaggctaag tttcgcgtgc gcttgcgggc gagtcaggaa cgaaaaactc tgcgtgagaa
42180caagtggaaa atcgagggat agcagcgcgt tgagcatgcc cggccgtgtt tttgcagggt
42240attcgcgaaa cgaatagatg gatccaacgt aactgtcttt tggcgttctg atctcgagtc
42300ctcgcttgcc gcaaatgact ctgtcggtat aaatcgaagc gccgagtgag ccgctgacga
42360ccggaaccgg tgtgaaccga ccagtcatga tcaaccgtag cgcttcgcca atttcggtga
42420agagcacacc ctgcttctcg cggatgccaa gacgatgcag gccatacgct ttaagagagc
42480cagcgacaac atgccaaaga tcttccatgt tcctgatctg gcccgtgaga tcgttttccc
42540tttttccgct tagcttggtg aacctcctct ttaccttccc taaagccgcc tgtgggtaga
42600caatcaacgt aaggaagtgt tcattgcgga ggagttggcc ggagagcacg cgctgttcaa
42660aagcttcgtt caggctagcg gcgaaaacac tacggaagtg tcgcggcgcc gatgatggca
42720cgtcggcatg acgtacgagg tgagcatata ttgacacatg atcatcagcg atattgcgca
42780acagcgtgtt gaacgcacga caacgcgcat tgcgcatttc agtttcctca agctcgaatg
42840caacgccatc aattctcgca atggtcatga tcgatccgtc ttcaagaagg acgatatggt
42900cgctgaggtg gccaatataa gggagataga tctcaccgga tctttcggtc gttccactcg
42960cgccgagcat cacaccattc ctctccctcg tgggggaacc ctaattggat ttgggctaac
43020agtagcgccc ccccaaactg cactatcaat gcttcttccc gcggtccgca aaaatagcag
43080gacgacgctc gccgcattgt agtctcgctc cacgatgagc cgggctgcaa accataacgg
43140cacgagaacg acttcgtaga gcgggttctg aacgataacg atgacaaagc cggcgaacat
43200catgaataac cctgccaatg tcagtggcac cccaagaaac aatgcgggcc gtgtggctgc
43260gaggtaaagg gtcgattctt ccaaacgatc agccatcaac taccgccagt gagcgtttgg
43320ccgaggaagc tcgccccaaa catgataaca atgccgccga cgacgccggc aaccagccca
43380agcgaagccc gcccgaacat ccaggagatc ccgatagcga caatgccgag aacagcgagt
43440gactggccga acggaccaag gataaacgtg catatattgt taaccattgt ggcggggtca
43500gtgccgccac ccgcagattg cgctgcggcg ggtccggatg aggaaatgct ccatgcaatt
43560gcaccgcaca agcttggggc gcagctcgat atcacgcgca tcatcgcatt cgagagcgag
43620aggcgattta gatgtaaacg gtatctctca aagcatcgca tcaatgcgca cctccttagt
43680ataagtcgaa taagacttga ttgtcgtctg cggatttgcc gttgtcctgg tgtggcggtg
43740gcggagcgat taaaccgcca gcgccatcct cctgcgagcg gcgctgatat gacccccaaa
43800catcccacgt ctcttcggat tttagcgcct cgtgatcgtc ttttggaggc tcgattaacg
43860cgggcaccag cgattgagca gctgtttcaa cttttcgcac gtagccgttt gcaaaaccgc
43920cgatgaaatt accggtgttg taagcggaga tcgcccgacg aagcgcaaat tgcttctcgt
43980caatcgtttc gccgcctgca taacgacttt tcagcatgtt tgcagcggca gataatgatg
44040tgcacgcctg gagcgcaccg tcaggtgtca gaccgagcat agaaaaattt cgagagttta
44100tttgcatgag gccaacatcc agcgaatgcc gtgcatcgag acggtgcctg acgacttggg
44160ttgcttggct gtgatcttgc cagtgaagcg tttcgccggt cgtgttgtca tgaatcgcta
44220aaggatcaaa gcgactctcc accttagcta tcgccgcaag cgtagatgtc gcaactgatg
44280gggcacactt gcgagcaaca tggtcaaact cagcagatga gagtggcgtg gcaaggctcg
44340acgaacagaa ggagaccatc aaggcaagag aaagcgaccc cgatctctta agcatacctt
44400atctccttag ctcgcaacta acaccgcctc tcccgttgga agaagtgcgt tgttttatgt
44460tgaagattat cgggagggtc ggttactcga aaattttcaa ttgcttcttt atgatttcaa
44520ttgaagcgag aaacctcgcc cggcgtcttg gaacgcaaca tggaccgaga accgcgcatc
44580catgactaag caaccggatc gacctattca ggccgcagtt ggtcaggtca ggctcagaac
44640gaaaatgctc ggcgaggtta cgctgtctgt aaacccattc gatgaacggg aagcttcctt
44700ccgattgctc ttggcaggaa tattggccca tgcctgcttg cgctttgcaa atgctcttat
44760cgcgttggta tcatatgcct tgtccgccag cagaaacgca ctctaagcga ttatttgtaa
44820aaatgtttcg gtcatgcggc ggtcatgggc ttgacccgct gtcagcgcaa gacggatcgg
44880tcaaccgtcg gcatcgacaa cagcgtgaat cttggtggtc aaaccgccac gggaacgtcc
44940catacagcca tcgtcttgat cccgctgttt cccgtcgccg catgttggtg gacgcggaca
45000caggaactgt caatcatgac gacattctat cgaaagcctt ggaaatcaca ctcagaatat
45060gatcccagac gtctgcctca cgccatcgta caaagcgatt gtagcaggtt gtacaggaac
45120cgtatcgatc aggaacgtct gcccagggcg ggcccgtccg gaagcgccac aagatgacat
45180tgatcacccg cgtcaacgcg cggcacgcga cgcggcttat ttgggaacaa aggactgaac
45240aacagtccat tcgaaatcgg tgacatcaaa gcggggacgg gttatcagtg gcctccaagt
45300caagcctcaa tgaatcaaaa tcagaccgat ttgcaaacct gatttatgag tgtgcggcct
45360aaatgatgaa atcgtccttc tagatcgcct ccgtggtgta gcaacacctc gcagtatcgc
45420cgtgctgacc ttggccaggg aattgactgg caagggtgct ttcacatgac cgctcttttg
45480gccgcgatag atgatttcgt tgctgctttg ggcacgtaga aggagagaag tcatatcgga
45540gaaattcctc ctggcgcgag agcctgctct atcgcgacgg catcccactg tcgggaacag
45600accggatcat tcacgaggcg aaagtcgtca acacatgcgt tataggcatc ttcccttgaa
45660ggatgatctt gttgctgcca atctggaggt gcggcagccg caggcagatg cgatctcagc
45720gcaacttgcg gcaaaacatc tcactcacct gaaaaccact agcgagtctc gcgatcagac
45780gaaggccttt tacttaacga cacaatatcc gatgtctgca tcacaggcgt cgctatccca
45840gtcaatacta aagcggtgca ggaactaaag attactgatg acttaggcgt gccacgaggc
45900ctgagacgac gcgcgtagac agttttttga aatcattatc aaagtgatgg cctccgctga
45960agcctatcac ctctgcgccg gtctgtcgga gagatgggca agcattatta cggtcttcgc
46020gcccgtacat gcattggacg attgcagggt caatggatct gagatcatcc agaggattgc
46080cgcccttacc ttccgtttcg agttggagcc agcccctaaa tgagacgaca tagtcgactt
46140gatgtgacaa tgccaagaga gagatttgct taacccgatt tttttgctca agcgtaagcc
46200tattgaagct tgccggcatg acgtccgcgc cgaaagaata tcctacaagt aaaacattct
46260gcacaccgaa atgcttggtg tagacatcga ttatgtgacc aagatcctta gcagtttcgc
46320ttggggaccg ctccgaccag aaataccgaa gtgaactgac gccaatgaca ggaatccctt
46380ccgtctgcag ataggtacca tcgatagatc tgctgcctcg cgcgtttcgg tgatgacggt
46440gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
46500gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
46560atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
46620agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
46680aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
46740ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
46800gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
46860aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
46920gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
46980ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
47040cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
47100cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
47160gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
47220cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
47280agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
47340ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
47400ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
47460gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
47520cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
47580attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
47640accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
47700ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
47760gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
47820agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
47880ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
47940ttgttgccat tgctgcaggg gggggggggg ggggggactt ccattgttca ttccacggac
48000aaaaacagag aaaggaaacg acagaggcca aaaagcctcg ctttcagcac ctgtcgtttc
48060ctttcttttc agagggtatt ttaaataaaa acattaagtt atgacgaaga agaacggaaa
48120cgccttaaac cggaaaattt tcataaatag cgaaaacccg cgaggtcgcc gccccgtagt
48180cggatcaccg gaaaggaccc gtaaagtgat aatgattatc atctacatat cacaacgtgc
48240gtggaggcca tcaaaccacg tcaaataatc aattatgacg caggtatcgt attaattgat
48300ctgcatcaac ttaacgtaaa aacaacttca gacaatacaa atcagcgaca ctgaatacgg
48360ggcaacctca tgtccccccc cccccccccc ctgcaggcat cgtggtgtca cgctcgtcgt
48420ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
48480tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
48540ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
48600ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
48660tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
48720gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
48780taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
48840cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
48900agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
48960gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
49020ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
49080ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
49140aagaattggt cgacgatctt gctgcgttcg gatattttcg tggagttccc gccacagacc
49200cggattgaag gcgagatcca gcaactcgcg ccagatcatc ctgtgacgga actttggcgc
49260gtgatgactg gccaggacgt cggccgaaag agcgacaagc agatcacgct tttcgacagc
49320gtcggatttg cgatcgagga tttttcggcg ctgcgctacg tccgcgaccg cgttgaggga
49380tcaagccaca gcagcccact cgaccttcta gccgacccag acgagccaag ggatcttttt
49440ggaatgctgc tccgtcgtca ggctttccga cgtttgggtg gttgaacaga agtcattatc
49500gtacggaatg ccaagcactc ccgaggggaa ccctgtggtt ggcatgcaca tacaaatgga
49560cgaacggata aaccttttca cgccctttta aatatccgtt attctaataa acgctctttt
49620ctcttaggtt tacccgccaa tatatcctgt caaacactga tagtttaaac tgaaggcggg
49680aaacgacaat ctgatcatga gcggagaatt aagggagtca cgttatgacc cccgccgatg
49740acgcgggaca agccgtttta cgtttggaac tgacagaacc gcaacgttga aggagccact
49800cagcaagctg gtacgattgt aatacgactc actatagggc gaattgagcg ctgtttaaac
49860gctcttcaac tggaagagcg gttacccgga ccgaagcttg catgcctgca g
49911736909DNAartificial sequencevector 7tctagagctc gttcctcgag gcctcgaggc
ctcgaggaac ggtacctgcg gggaagctta 60caataatgtg tgttgttaag tcttgttgcc
tgtcatcgtc tgactgactt tcgtcataaa 120tcccggcctc cgtaacccag ctttgggcaa
gctcacggat ttgatccggc ggaacgggaa 180tatcgagatg ccgggctgaa cgctgcagtt
ccagctttcc ctttcgggac aggtactcca 240gctgattgat tatctgctga agggtcttgg
ttccacctcc tggcacaatg cgaatgatta 300cttgagcgcg atcgggcatc caattttctc
ccgtcaggtg cgtggtcaag tgctacaagg 360cacctttcag taacgagcga ccgtcgatcc
gtcgccggga tacggacaaa atggagcgca 420gtagtccatc gagggcggcg aaagcctcgc
caaaagcaat acgttcatct cgcacagcct 480ccagatccga tcgagggtct tcggcgtagg
cagatagaag catggataca ttgcttgaga 540gtattccgat ggactgaagt atggcttcca
tcttttctcg tgtgtctgca tctatttcga 600gaaagccccc gatgcggcgc accgcaacgc
gaattgccat actatccgaa agtcccagca 660ggcgcgcttg ataggaaaag gtttcatact
cggccgatcg cagacgggca ctcacgacct 720tgaacccttc aactttcagg gatcgatgct
ggttgatggt agtctcactc gacgtggctc 780tggtgtgttt tgacatagct tcctccaaag
aaagcggaag gtctggatac tccagcacga 840aatgtgcccg ggtagacgga tggaagtcta
gccctgctca atatgaaatc aacagtacat 900ttacagtcaa tactgaatat acttgctaca
tttgcaattg tcttataacg aatgtgaaat 960aaaaatagtg taacaacgct tttactcatc
gataatcaca aaaacattta tacgaacaaa 1020aatacaaatg cactccggtt tcacaggata
ggcgggatca gaatatgcaa cttttgacgt 1080tttgttcttt caaagggggt gctggcaaaa
ccaccgcact catgggcctt tgcgctgctt 1140tggcaaatga cggtaaacga gtggccctct
ttgatgccga cgaaaaccgg cctctgacgc 1200gatggagaga aaacgcctta caaagcagta
ctgggatcct cgctgtgaag tctattccgc 1260cgacgaaatg ccccttcttg aagcagccta
tgaaaatgcc gagctcgaag gatttgatta 1320tgcgttggcc gatacgcgtg gcggctcgag
cgagctcaac aacacaatca tcgctagctc 1380aaacctgctt ctgatcccca ccatgctaac
gccgctcgac atcgatgagg cactatctac 1440ctaccgctac gtcatcgagc tgctgttgag
tgaaaatttg gcaattccta cagctgtttt 1500gcgccaacgc gtcccggtcg gccgattgac
aacatcgcaa cgcaggatgt cagagacgct 1560agagagcctt ccagttgtac cgtctcccat
gcatgaaaga gatgcatttg ccgcgatgaa 1620agaacgcggc atgttgcatc ttacattact
aaacacggga actgatccga cgatgcgcct 1680catagagagg aatcttcgga ttgcgatgga
ggaagtcgtg gtcatttcga aactgatcag 1740caaaatcttg gaggcttgaa gatggcaatt
cgcaagcccg cattgtcggt cggcgaagca 1800cggcggcttg ctggtgctcg acccgagatc
caccatccca acccgacact tgttccccag 1860aagctggacc tccagcactt gcctgaaaaa
gccgacgaga aagaccagca acgtgagcct 1920ctcgtcgccg atcacattta cagtcccgat
cgacaactta agctaactgt ggatgccctt 1980agtccacctc cgtccccgaa aaagctccag
gtttttcttt cagcgcgacc gcccgcgcct 2040caagtgtcga aaacatatga caacctcgtt
cggcaataca gtccctcgaa gtcgctacaa 2100atgattttaa ggcgcgcgtt ggacgatttc
gaaagcatgc tggcagatgg atcatttcgc 2160gtggccccga aaagttatcc gatcccttca
actacagaaa aatccgttct cgttcagacc 2220tcacgcatgt tcccggttgc gttgctcgag
gtcgctcgaa gtcattttga tccgttgggg 2280ttggagaccg ctcgagcttt cggccacaag
ctggctaccg ccgcgctcgc gtcattcttt 2340gctggagaga agccatcgag caattggtga
agagggacct atcggaaccc ctcaccaaat 2400attgagtgta ggtttgaggc cgctggccgc
gtcctcagtc accttttgag ccagataatt 2460aagagccaaa tgcaattggc tcaggctgcc
atcgtccccc cgtgcgaaac ctgcacgtcc 2520gcgtcaaaga aataaccggc acctcttgct
gtttttatca gttgagggct tgacggatcc 2580gcctcaagtt tgcggcgcag ccgcaaaatg
agaacatcta tactcctgtc gtaaacctcc 2640tcgtcgcgta ctcgactggc aatgagaagt
tgctcgcgcg atagaacgtc gcggggtttc 2700tctaaaaacg cgaggagaag attgaactca
cctgccgtaa gtttcacctc accgccagct 2760tcggacatca agcgacgttg cctgagatta
agtgtccagt cagtaaaaca aaaagaccgt 2820cggtctttgg agcggacaac gttggggcgc
acgcgcaagg caacccgaat gcgtgcaaga 2880aactctctcg tactaaacgg cttagcgata
aaatcacttg ctcctagctc gagtgcaaca 2940actttatccg tctcctcaag gcggtcgcca
ctgataatta tgattggaat atcagacttt 3000gccgccagat ttcgaacgat ctcaagccca
tcttcacgac ctaaatttag atcaacaacc 3060acgacatcga ccgtcgcgga agagagtact
ctagtgaact gggtgctgtc ggctaccgcg 3120gtcactttga aggcgtggat cgtaaggtat
tcgataataa gatgccgcat agcgacatcg 3180tcatcgataa gaagaacgtg tttcaacggc
tcacctttca atctaaaatc tgaacccttg 3240ttcacagcgc ttgagaaatt ttcacgtgaa
ggatgtacaa tcatctccag ctaaatgggc 3300agttcgtcag aattgcggct gaccgcggat
gacgaaaatg cgaaccaagt atttcaattt 3360tatgacaaaa gttctcaatc gttgttacaa
gtgaaacgct tcgaggttac agctactatt 3420gattaaggag atcgcctatg gtctcgcccc
ggcgtcgtgc gtccgccgcg agccagatct 3480cgcctacttc ataaacgtcc tcataggcac
ggaatggaat gatgacatcg atcgccgtag 3540agagcatgtc aatcagtgtg cgatcttcca
agctagcacc ttgggcgcta cttttgacaa 3600gggaaaacag tttcttgaat ccttggattg
gattcgcgcc gtgtattgtt gaaatcgatc 3660ccggatgtcc cgagacgact tcactcagat
aagcccatgc tgcatcgtcg cgcatctcgc 3720caagcaatat ccggtccggc cgcatacgca
gacttgcttg gagcaagtgc tcggcgctca 3780cagcacccag cccagcaccg ttcttggagt
agagtagtct aacatgatta tcgtgtggaa 3840tgacgagttc gagcgtatct tctatggtga
ttagcctttc ctgggggggg atggcgctga 3900tcaaggtctt gctcattgtt gtcttgccgc
ttccggtagg gccacatagc aacatcgtca 3960gtcggctgac gacgcatgcg tgcagaaacg
cttccaaatc cccgttgtca aaatgctgaa 4020ggatagcttc atcatcctga ttttggcgtt
tccttcgtgt ctgccactgg ttccacctcg 4080aagcatcata acgggaggag acttctttaa
gaccagaaac acgcgagctt ggccgtcgaa 4140tggtcaagct gacggtgccc gagggaacgg
tcggcggcag acagatttgt agtcgttcac 4200caccaggaag ttcagtggcg cagagggggt
tacgtggtcc gacatcctgc tttctcagcg 4260cgcccgctaa aatagcgata tcttcaagat
catcataaga gacgggcaaa ggcatcttgg 4320taaaaatgcc ggcttggcgc acaaatgcct
ctccaggtcg attgatcgca atttcttcag 4380tcttcgggtc atcgagccat tccaaaatcg
gcttcagaag aaagcgtagt tgcggatcca 4440cttccattta caatgtatcc tatctctaag
cggaaatttg aattcattaa gagcggcggt 4500tcctcccccg cgtggcgccg ccagtcaggc
ggagctggta aacaccaaag aaatcgaggt 4560cccgtgctac gaaaatggaa acggtgtcac
cctgattctt cttcagggtt ggcggtatgt 4620tgatggttgc cttaagggct gtctcagttg
tctgctcacc gttattttga aagctgttga 4680agctcatccc gccacccgag ctgccggcgt
aggtgctagc tgcctggaag gcgccttgaa 4740caacactcaa gagcatagct ccgctaaaac
gctgccagaa gtggctgtcg accgagcccg 4800gcaatcctga gcgaccgagt tcgtccgcgc
ttggcgatgt taacgagatc atcgcatggt 4860caggtgtctc ggcgcgatcc cacaacacaa
aaacgcgccc atctccctgt tgcaagccac 4920gctgtatttc gccaacaacg gtggtgccac
gatcaagaag cacgatattg ttcgttgttc 4980cacgaatatc ctgaggcaag acacacttta
catagcctgc caaatttgtg tcgattgcgg 5040tttgcaagat gcacggaatt attgtccctt
gcgttaccat aaaatcgggg tgcggcaaga 5100gcgtggcgct gctgggctgc agctcggtgg
gtttcatacg tatcgacaaa tcgttctcgc 5160cggacacttc gccattcggc aaggagttgt
cgtcacgctt gccttcttgt cttcggcccg 5220tgtcgccctg aatggcgcgt ttgctgaccc
cttgatcgcc gctgctatat gcaaaaatcg 5280gtgtttcttc cggccgtggc tcatgccgct
ccggttcgcc cctcggcggt agaggagcag 5340caggctgaac agcctcttga accgctggag
gatccggcgg cacctcaatc ggagctggat 5400gaaatggctt ggtgtttgtt gcgatcaaag
ttgacggcga tgcgttctca ttcaccttct 5460tttggcgccc acctagccaa atgaggctta
atgataacgc gagaacgaca cctccgacga 5520tcaatttctg agaccccgaa agacgccggc
gatgtttgtc ggagaccagg gatccagatg 5580catcaacctc atgtgccgct tgctgactat
cgttattcat cccttcgccc ccttcaggac 5640gcgtttcaca tcgggcctca ccgtgcccgt
ttgcggcctt tggccaacgg gatcgtaagc 5700ggtgttccag atacatagta ctgtgtggcc
atccctcaga cgccaacctc gggaaaccga 5760agaaatctcg acatcgctcc ctttaactga
atagttggca acagcttcct tgccatcagg 5820attgatggtg tagatggagg gtatgcgtac
attgcccgga aagtggaata ccgtcgtaaa 5880tccattgtcg aagacttcga gtggcaacag
cgaacgatcg ccttgggcga cgtagtgcca 5940attactgtcc gccgcaccaa gggctgtgac
aggctgatcc aataaattct cagctttccg 6000ttgatattgt gcttccgcgt gtagtctgtc
cacaacagcc ttctgttgtg cctcccttcg 6060ccgagccgcc gcatcgtcgg cggggtaggc
gaattggacg ctgtaataga gatcgggctg 6120ctctttatcg aggtgggaca gagtcttgga
acttatactg aaaacataac ggcgcatccc 6180ggagtcgctt gcggttagca cgattactgg
ctgaggcgtg aggacctggc ttgccttgaa 6240aaatagataa tttccccgcg gtagggctgc
tagatctttg ctatttgaaa cggcaaccgc 6300tgtcaccgtt tcgttcgtgg cgaatgttac
gaccaaagta gctccaaccg ccgtcgagag 6360gcgcaccact tgatcgggat tgtaagccaa
ataacgcatg cgcggatcta gcttgcccgc 6420cattggagtg tcttcagcct ccgcaccagt
cgcagcggca aataaacatg ctaaaatgaa 6480aagtgctttt ctgatcatgg ttcgctgtgg
cctacgtttg aaacggtatc ttccgatgtc 6540tgataggagg tgacaaccag acctgccggg
ttggttagtc tcaatctgcc gggcaagctg 6600gtcacctttt cgtagcgaac tgtcgcggtc
cacgtactca ccacaggcat tttgccgtca 6660acgacgaggg tccttttata gcgaatttgc
tgcgtgcttg gagttacatc atttgaagcg 6720atgtgctcga cctccaccct gccgcgtttg
ccaagaatga cttgaggcga actgggattg 6780ggatagttga agaattgctg gtaatcctgg
cgcactgttg gggcactgaa gttcgatacc 6840aggtcgtagg cgtactgagc ggtgtcggca
tcataactct cgcgcaggcg aacgtactcc 6900cacaatgagg cgttaacgac ggcctcctct
tgagttgcag gcaatcgcga gacagacacc 6960tcgctgtcaa cggtgccgtc cggccgtatc
catagatata cgggcacaag cctgctcaac 7020ggcaccattg tggctatagc gaacgcttga
gcaacatttc ccaaaatcgc gatagctgcg 7080acagctgcaa tgagtttgga gagacgtcgc
gccgatttcg ctcgcgcggt ttgaaaggct 7140tctacttcct tatagtgctc ggcaaggctt
tcgcgcgcca ctagcatggc atattcaggc 7200cccgtcatag cgtccacccg aattgccgag
ctgaagatct gacggagtag gctgccatcg 7260ccccacattc agcgggaaga tcgggccttt
gcagctcgct aatgtgtcgt ttgtctggca 7320gccgctcaaa gcgacaacta ggcacagcag
gcaatacttc atagaattct ccattgaggc 7380gaatttttgc gcgacctagc ctcgctcaac
ctgagcgaag cgacggtaca agctgctggc 7440agattgggtt gcgccgctcc agtaactgcc
tccaatgttg ccggcgatcg ccggcaaagc 7500gacaatgagc gcatcccctg tcagaaaaaa
catatcgagt tcgtaaagac caatgatctt 7560ggccgcggtc gtaccggcga aggtgattac
accaagcata agggtgagcg cagtcgcttc 7620ggttaggatg acgatcgttg ccacgaggtt
taagaggaga agcaagagac cgtaggtgat 7680aagttgcccg atccacttag ctgcgatgtc
ccgcgtgcga tcaaaaatat atccgacgag 7740gatcagaggc ccgatcgcga gaagcacttt
cgtgagaatt ccaacggcgt cgtaaactcc 7800gaaggcagac cagagcgtgc cgtaaaggac
ccactgtgcc ccttggaaag caaggatgtc 7860ctggtcgttc atcggaccga tttcggatgc
gattttctga aaaacggcct gggtcacggc 7920gaacattgta tccaactgtg ccggaacagt
ctgcagaggc aagccggtta cactaaactg 7980ctgaacaaag tttgggaccg tcttttcgaa
gatggaaacc acatagtctt ggtagttagc 8040ctgcccaaca attagagcaa caacgatggt
gaccgtgatc acccgagtga taccgctacg 8100ggtatcgact tcgccgcgta tgactaaaat
accctgaaca ataatccaaa gagtgacaca 8160ggcgatcaat ggcgcactca ccgcctcctg
gatagtctca agcatcgagt ccaagcctgt 8220cgtgaaggct acatcgaaga tcgtatgaat
ggccgtaaac ggcgccggaa tcgtgaaatt 8280catcgattgg acctgaactt gactggtttg
tcgcataatg ttggataaaa tgagctcgca 8340ttcggcgagg atgcgggcgg atgaacaaat
cgcccagcct taggggaggg caccaaagat 8400gacagcggtc ttttgatgct ccttgcgttg
agcggccgcc tcttccgcct cgtgaaggcc 8460ggcctgcgcg gtagtcatcg ttaataggct
tgtcgcctgt acattttgaa tcattgcgtc 8520atggatctgc ttgagaagca aaccattggt
cacggttgcc tgcatgatat tgcgagatcg 8580ggaaagctga gcagacgtat cagcattcgc
cgtcaagcgt ttgtccatcg tttccagatt 8640gtcagccgca atgccagcgc tgtttgcgga
accggtgatc tgcgatcgca acaggtccgc 8700ttcagcatca ctacccacga ctgcacgatc
tgtatcgctg gtgatcgcac gtgccgtggt 8760cgacattggc attcgcggcg aaaacatttc
attgtctagg tccttcgtcg aaggatactg 8820atttttctgg ttgagcgaag tcagtagtcc
agtaacgccg taggccgacg tcaacatcgt 8880aaccatcgct atagtctgag tgagattctc
cgcagtcgcg agcgcagtcg cgagcgtctc 8940agcctccgtt gccgggtcgc taacaacaaa
ctgcgcccgc gcgggctgaa tatatagaaa 9000gctgcaggtc aaaactgttg caataagttg
cgtcgtcttc atcgtttcct accttatcaa 9060tcttctgcct cgtggtgacg ggccatgaat
tcgctgagcc agccagatga gttgccttct 9120tgtgcctcgc gtagtcgagt tgcaaagcgc
accgtgttgg cacgccccga aagcacggcg 9180acatattcac gcatatcccg cagatcaaat
tcgcagatga cgcttccact ttctcgttta 9240agaagaaact tacggctgcc gaccgtcatg
tcttcacgga tcgcctgaaa ttccttttcg 9300gtacatttca gtccatcgac ataagccgat
cgatctgcgg ttggtgatgg atagaaaatc 9360ttcgtcatac attgcgcaac caagctggct
cctagcggcg attccagaac atgctctggt 9420tgctgcgttg ccagtattag catcccgttg
ttttttcgaa cggtcaggag gaatttgtcg 9480acgacagtcg aaaatttagg gtttaacaaa
taggcgcgaa actcatcgca gctcatcaca 9540aaacggcggc cgtcgatcat ggctccaatc
cgatgcagga gatatgctgc agcgggagcg 9600catacttcct cgtattcgag aagatgcgtc
atgtcgaagc cggtaatcga cggatctaac 9660tttacttcgt caacttcgcc gtcaaatgcc
cagccaagcg catggccccg gcaccagcgt 9720tggagccgcg ctcctgcgcc ttcggcgggc
ccatgcaaca aaaattcacg taaccccgcg 9780attgaacgca tttgtggatc aaacgagagc
tgacgatgga taccacggac cagacggcgg 9840ttctcttccg gagaaatccc accccgacca
tcactctcga tgagagccac gatccattcg 9900cgcagaaaat cgtgtgaggc tgctgtgttt
tctaggccac gcaacggcgc caacccgctg 9960ggtgtgcctc tgtgaagtgc caaatatgtt
cctcctgtgg cgcgaaccag caattcgcca 10020ccccggtcct tgtcaaagaa cacgaccgta
cctgcacggt cgaccatgct ctgttcgagc 10080atggctagaa caaacatcat gagcgtcgtc
ttacccctcc cgataggccc gaatattgcc 10140gtcatgccaa catcgtgctc atgcgggata
tagtcgaaag gcgttccgcc attggtacga 10200aatcgggcaa tcgcgttgcc ccagtggcct
gagctggcgc cctctggaaa gttttcgaaa 10260gagacaaacc ctgcgaaatt gcgtgaagtg
attgcgccag ggcgtgtgcg ccacttaaaa 10320ttccccggca attgggacca ataggccgct
tccataccaa taccttcttg gacaaccacg 10380gcacctgcat ccgccattcg tgtccgagcc
cgcgcgcccc tgtccccaag actattgaga 10440tcgtctgcat agacgcaaag gctcaaatga
tgtgagccca taacgaattc gttgctcgca 10500agtgcgtcct cagcctcgga taatttgccg
atttgagtca cggctttatc gccggaactc 10560agcatctggc tcgatttgag gctaagtttc
gcgtgcgctt gcgggcgagt caggaacgaa 10620aaactctgcg tgagaacaag tggaaaatcg
agggatagca gcgcgttgag catgcccggc 10680cgtgtttttg cagggtattc gcgaaacgaa
tagatggatc caacgtaact gtcttttggc 10740gttctgatct cgagtcctcg cttgccgcaa
atgactctgt cggtataaat cgaagcgccg 10800agtgagccgc tgacgaccgg aaccggtgtg
aaccgaccag tcatgatcaa ccgtagcgct 10860tcgccaattt cggtgaagag cacaccctgc
ttctcgcgga tgccaagacg atgcaggcca 10920tacgctttaa gagagccagc gacaacatgc
caaagatctt ccatgttcct gatctggccc 10980gtgagatcgt tttccctttt tccgcttagc
ttggtgaacc tcctctttac cttccctaaa 11040gccgcctgtg ggtagacaat caacgtaagg
aagtgttcat tgcggaggag ttggccggag 11100agcacgcgct gttcaaaagc ttcgttcagg
ctagcggcga aaacactacg gaagtgtcgc 11160ggcgccgatg atggcacgtc ggcatgacgt
acgaggtgag catatattga cacatgatca 11220tcagcgatat tgcgcaacag cgtgttgaac
gcacgacaac gcgcattgcg catttcagtt 11280tcctcaagct cgaatgcaac gccatcaatt
ctcgcaatgg tcatgatcga tccgtcttca 11340agaaggacga tatggtcgct gaggtggcca
atataaggga gatagatctc accggatctt 11400tcggtcgttc cactcgcgcc gagcatcaca
ccattcctct ccctcgtggg ggaaccctaa 11460ttggatttgg gctaacagta gcgccccccc
aaactgcact atcaatgctt cttcccgcgg 11520tccgcaaaaa tagcaggacg acgctcgccg
cattgtagtc tcgctccacg atgagccggg 11580ctgcaaacca taacggcacg agaacgactt
cgtagagcgg gttctgaacg ataacgatga 11640caaagccggc gaacatcatg aataaccctg
ccaatgtcag tggcacccca agaaacaatg 11700cgggccgtgt ggctgcgagg taaagggtcg
attcttccaa acgatcagcc atcaactacc 11760gccagtgagc gtttggccga ggaagctcgc
cccaaacatg ataacaatgc cgccgacgac 11820gccggcaacc agcccaagcg aagcccgccc
gaacatccag gagatcccga tagcgacaat 11880gccgagaaca gcgagtgact ggccgaacgg
accaaggata aacgtgcata tattgttaac 11940cattgtggcg gggtcagtgc cgccacccgc
agattgcgct gcggcgggtc cggatgagga 12000aatgctccat gcaattgcac cgcacaagct
tggggcgcag ctcgatatca cgcgcatcat 12060cgcattcgag agcgagaggc gatttagatg
taaacggtat ctctcaaagc atcgcatcaa 12120tgcgcacctc cttagtataa gtcgaataag
acttgattgt cgtctgcgga tttgccgttg 12180tcctggtgtg gcggtggcgg agcgattaaa
ccgccagcgc catcctcctg cgagcggcgc 12240tgatatgacc cccaaacatc ccacgtctct
tcggatttta gcgcctcgtg atcgtctttt 12300ggaggctcga ttaacgcggg caccagcgat
tgagcagctg tttcaacttt tcgcacgtag 12360ccgtttgcaa aaccgccgat gaaattaccg
gtgttgtaag cggagatcgc ccgacgaagc 12420gcaaattgct tctcgtcaat cgtttcgccg
cctgcataac gacttttcag catgtttgca 12480gcggcagata atgatgtgca cgcctggagc
gcaccgtcag gtgtcagacc gagcatagaa 12540aaatttcgag agtttatttg catgaggcca
acatccagcg aatgccgtgc atcgagacgg 12600tgcctgacga cttgggttgc ttggctgtga
tcttgccagt gaagcgtttc gccggtcgtg 12660ttgtcatgaa tcgctaaagg atcaaagcga
ctctccacct tagctatcgc cgcaagcgta 12720gatgtcgcaa ctgatggggc acacttgcga
gcaacatggt caaactcagc agatgagagt 12780ggcgtggcaa ggctcgacga acagaaggag
accatcaagg caagagaaag cgaccccgat 12840ctcttaagca taccttatct ccttagctcg
caactaacac cgcctctccc gttggaagaa 12900gtgcgttgtt ttatgttgaa gattatcggg
agggtcggtt actcgaaaat tttcaattgc 12960ttctttatga tttcaattga agcgagaaac
ctcgcccggc gtcttggaac gcaacatgga 13020ccgagaaccg cgcatccatg actaagcaac
cggatcgacc tattcaggcc gcagttggtc 13080aggtcaggct cagaacgaaa atgctcggcg
aggttacgct gtctgtaaac ccattcgatg 13140aacgggaagc ttccttccga ttgctcttgg
caggaatatt ggcccatgcc tgcttgcgct 13200ttgcaaatgc tcttatcgcg ttggtatcat
atgccttgtc cgccagcaga aacgcactct 13260aagcgattat ttgtaaaaat gtttcggtca
tgcggcggtc atgggcttga cccgctgtca 13320gcgcaagacg gatcggtcaa ccgtcggcat
cgacaacagc gtgaatcttg gtggtcaaac 13380cgccacggga acgtcccata cagccatcgt
cttgatcccg ctgtttcccg tcgccgcatg 13440ttggtggacg cggacacagg aactgtcaat
catgacgaca ttctatcgaa agccttggaa 13500atcacactca gaatatgatc ccagacgtct
gcctcacgcc atcgtacaaa gcgattgtag 13560caggttgtac aggaaccgta tcgatcagga
acgtctgccc agggcgggcc cgtccggaag 13620cgccacaaga tgacattgat cacccgcgtc
aacgcgcggc acgcgacgcg gcttatttgg 13680gaacaaagga ctgaacaaca gtccattcga
aatcggtgac atcaaagcgg ggacgggtta 13740tcagtggcct ccaagtcaag cctcaatgaa
tcaaaatcag accgatttgc aaacctgatt 13800tatgagtgtg cggcctaaat gatgaaatcg
tccttctaga tcgcctccgt ggtgtagcaa 13860cacctcgcag tatcgccgtg ctgaccttgg
ccagggaatt gactggcaag ggtgctttca 13920catgaccgct cttttggccg cgatagatga
tttcgttgct gctttgggca cgtagaagga 13980gagaagtcat atcggagaaa ttcctcctgg
cgcgagagcc tgctctatcg cgacggcatc 14040ccactgtcgg gaacagaccg gatcattcac
gaggcgaaag tcgtcaacac atgcgttata 14100ggcatcttcc cttgaaggat gatcttgttg
ctgccaatct ggaggtgcgg cagccgcagg 14160cagatgcgat ctcagcgcaa cttgcggcaa
aacatctcac tcacctgaaa accactagcg 14220agtctcgcga tcagacgaag gccttttact
taacgacaca atatccgatg tctgcatcac 14280aggcgtcgct atcccagtca atactaaagc
ggtgcaggaa ctaaagatta ctgatgactt 14340aggcgtgcca cgaggcctga gacgacgcgc
gtagacagtt ttttgaaatc attatcaaag 14400tgatggcctc cgctgaagcc tatcacctct
gcgccggtct gtcggagaga tgggcaagca 14460ttattacggt cttcgcgccc gtacatgcat
tggacgattg cagggtcaat ggatctgaga 14520tcatccagag gattgccgcc cttaccttcc
gtttcgagtt ggagccagcc cctaaatgag 14580acgacatagt cgacttgatg tgacaatgcc
aagagagaga tttgcttaac ccgatttttt 14640tgctcaagcg taagcctatt gaagcttgcc
ggcatgacgt ccgcgccgaa agaatatcct 14700acaagtaaaa cattctgcac accgaaatgc
ttggtgtaga catcgattat gtgaccaaga 14760tccttagcag tttcgcttgg ggaccgctcc
gaccagaaat accgaagtga actgacgcca 14820atgacaggaa tcccttccgt ctgcagatag
gtaccatcga tagatctgct gcctcgcgcg 14880tttcggtgat gacggtgaaa acctctgaca
catgcagctc ccggagacgg tcacagcttg 14940tctgtaagcg gatgccggga gcagacaagc
ccgtcagggc gcgtcagcgg gtgttggcgg 15000gtgtcggggc gcagccatga cccagtcacg
tagcgatagc ggagtgtata ctggcttaac 15060tatgcggcat cagagcagat tgtactgaga
gtgcaccata tgcggtgtga aataccgcac 15120agatgcgtaa ggagaaaata ccgcatcagg
cgctcttccg cttcctcgct cactgactcg 15180ctgcgctcgg tcgttcggct gcggcgagcg
gtatcagctc actcaaaggc ggtaatacgg 15240ttatccacag aatcagggga taacgcagga
aagaacatgt gagcaaaagg ccagcaaaag 15300gccaggaacc gtaaaaaggc cgcgttgctg
gcgtttttcc ataggctccg cccccctgac 15360gagcatcaca aaaatcgacg ctcaagtcag
aggtggcgaa acccgacagg actataaaga 15420taccaggcgt ttccccctgg aagctccctc
gtgcgctctc ctgttccgac cctgccgctt 15480accggatacc tgtccgcctt tctcccttcg
ggaagcgtgg cgctttctca tagctcacgc 15540tgtaggtatc tcagttcggt gtaggtcgtt
cgctccaagc tgggctgtgt gcacgaaccc 15600cccgttcagc ccgaccgctg cgccttatcc
ggtaactatc gtcttgagtc caacccggta 15660agacacgact tatcgccact ggcagcagcc
actggtaaca ggattagcag agcgaggtat 15720gtaggcggtg ctacagagtt cttgaagtgg
tggcctaact acggctacac tagaaggaca 15780gtatttggta tctgcgctct gctgaagcca
gttaccttcg gaaaaagagt tggtagctct 15840tgatccggca aacaaaccac cgctggtagc
ggtggttttt ttgtttgcaa gcagcagatt 15900acgcgcagaa aaaaaggatc tcaagaagat
cctttgatct tttctacggg gtctgacgct 15960cagtggaacg aaaactcacg ttaagggatt
ttggtcatga gattatcaaa aaggatcttc 16020acctagatcc ttttaaatta aaaatgaagt
tttaaatcaa tctaaagtat atatgagtaa 16080acttggtctg acagttacca atgcttaatc
agtgaggcac ctatctcagc gatctgtcta 16140tttcgttcat ccatagttgc ctgactcccc
gtcgtgtaga taactacgat acgggagggc 16200ttaccatctg gccccagtgc tgcaatgata
ccgcgagacc cacgctcacc ggctccagat 16260ttatcagcaa taaaccagcc agccggaagg
gccgagcgca gaagtggtcc tgcaacttta 16320tccgcctcca tccagtctat taattgttgc
cgggaagcta gagtaagtag ttcgccagtt 16380aatagtttgc gcaacgttgt tgccattgct
gcaggggggg gggggggggg gttccattgt 16440tcattccacg gacaaaaaca gagaaaggaa
acgacagagg ccaaaaagct cgctttcagc 16500acctgtcgtt tcctttcttt tcagagggta
ttttaaataa aaacattaag ttatgacgaa 16560gaagaacgga aacgccttaa accggaaaat
tttcataaat agcgaaaacc cgcgaggtcg 16620ccgccccgta acctgtcgga tcaccggaaa
ggacccgtaa agtgataatg attatcatct 16680acatatcaca acgtgcgtgg aggccatcaa
accacgtcaa ataatcaatt atgacgcagg 16740tatcgtatta attgatctgc atcaacttaa
cgtaaaaaca acttcagaca atacaaatca 16800gcgacactga atacggggca acctcatgtc
cccccccccc ccccccctgc aggcatcgtg 16860gtgtcacgct cgtcgtttgg tatggcttca
ttcagctccg gttcccaacg atcaaggcga 16920gttacatgat cccccatgtt gtgcaaaaaa
gcggttagct ccttcggtcc tccgatcgtt 16980gtcagaagta agttggccgc agtgttatca
ctcatggtta tggcagcact gcataattct 17040cttactgtca tgccatccgt aagatgcttt
tctgtgactg gtgagtactc aaccaagtca 17100ttctgagaat agtgtatgcg gcgaccgagt
tgctcttgcc cggcgtcaac acgggataat 17160accgcgccac atagcagaac tttaaaagtg
ctcatcattg gaaaacgttc ttcggggcga 17220aaactctcaa ggatcttacc gctgttgaga
tccagttcga tgtaacccac tcgtgcaccc 17280aactgatctt cagcatcttt tactttcacc
agcgtttctg ggtgagcaaa aacaggaagg 17340caaaatgccg caaaaaaggg aataagggcg
acacggaaat gttgaatact catactcttc 17400ctttttcaat attattgaag catttatcag
ggttattgtc tcatgagcgg atacatattt 17460gaatgtattt agaaaaataa acaaataggg
gttccgcgca catttccccg aaaagtgcca 17520cctgacgtct aagaaaccat tattatcatg
acattaacct ataaaaatag gcgtatcacg 17580aggccctttc gtcttcaaga attcggagct
tttgccattc tcaccggatt cagtcgtcac 17640tcatggtgat ttctcacttg ataaccttat
ttttgacgag gggaaattaa taggttgtat 17700tgatgttgga cgagtcggaa tcgcagaccg
ataccaggat cttgccatcc tatggaactg 17760cctcggtgag ttttctcctt cattacagaa
acggcttttt caaaaatatg gtattgataa 17820tcctgatatg aataaattgc agtttcattt
gatgctcgat gagtttttct aatcagaatt 17880ggttaattgg ttgtaacact ggcagagcat
tacgctgact tgacgggacg gcggctttgt 17940tgaataaatc gaacttttgc tgagttgaag
gatcagatca cgcatcttcc cgacaacgca 18000gaccgttccg tggcaaagca aaagttcaaa
atcaccaact ggtccaccta caacaaagct 18060ctcatcaacc gtggctccct cactttctgg
ctggatgatg gggcgattca ggcctggtat 18120gagtcagcaa caccttcttc acgaggcaga
cctcagcgcc agaaggccgc cagagaggcc 18180gagcgcggcc gtgaggcttg gacgctaggg
cagggcatga aaaagcccgt agcgggctgc 18240tacgggcgtc tgacgcggtg gaaaggggga
ggggatgttg tctacatggc tctgctgtag 18300tgagtgggtt gcgctccggc agcggtcctg
atcaatcgtc accctttctc ggtccttcaa 18360cgttcctgac aacgagcctc cttttcgcca
atccatcgac aatcaccgcg agtccctgct 18420cgaacgctgc gtccggaccg gcttcgtcga
aggcgtctat cgcggcccgc aacagcggcg 18480agagcggagc ctgttcaacg gtgccgccgc
gctcgccggc atcgctgtcg ccggcctgct 18540cctcaagcac ggccccaaca gtgaagtagc
tgattgtcat cagcgcattg acggcgtccc 18600cggccgaaaa acccgcctcg cagaggaagc
gaagctgcgc gtcggccgtt tccatctgcg 18660gtgcgcccgg tcgcgtgccg gcatggatgc
gcgcgccatc gcggtaggcg agcagcgcct 18720gcctgaagct gcgggcattc ccgatcagaa
atgagcgcca gtcgtcgtcg gctctcggca 18780ccgaatgcgt atgattctcc gccagcatgg
cttcggccag tgcgtcgagc agcgcccgct 18840tgttcctgaa gtgccagtaa agcgccggct
gctgaacccc caaccgttcc gccagtttgc 18900gtgtcgtcag accgtctacg ccgacctcgt
tcaacaggtc cagggcggca cggatcactg 18960tattcggctg caactttgtc atgcttgaca
ctttatcact gataaacata atatgtccac 19020caacttatca gtgataaaga atccgcgcgt
tcaatcggac cagcggaggc tggtccggag 19080gccagacgtg aaacccaaca tacccctgat
cgtaattctg agcactgtcg cgctcgacgc 19140tgtcggcatc ggcctgatta tgccggtgct
gccgggcctc ctgcgcgatc tggttcactc 19200gaacgacgtc accgcccact atggcattct
gctggcgctg tatgcgttgg tgcaatttgc 19260ctgcgcacct gtgctgggcg cgctgtcgga
tcgtttcggg cggcggccaa tcttgctcgt 19320ctcgctggcc ggcgccactg tcgactacgc
catcatggcg acagcgcctt tcctttgggt 19380tctctatatc gggcggatcg tggccggcat
caccggggcg actggggcgg tagccggcgc 19440ttatattgcc gatatcactg atggcgatga
gcgcgcgcgg cacttcggct tcatgagcgc 19500ctgtttcggg ttcgggatgg tcgcgggacc
tgtgctcggt gggctgatgg gcggtttctc 19560cccccacgct ccgttcttcg ccgcggcagc
cttgaacggc ctcaatttcc tgacgggctg 19620tttccttttg ccggagtcgc acaaaggcga
acgccggccg ttacgccggg aggctctcaa 19680cccgctcgct tcgttccggt gggcccgggg
catgaccgtc gtcgccgccc tgatggcggt 19740cttcttcatc atgcaacttg tcggacaggt
gccggccgcg ctttgggtca ttttcggcga 19800ggatcgcttt cactgggacg cgaccacgat
cggcatttcg cttgccgcat ttggcattct 19860gcattcactc gcccaggcaa tgatcaccgg
ccctgtagcc gcccggctcg gcgaaaggcg 19920ggcactcatg ctcggaatga ttgccgacgg
cacaggctac atcctgcttg ccttcgcgac 19980acggggatgg atggcgttcc cgatcatggt
cctgcttgct tcgggtggca tcggaatgcc 20040ggcgctgcaa gcaatgttgt ccaggcaggt
ggatgaggaa cgtcaggggc agctgcaagg 20100ctcactggcg gcgctcacca gcctgacctc
gatcgtcgga cccctcctct tcacggcgat 20160ctatgcggct tctataacaa cgtggaacgg
gtgggcatgg attgcaggcg ctgccctcta 20220cttgctctgc ctgccggcgc tgcgtcgcgg
gctttggagc ggcgcagggc aacgagccga 20280tcgctgatcg tggaaacgat aggcctatgc
catgcgggtc aaggcgactt ccggcaagct 20340atacgcgccc taggagtgcg gttggaacgt
tggcccagcc agatactccc gatcacgagc 20400aggacgccga tgatttgaag cgcactcagc
gtctgatcca agaacaacca tcctagcaac 20460acggcggtcc ccgggctgag aaagcccagt
aaggaaacaa ctgtaggttc gagtcgcgag 20520atcccccgga accaaaggaa gtaggttaaa
cccgctccga tcaggccgag ccacgccagg 20580ccgagaacat tggttcctgt aggcatcggg
attggcggat caaacactaa agctactgga 20640acgagcagaa gtcctccggc cgccagttgc
caggcggtaa aggtgagcag aggcacggga 20700ggttgccact tgcgggtcag cacggttccg
aacgccatgg aaaccgcccc cgccaggccc 20760gctgcgacgc cgacaggatc tagcgctgcg
tttggtgtca acaccaacag cgccacgccc 20820gcagttccgc aaatagcccc caggaccgcc
atcaatcgta tcgggctacc tagcagagcg 20880gcagagatga acacgaccat cagcggctgc
acagcgccta ccgtcgccgc gaccccgccc 20940ggcaggcggt agaccgaaat aaacaacaag
ctccagaata gcgaaatatt aagtgcgccg 21000aggatgaaga tgcgcatcca ccagattccc
gttggaatct gtcggacgat catcacgagc 21060aataaacccg ccggcaacgc ccgcagcagc
ataccggcga cccctcggcc tcgctgttcg 21120ggctccacga aaacgccgga cagatgcgcc
ttgtgagcgt ccttggggcc gtcctcctgt 21180ttgaagaccg acagcccaat gatctcgccg
tcgatgtagg cgccgaatgc cacggcatct 21240cgcaaccgtt cagcgaacgc ctccatgggc
tttttctcct cgtgctcgta aacggacccg 21300aacatctctg gagctttctt cagggccgac
aatcggatct cgcggaaatc ctgcacgtcg 21360gccgctccaa gccgtcgaat ctgagcctta
atcacaattg tcaattttaa tcctctgttt 21420atcggcagtt cgtagagcgc gccgtgcgtc
ccgagcgata ctgagcgaag caagtgcgtc 21480gagcagtgcc cgcttgttcc tgaaatgcca
gtaaagcgct ggctgctgaa cccccagccg 21540gaactgaccc cacaaggccc tagcgtttgc
aatgcaccag gtcatcattg acccaggcgt 21600gttccaccag gccgctgcct cgcaactctt
cgcaggcttc gccgacctgc tcgcgccact 21660tcttcacgcg ggtggaatcc gatccgcaca
tgaggcggaa ggtttccagc ttgagcgggt 21720acggctcccg gtgcgagctg aaatagtcga
acatccgtcg ggccgtcggc gacagcttgc 21780ggtacttctc ccatatgaat ttcgtgtagt
ggtcgccagc aaacagcacg acgatttcct 21840cgtcgatcag gacctggcaa cgggacgttt
tcttgccacg gtccaggacg cggaagcggt 21900gcagcagcga caccgattcc aggtgcccaa
cgcggtcgga cgtgaagccc atcgccgtcg 21960cctgtaggcg cgacaggcat tcctcggcct
tcgtgtaata ccggccattg atcgaccagc 22020ccaggtcctg gcaaagctcg tagaacgtga
aggtgatcgg ctcgccgata ggggtgcgct 22080tcgcgtactc caacacctgc tgccacacca
gttcgtcatc gtcggcccgc agctcgacgc 22140cggtgtaggt gatcttcacg tccttgttga
cgtggaaaat gaccttgttt tgcagcgcct 22200cgcgcgggat tttcttgttg cgcgtggtga
acagggcaga gcgggccgtg tcgtttggca 22260tcgctcgcat cgtgtccggc cacggcgcaa
tatcgaacaa ggaaagctgc atttccttga 22320tctgctgctt cgtgtgtttc agcaacgcgg
cctgcttggc ctcgctgacc tgttttgcca 22380ggtcctcgcc ggcggttttt cgcttcttgg
tcgtcatagt tcctcgcgtg tcgatggtca 22440tcgacttcgc caaacctgcc gcctcctgtt
cgagacgacg cgaacgctcc acggcggccg 22500atggcgcggg cagggcaggg ggagccagtt
gcacgctgtc gcgctcgatc ttggccgtag 22560cttgctggac catcgagccg acggactgga
aggtttcgcg gggcgcacgc atgacggtgc 22620ggcttgcgat ggtttcggca tcctcggcgg
aaaaccccgc gtcgatcagt tcttgcctgt 22680atgccttccg gtcaaacgtc cgattcattc
accctccttg cgggattgcc ccgactcacg 22740ccggggcaat gtgcccttat tcctgatttg
acccgcctgg tgccttggtg tccagataat 22800ccaccttatc ggcaatgaag tcggtcccgt
agaccgtctg gccgtccttc tcgtacttgg 22860tattccgaat cttgccctgc acgaatacca
gcgacccctt gcccaaatac ttgccgtggg 22920cctcggcctg agagccaaaa cacttgatgc
ggaagaagtc ggtgcgctcc tgcttgtcgc 22980cggcatcgtt gcgccactct tcattaaccg
ctatatcgaa aattgcttgc ggcttgttag 23040aattgccatg acgtacctcg gtgtcacggg
taagattacc gataaactgg aactgattat 23100ggctcatatc gaaagtctcc ttgagaaagg
agactctagt ttagctaaac attggttccg 23160ctgtcaagaa ctttagcggc taaaattttg
cgggccgcga ccaaaggtgc gaggggcggc 23220ttccgctgtg tacaaccaga tatttttcac
caacatcctt cgtctgctcg atgagcgggg 23280catgacgaaa catgagctgt cggagagggc
aggggtttca atttcgtttt tatcagactt 23340aaccaacggt aaggccaacc cctcgttgaa
ggtgatggag gccattgccg acgccctgga 23400aactccccta cctcttctcc tggagtccac
cgaccttgac cgcgaggcac tcgcggagat 23460tgcgggtcat cctttcaaga gcagcgtgcc
gcccggatac gaacgcatca gtgtggtttt 23520gccgtcacat aaggcgttta tcgtaaagaa
atggggcgac gacacccgaa aaaagctgcg 23580tggaaggctc tgacgccaag ggttagggct
tgcacttcct tctttagccg ctaaaacggc 23640cccttctctg cgggccgtcg gctcgcgcat
catatcgaca tcctcaacgg aagccgtgcc 23700gcgaatggca tcgggcgggt gcgctttgac
agttgttttc tatcagaacc cctacgtcgt 23760gcggttcgat tagctgtttg tcttgcaggc
taaacacttt cggtatatcg tttgcctgtg 23820cgataatgtt gctaatgatt tgttgcgtag
gggttactga aaagtgagcg ggaaagaaga 23880gtttcagacc atcaaggagc gggccaagcg
caagctggaa cgcgacatgg gtgcggacct 23940gttggccgcg ctcaacgacc cgaaaaccgt
tgaagtcatg ctcaacgcgg acggcaaggt 24000gtggcacgaa cgccttggcg agccgatgcg
gtacatctgc gacatgcggc ccagccagtc 24060gcaggcgatt atagaaacgg tggccggatt
ccacggcaaa gaggtcacgc ggcattcgcc 24120catcctggaa ggcgagttcc ccttggatgg
cagccgcttt gccggccaat tgccgccggt 24180cgtggccgcg ccaacctttg cgatccgcaa
gcgcgcggtc gccatcttca cgctggaaca 24240gtacgtcgag gcgggcatca tgacccgcga
gcaatacgag gtcattaaaa gcgccgtcgc 24300ggcgcatcga aacatcctcg tcattggcgg
tactggctcg ggcaagacca cgctcgtcaa 24360cgcgatcatc aatgaaatgg tcgccttcaa
cccgtctgag cgcgtcgtca tcatcgagga 24420caccggcgaa atccagtgcg ccgcagagaa
cgccgtccaa taccacacca gcatcgacgt 24480ctcgatgacg ctgctgctca agacaacgct
gcgtatgcgc cccgaccgca tcctggtcgg 24540tgaggtacgt ggccccgaag cccttgatct
gttgatggcc tggaacaccg ggcatgaagg 24600aggtgccgcc accctgcacg caaacaaccc
caaagcgggc ctgagccggc tcgccatgct 24660tatcagcatg cacccggatt caccgaaacc
cattgagccg ctgattggcg aggcggttca 24720tgtggtcgtc catatcgcca ggacccctag
cggccgtcga gtgcaagaaa ttctcgaagt 24780tcttggttac gagaacggcc agtacatcac
caaaaccctg taaggagtat ttccaatgac 24840aacggctgtt ccgttccgtc tgaccatgaa
tcgcggcatt ttgttctacc ttgccgtgtt 24900cttcgttctc gctctcgcgt tatccgcgca
tccggcgatg gcctcggaag gcaccggcgg 24960cagcttgcca tatgagagct ggctgacgaa
cctgcgcaac tccgtaaccg gcccggtggc 25020cttcgcgctg tccatcatcg gcatcgtcgt
cgccggcggc gtgctgatct tcggcggcga 25080actcaacgcc ttcttccgaa ccctgatctt
cctggttctg gtgatggcgc tgctggtcgg 25140cgcgcagaac gtgatgagca ccttcttcgg
tcgtggtgcc gaaatcgcgg ccctcggcaa 25200cggggcgctg caccaggtgc aagtcgcggc
ggcggatgcc gtgcgtgcgg tagcggctgg 25260acggctcgcc taatcatggc tctgcgcacg
atccccatcc gtcgcgcagg caaccgagaa 25320aacctgttca tgggtggtga tcgtgaactg
gtgatgttct cgggcctgat ggcgtttgcg 25380ctgattttca gcgcccaaga gctgcgggcc
accgtggtcg gtctgatcct gtggttcggg 25440gcgctctatg cgttccgaat catggcgaag
gccgatccga agatgcggtt cgtgtacctg 25500cgtcaccgcc ggtacaagcc gtattacccg
gcccgctcga ccccgttccg cgagaacacc 25560aatagccaag ggaagcaata ccgatgatcc
aagcaattgc gattgcaatc gcgggcctcg 25620gcgcgcttct gttgttcatc ctctttgccc
gcatccgcgc ggtcgatgcc gaactgaaac 25680tgaaaaagca tcgttccaag gacgccggcc
tggccgatct gctcaactac gccgctgtcg 25740tcgatgacgg cgtaatcgtg ggcaagaacg
gcagctttat ggctgcctgg ctgtacaagg 25800gcgatgacaa cgcaagcagc accgaccagc
agcgcgaagt agtgtccgcc cgcatcaacc 25860aggccctcgc gggcctggga agtgggtgga
tgatccatgt ggacgccgtg cggcgtcctg 25920ctccgaacta cgcggagcgg ggcctgtcgg
cgttccctga ccgtctgacg gcagcgattg 25980aagaagagcg ctcggtcttg ccttgctcgt
cggtgatgta cttcaccagc tccgcgaagt 26040cgctcttctt gatggagcgc atggggacgt
gcttggcaat cacgcgcacc ccccggccgt 26100tttagcggct aaaaaagtca tggctctgcc
ctcgggcgga ccacgcccat catgaccttg 26160ccaagctcgt cctgcttctc ttcgatcttc
gccagcaggg cgaggatcgt ggcatcaccg 26220aaccgcgccg tgcgcgggtc gtcggtgagc
cagagtttca gcaggccgcc caggcggccc 26280aggtcgccat tgatgcgggc cagctcgcgg
acgtgctcat agtccacgac gcccgtgatt 26340ttgtagccct ggccgacggc cagcaggtag
gccgacaggc tcatgccggc cgccgccgcc 26400ttttcctcaa tcgctcttcg ttcgtctgga
aggcagtaca ccttgatagg tgggctgccc 26460ttcctggttg gcttggtttc atcagccatc
cgcttgccct catctgttac gccggcggta 26520gccggccagc ctcgcagagc aggattcccg
ttgagcaccg ccaggtgcga ataagggaca 26580gtgaagaagg aacacccgct cgcgggtggg
cctacttcac ctatcctgcc cggctgacgc 26640cgttggatac accaaggaaa gtctacacga
accctttggc aaaatcctgt atatcgtgcg 26700aaaaaggatg gatataccga aaaaatcgct
ataatgaccc cgaagcaggg ttatgcagcg 26760gaaaagcgct gcttccctgc tgttttgtgg
aatatctacc gactggaaac aggcaaatgc 26820aggaaattac tgaactgagg ggacaggcga
gagacgatgc caaagagcta caccgacgag 26880ctggccgagt gggttgaatc ccgcgcggcc
aagaagcgcc ggcgtgatga ggctgcggtt 26940gcgttcctgg cggtgagggc ggatgtcgag
gcggcgttag cgtccggcta tgcgctcgtc 27000accatttggg agcacatgcg ggaaacgggg
aaggtcaagt tctcctacga gacgttccgc 27060tcgcacgcca ggcggcacat caaggccaag
cccgccgatg tgcccgcacc gcaggccaag 27120gctgcggaac ccgcgccggc acccaagacg
ccggagccac ggcggccgaa gcaggggggc 27180aaggctgaaa agccggcccc cgctgcggcc
ccgaccggct tcaccttcaa cccaacaccg 27240gacaaaaagg atctactgta atggcgaaaa
ttcacatggt tttgcagggc aagggcgggg 27300tcggcaagtc ggccatcgcc gcgatcattg
cgcagtacaa gatggacaag gggcagacac 27360ccttgtgcat cgacaccgac ccggtgaacg
cgacgttcga gggctacaag gccctgaacg 27420tccgccggct gaacatcatg gccggcgacg
aaattaactc gcgcaacttc gacaccctgg 27480tcgagctgat tgcgccgacc aaggatgacg
tggtgatcga caacggtgcc agctcgttcg 27540tgcctctgtc gcattacctc atcagcaacc
aggtgccggc tctgctgcaa gaaatggggc 27600atgagctggt catccatacc gtcgtcaccg
gcggccaggc tctcctggac acggtgagcg 27660gcttcgccca gctcgccagc cagttcccgg
ccgaagcgct tttcgtggtc tggctgaacc 27720cgtattgggg gcctatcgag catgagggca
agagctttga gcagatgaag gcgtacacgg 27780ccaacaaggc ccgcgtgtcg tccatcatcc
agattccggc cctcaaggaa gaaacctacg 27840gccgcgattt cagcgacatg ctgcaagagc
ggctgacgtt cgaccaggcg ctggccgatg 27900aatcgctcac gatcatgacg cggcaacgcc
tcaagatcgt gcggcgcggc ctgtttgaac 27960agctcgacgc ggcggccgtg ctatgagcga
ccagattgaa gagctgatcc gggagattgc 28020ggccaagcac ggcatcgccg tcggccgcga
cgacccggtg ctgatcctgc ataccatcaa 28080cgcccggctc atggccgaca gtgcggccaa
gcaagaggaa atccttgccg cgttcaagga 28140agagctggaa gggatcgccc atcgttgggg
cgaggacgcc aaggccaaag cggagcggat 28200gctgaacgcg gccctggcgg ccagcaagga
cgcaatggcg aaggtaatga aggacagcgc 28260cgcgcaggcg gccgaagcga tccgcaggga
aatcgacgac ggccttggcc gccagctcgc 28320ggccaaggtc gcggacgcgc ggcgcgtggc
gatgatgaac atgatcgccg gcggcatggt 28380gttgttcgcg gccgccctgg tggtgtgggc
ctcgttatga atcgcagagg cgcagatgaa 28440aaagcccggc gttgccgggc tttgtttttg
cgttagctgg gcttgtttga caggcccaag 28500ctctgactgc gcccgcgctc gcgctcctgg
gcctgtttct tctcctgctc ctgcttgcgc 28560atcagggcct ggtgccgtcg ggctgcttca
cgcatcgaat cccagtcgcc ggccagctcg 28620ggatgctccg cgcgcatctt gcgcgtcgcc
agttcctcga tcttgggcgc gtgaatgccc 28680atgccttcct tgatttcgcg caccatgtcc
agccgcgtgt gcagggtctg caagcgggct 28740tgctgttggg cctgctgctg ctgccaggcg
gcctttgtac gcggcaggga cagcaagccg 28800ggggcattgg actgtagctg ctgcaaacgc
gcctgctgac ggtctacgag ctgttctagg 28860cggtcctcga tgcgctccac ctggtcatgc
tttgcctgca cgtagagcgc aagggtctgc 28920tggtaggtct gctcgatggg cgcggattct
aagagggcct gctgttccgt ctcggcctcc 28980tgggccgcct gtagcaaatc ctcgccgctg
ttgccgctgg actgctttac tgccggggac 29040tgctgttgcc ctgctcgcgc cgtcgtcgca
gttcggcttg cccccactcg attgactgct 29100tcatttcgag ccgcagcgat gcgatctcgg
attgcgtcaa cggacggggc agcgcggagg 29160tgtccggctt ctccttgggt gagtcggtcg
atgccatagc caaaggtttc cttccaaaat 29220gcgtccattg ctggaccgtg tttctcattg
atgcccgcaa gcatcttcgg cttgaccgcc 29280aggtcaagcg cgccttcatg ggcggtcatg
acggacgccg ccatgacctt gccgccgttg 29340ttctcgatgt agccgcgtaa tgaggcaatg
gtgccgccca tcgtcagcgt gtcatcgaca 29400acgatgtact tctggccggg gatcacctcc
ccctcgaaag tcgggttgaa cgccaggcga 29460tgatctgaac cggctccggt tcgggcgacc
ttctcccgct gcacaatgtc cgtttcgacc 29520tcaaggccaa ggcggtcggc cagaacgacc
gccatcatgg ccggaatctt gttgttcccc 29580gccgcctcga cggcgaggac tggaacgatg
cggggcttgt cgtcgccgat cagcgtcttg 29640agctgggcaa cagtgtcgtc cgaaatcagg
cgctcgacca aattaagcgc cgcttccgcg 29700tcgccctgct tcgcagcctg gtattcaggc
tcgttggtca aagaaccaag gtcgccgttg 29760cgaaccacct tcgggaagtc tccccacggt
gcgcgctcgg ctctgctgta gctgctcaag 29820acgcctccct ttttagccgc taaaactcta
acgagtgcgc ccgcgactca acttgacgct 29880ttcggcactt acctgtgcct tgccacttgc
gtcataggtg atgcttttcg cactcccgat 29940ttcaggtact ttatcgaaat ctgaccgggc
gtgcattaca aagttcttcc ccacctgttg 30000gtaaatgctg ccgctatctg cgtggacgat
gctgccgtcg tggcgctgcg acttatcggc 30060cttttgggcc atatagatgt tgtaaatgcc
aggtttcagg gccccggctt tatctacctt 30120ctggttcgtc catgcgcctt ggttctcggt
ctggacaatt ctttgcccat tcatgaccag 30180gaggcggtgt ttcattgggt gactcctgac
ggttgcctct ggtgttaaac gtgtcctggt 30240cgcttgccgg ctaaaaaaaa gccgacctcg
gcagttcgag gccggctttc cctagagccg 30300ggcgcgtcaa ggttgttcca tctattttag
tgaactgcgt tcgatttatc agttactttc 30360ctcccgcttt gtgtttcctc ccactcgttt
ccgcgtctag ccgacccctc aacatagcgg 30420cctcttcttg ggctgccttt gcctcttgcc
gcgcttcgtc acgctcggct tgcaccgtcg 30480taaagcgctc ggcctgcctg gccgcctctt
gcgccgccaa cttcctttgc tcctggtggg 30540cctcggcgtc ggcctgcgcc ttcgctttca
ccgctgccaa ctccgtgcgc aaactctccg 30600cttcgcgcct ggtggcgtcg cgctcgccgc
gaagcgcctg catttcctgg ttggccgcgt 30660ccagggtctt gcggctctct tctttgaatg
cgcgggcgtc ctggtgagcg tagtccagct 30720cggcgcgcag ctcctgcgct cgacgctcca
cctcgtcggc ccgctgcgtc gccagcgcgg 30780cccgctgctc ggctcctgcc agggcggtgc
gtgcttcggc cagggcttgc cgctggcgtg 30840cggccagctc ggccgcctcg gcggcctgct
gctctagcaa tgtaacgcgc gcctgggctt 30900cttccagctc gcgggcctgc gcctcgaagg
cgtcggccag ctccccgcgc acggcttcca 30960actcgttgcg ctcacgatcc cagccggctt
gcgctgcctg caacgattca ttggcaaggg 31020cctgggcggc ttgccagagg gcggccacgg
cctggttgcc ggcctgctgc accgcgtccg 31080gcacctggac tgccagcggg gcggcctgcg
ccgtgcgctg gcgtcgccat tcgcgcatgc 31140cggcgctggc gtcgttcatg ttgacgcggg
cggccttacg cactgcatcc acggtcggga 31200agttctcccg gtcgccttgc tcgaacagct
cgtccgcagc cgcaaaaatg cggtcgcgcg 31260tctctttgtt cagttccatg ttggctccgg
taattggtaa gaataataat actcttacct 31320accttatcag cgcaagagtt tagctgaaca
gttctcgact taacggcagg ttttttagcg 31380gctgaagggc aggcaaaaaa agccccgcac
ggtcggcggg ggcaaagggt cagcgggaag 31440gggattagcg ggcgtcgggc ttcttcatgc
gtcggggccg cgcttcttgg gatggagcac 31500gacgaagcgc gcacgcgcat cgtcctcggc
cctatcggcc cgcgtcgcgg tcaggaactt 31560gtcgcgcgct aggtcctccc tggtgggcac
caggggcatg aactcggcct gctcgatgta 31620ggtccactcc atgaccgcat cgcagtcgag
gccgcgttcc ttcaccgtct cttgcaggtc 31680gcggtacgcc cgctcgttga gcggctggta
acgggccaat tggtcgtaaa tggctgtcgg 31740ccatgagcgg cctttcctgt tgagccagca
gccgacgacg aagccggcaa tgcaggcccc 31800tggcacaacc aggccgacgc cgggggcagg
ggatggcagc agctcgccaa ccaggaaccc 31860cgccgcgatg atgccgatgc cggtcaacca
gcccttgaaa ctatccggcc ccgaaacacc 31920cctgcgcatt gcctggatgc tgcgccggat
agcttgcaac atcaggagcc gtttcttttg 31980ttcgtcagtc atggtccgcc ctcaccagtt
gttcgtatcg gtgtcggacg aactgaaatc 32040gcaagagctg ccggtatcgg tccagccgct
gtccgtgtcg ctgctgccga agcacggcga 32100ggggtccgcg aacgccgcag acggcgtatc
cggccgcagc gcatcgccca gcatggcccc 32160ggtcagcgag ccgccggcca ggtagcccag
catggtgctg ttggtcgccc cggccaccag 32220ggccgacgtg acgaaatcgc cgtcattccc
tctggattgt tcgctgctcg gcggggcagt 32280gcgccgcgcc ggcggcgtcg tggatggctc
gggttggctg gcctgcgacg gccggcgaaa 32340ggtgcgcagc agctcgttat cgaccggctg
cggcgtcggg gccgccgcct tgcgctgcgg 32400tcggtgttcc ttcttcggct cgcgcagctt
gaacagcatg atcgcggaaa ccagcagcaa 32460cgccgcgcct acgcctcccg cgatgtagaa
cagcatcgga ttcattcttc ggtcctcctt 32520gtagcggaac cgttgtctgt gcggcgcggg
tggcccgcgc cgctgtcttt ggggatcagc 32580cctcgatgag cgcgaccagt ttcacgtcgg
caaggttcgc ctcgaactcc tggccgtcgt 32640cctcgtactt caaccaggca tagccttccg
ccggcggccg acggttgagg ataaggcggg 32700cagggcgctc gtcgtgctcg acctggacga
tggccttttt cagcttgtcc gggtccggct 32760ccttcgcgcc cttttccttg gcgtccttac
cgtcctggtc gccgtcctcg ccgtcctggc 32820cgtcgccggc ctccgcgtca cgctcggcat
cagtctggcc gttgaaggca tcgacggtgt 32880tgggatcgcg gcccttctcg tccaggaact
cgcgcagcag cttgaccgtg ccgcgcgtga 32940tttcctgggt gtcgtcgtca agccacgcct
cgacttcctc cgggcgcttc ttgaaggccg 33000tcaccagctc gttcaccacg gtcacgtcgc
gcacgcggcc ggtgttgaac gcatcggcga 33060tcttctccgg caggtccagc agcgtgacgt
gctgggtgat gaacgccggc gacttgccga 33120tttccttggc gatatcgcct ttcttcttgc
ccttcgccag ctcgcggcca atgaagtcgg 33180caatttcgcg cggggtcagc tcgttgcgtt
gcaggttctc gataacctgg tcggcttcgt 33240tgtagtcgtt gtcgatgaac gccgggatgg
acttcttgcc ggcccacttc gagccacggt 33300agcggcgggc gccgtgattg atgatatagc
ggcccggctg ctcctggttc tcgcgcaccg 33360aaatgggtga cttcaccccg cgctctttga
tcgtggcacc gatttccgcg atgctctccg 33420gggaaaagcc ggggttgtcg gccgtccgcg
gctgatgcgg atcttcgtcg atcaggtcca 33480ggtccagctc gatagggccg gaaccgccct
gagacgccgc aggagcgtcc aggaggctcg 33540acaggtcgcc gatgctatcc aaccccaggc
cggacggctg cgccgcgcct gcggcttcct 33600gagcggccgc agcggtgttt ttcttggtgg
tcttggcttg agccgcagtc attgggaaat 33660ctccatcttc gtgaacacgt aatcagccag
ggcgcgaacc tctttcgatg ccttgcgcgc 33720ggccgttttc ttgatcttcc agaccggcac
accggatgcg agggcatcgg cgatgctgct 33780gcgcaggcca acggtggccg gaatcatcat
cttggggtac gcggccagca gctcggcttg 33840gtggcgcgcg tggcgcggat tccgcgcatc
gaccttgctg ggcaccatgc caaggaattg 33900cagcttggcg ttcttctggc gcacgttcgc
aatggtcgtg accatcttct tgatgccctg 33960gatgctgtac gcctcaagct cgatggggga
cagcacatag tcggccgcga agagggcggc 34020cgccaggccg acgccaaggg tcggggccgt
gtcgatcagg cacacgtcga agccttggtt 34080cgccagggcc ttgatgttcg ccccgaacag
ctcgcgggcg tcgtccagcg acagccgttc 34140ggcgttcgcc agtaccgggt tggactcgat
gagggcgagg cgcgcggcct ggccgtcgcc 34200ggctgcgggt gcggtttcgg tccagccgcc
ggcagggaca gcgccgaaca gcttgcttgc 34260atgcaggccg gtagcaaagt ccttgagcgt
gtaggacgca ttgccctggg ggtccaggtc 34320gatcacggca acccgcaagc cgcgctcgaa
aaagtcgaag gcaagatgca caagggtcga 34380agtcttgccg acgccgcctt tctggttggc
cgtgaccaaa gttttcatcg tttggtttcc 34440tgttttttct tggcgtccgc ttcccacttc
cggacgatgt acgcctgatg ttccggcaga 34500accgccgtta cccgcgcgta cccctcgggc
aagttcttgt cctcgaacgc ggcccacacg 34560cgatgcaccg cttgcgacac tgcgcccctg
gtcagtccca gcgacgttgc gaacgtcgcc 34620tgtggcttcc catcgactaa gacgccccgc
gctatctcga tggtctgctg ccccacttcc 34680agcccctgga tcgcctcctg gaactggctt
tcggtaagcc gtttcttcat ggataacacc 34740cataatttgc tccgcgcctt ggttgaacat
agcggtgaca gccgccagca catgagagaa 34800gtttagctaa acatttctcg cacgtcaaca
cctttagccg ctaaaactcg tccttggcgt 34860aacaaaacaa aagcccggaa accgggcttt
cgtctcttgc cgcttatggc tctgcacccg 34920gctccatcac caacaggtcg cgcacgcgct
tcactcggtt gcggatcgac actgccagcc 34980caacaaagcc ggttgccgcc gccgccagga
tcgcgccgat gatgccggcc acaccggcca 35040tcgcccacca ggtcgccgcc ttccggttcc
attcctgctg gtactgcttc gcaatgctgg 35100acctcggctc accataggct gaccgctcga
tggcgtatgc cgcttctccc cttggcgtaa 35160aacccagcgc cgcaggcggc attgccatgc
tgcccgccgc tttcccgacc acgacgcgcg 35220caccaggctt gcggtccaga ccttcggcca
cggcgagctg cgcaaggaca taatcagccg 35280ccgacttggc tccacgcgcc tcgatcagct
cttgcactcg cgcgaaatcc ttggcctcca 35340cggccgccat gaatcgcgca cgcggcgaag
gctccgcagg gccggcgtcg tgatcgccgc 35400cgagaatgcc cttcaccaag ttcgacgaca
cgaaaatcat gctgacggct atcaccatca 35460tgcagacgga tcgcacgaac ccgctgaatt
gaacacgagc acggcacccg cgaccactat 35520gccaagaatg cccaaggtaa aaattgccgg
ccccgccatg aagtccgtga atgccccgac 35580ggccgaagtg aagggcaggc cgccacccag
gccgccgccc tcactgcccg gcacctggtc 35640gctgaatgtc gatgccagca cctgcggcac
gtcaatgctt ccgggcgtcg cgctcgggct 35700gatcgcccat cccgttactg ccccgatccc
ggcaatggca aggactgcca gcgctgccat 35760ttttggggtg aggccgttcg cggccgaggg
gcgcagcccc tggggggatg ggaggcccgc 35820gttagcgggc cgggagggtt cgagaagggg
gggcaccccc cttcggcgtg cgcggtcacg 35880cgcacagggc gcagccctgg ttaaaaacaa
ggtttataaa tattggttta aaagcaggtt 35940aaaagacagg ttagcggtgg ccgaaaaacg
ggcggaaacc cttgcaaatg ctggattttc 36000tgcctgtgga cagcccctca aatgtcaata
ggtgcgcccc tcatctgtca gcactctgcc 36060cctcaagtgt caaggatcgc gcccctcatc
tgtcagtagt cgcgcccctc aagtgtcaat 36120accgcagggc acttatcccc aggcttgtcc
acatcatctg tgggaaactc gcgtaaaatc 36180aggcgttttc gccgatttgc gaggctggcc
agctccacgt cgccggccga aatcgagcct 36240gcccctcatc tgtcaacgcc gcgccgggtg
agtcggcccc tcaagtgtca acgtccgccc 36300ctcatctgtc agtgagggcc aagttttccg
cgaggtatcc acaacgccgg cggccgcggt 36360gtctcgcaca cggcttcgac ggcgtttctg
gcgcgtttgc agggccatag acggccgcca 36420gcccagcggc gagggcaacc agcccggtga
gcgtcggaaa ggcgctggaa gccccgtagc 36480gacgcggaga ggggcgagac aagccaaggg
cgcaggctcg atgcgcagca cgacatagcc 36540ggttctcgca aggacgagaa tttccctgcg
gtgcccctca agtgtcaatg aaagtttcca 36600acgcgagcca ttcgcgagag ccttgagtcc
acgctagatg agagctttgt tgtaggtgga 36660ccagttggtg attttgaact tttgctttgc
cacggaacgg tctgcgttgt cgggaagatg 36720cgtgatctga tccttcaact cagcaaaagt
tcgatttatt caacaaagcc acgttgtgtc 36780tcaaaatctc tgatgttaca ttgcacaaga
taaaaatata tcatcatgaa caataaaact 36840gtctgcttac ataaacagta atacaagggg
tgttatgagc catattcaac gggaaacgtc 36900ttgctcgac
36909813019DNAartificial sequencevector
8gttacccgga ccgaagctta gcccgggcat gcctgcagtg cagcgtgacc cggtcgtgcc
60cctctctaga gataatgagc attgcatgtc taagttataa aaaattacca catatttttt
120ttgtcacact tgtttgaagt gcagtttatc tatctttata catatattta aactttactc
180tacgaataat ataatctata gtactacaat aatatcagtg ttttagagaa tcatataaat
240gaacagttag acatggtcta aaggacaatt gagtattttg acaacaggac tctacagttt
300tatcttttta gtgtgcatgt gttctccttt ttttttgcaa atagcttcac ctatataata
360cttcatccat tttattagta catccattta gggtttaggg ttaatggttt ttatagacta
420atttttttag tacatctatt ttattctatt ttagcctcta aattaagaaa actaaaactc
480tattttagtt tttttattta ataatttaga tataaaatag aataaaataa agtgactaaa
540aattaaacaa atacccttta agaaattaaa aaaactaagg aaacattttt cttgtttcga
600gtagataatg ccagcctgtt aaacgccgtc gacgagtcta acggacacca accagcgaac
660cagcagcgtc gcgtcgggcc aagcgaagca gacggcacgg catctctgtc gctgcctctg
720gacccctctc gagagttccg ctccaccgtt ggacttgctc cgctgtcggc atccagaaat
780tgcgtggcgg agcggcagac gtgagccggc acggcaggcg gcctcctcct cctctcacgg
840cacggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc
900gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca
960cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc
1020cgctcgtcct cccccccccc ccctctctac cttctctaga tcggcgttcc ggtccatggt
1080tagggcccgg tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc
1140cgtgctgcta gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa
1200cttgccagtg tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat
1260cgatttcatg attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt
1320caatatatgc cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt
1380gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga gtagaattct gtttcaaact
1440acctggtgga tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg
1500aattgaagat gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt
1560tactgatgca tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg
1620ggcggtcgtt cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt
1680tattaatttt ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg
1740atggaaatat cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac
1800atgatggcat atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat
1860aaacaagtat gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc
1920agctatatgt ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt
1980tcttttgtcg atgctcaccc tgttgtttgg tgttacttct gcaggtcgac tctagaggat
2040ccacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat
2100taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
2160cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa
2220tgtgtggatt ttgagttagg atttaaatac gcgttgatcc ggcttactaa aagccagata
2280acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac
2340ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac agttgacagc
2400gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa
2460ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag
2520ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg
2580gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt
2640gtggatgtac agagtgatat cattgacacg cccggtcgac ggatggtgat ccccctggcc
2700agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg
2760gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
2820gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
2880ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg tcgaccatag
2940tgactggata tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt
3000taatatattg atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt
3060gttaacctag acttgtccat cttctggatt ggccaactta attaatgtat gaaataaaag
3120gatgcacaca tagtgacatg ctaatcacta taatgtgggc atcaaagttg tgtgttatgt
3180gtaattacta gttatctgaa taaaagagaa agagatcatc catatttctt atcctaaatg
3240aatgtcacgt gtctttataa ttctttgatg aaccagatgc atttcattaa ccaaatccat
3300atacatataa atattaatca tatataatta atatcaattg ggttagcaaa acaaatctag
3360tctaggtgtg ttttgcgaat tgcggccgcc accgcggtgg agctcgaatt ccggtccggg
3420tcacctttgt ccaccaagat ggaactgcgg ccgctcatta attaagtcag gcgcgcctct
3480agttgaagac acgttcatgt cttcatcgta agaagacact cagtagtctt cggccagaat
3540ggccatctgg attcagcagg cctagaaggc catttaaatc ctgaggatct ggtcttccta
3600aggacccggg atatcggacc gattaaactt taattcggtc cgaagcttgc atgcctgcag
3660tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg tctaagttat
3720aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta tctatcttta
3780tacatatatt taaactttac tctacgaata atataatcta tagtactaca ataatatcag
3840tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa ttgagtattt
3900tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct ttttttttgc
3960aaatagcttc acctatataa tacttcatcc attttattag tacatccatt tagggtttag
4020ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta ttttagcctc
4080taaattaaga aaactaaaac tctattttag tttttttatt taataattta gatataaaat
4140agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta aaaaaactaa
4200ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg tcgacgagtc
4260taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag cagacggcac
4320ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg ttggacttgc
4380tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg gcacggcagg
4440cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc caccgctcct
4500tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc
4560aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc
4620ggcacctccg cttcaaggta cgccgctcgt cctccccccc ccccctctct accttctcta
4680gatcggcgtt ccggtccatg catggttagg gcccggtagt tctacttctg ttcatgtttg
4740tgttagatcc gtgtttgtgt tagatccgtg ctgctagcgt tcgtacacgg atgcgacctg
4800tacgtcagac acgttctgat tgctaacttg ccagtgtttc tctttgggga atcctgggat
4860ggctctagcc gttccgcaga cgggatcgat ttcatgattt tttttgtttc gttgcatagg
4920gtttggtttg cccttttcct ttatttcaat atatgccgtg cacttgtttg tcgggtcatc
4980ttttcatgct tttttttgtc ttggttgtga tgatgtggtc tggttgggcg gtcgttctag
5040atcggagtag aattctgttt caaactacct ggtggattta ttaattttgg atctgtatgt
5100gtgtgccata catattcata gttacgaatt gaagatgatg gatggaaata tcgatctagg
5160ataggtatac atgttgatgc gggttttact gatgcatata cagagatgct ttttgttcgc
5220ttggttgtga tgatgtggtg tggttgggcg gtcgttcatt cgttctagat cggagtagaa
5280tactgtttca aactacctgg tgtatttatt aattttggaa ctgtatgtgt gtgtcataca
5340tcttcatagt tacgagttta agatggatgg aaatatcgat ctaggatagg tatacatgtt
5400gatgtgggtt ttactgatgc atatacatga tggcatatgc agcatctatt catatgctct
5460aaccttgagt acctatctat tataataaac aagtatgttt tataattatt ttgatcttga
5520tatacttgga tgatggcata tgcagcagct atatgtggat ttttttagcc ctgccttcat
5580acgctattta tttgcttggt actgtttctt ttgtcgatgc tcaccctgtt gtttggtgtt
5640acttctgcag gtcgacttta acttagccta ggatccacac gacaccatgt cccccgagcg
5700ccgccccgtc gagatccgcc cggccaccgc cgccgacatg gccgccgtgt gcgacatcgt
5760gaaccactac atcgagacct ccaccgtgaa cttccgcacc gagccgcaga ccccgcagga
5820gtggatcgac gacctggagc gcctccagga ccgctacccg tggctcgtgg ccgaggtgga
5880gggcgtggtg gccggcatcg cctacgccgg cccgtggaag gcccgcaacg cctacgactg
5940gaccgtggag tccaccgtgt acgtgtccca ccgccaccag cgcctcggcc tcggctccac
6000cctctacacc cacctcctca agagcatgga ggcccagggc ttcaagtccg tggtggccgt
6060gatcggcctc ccgaacgacc cgtccgtgcg cctccacgag gccctcggct acaccgcccg
6120cggcaccctc cgcgccgccg gctacaagca cggcggctgg cacgacgtcg gcttctggca
6180gcgcgacttc gagctgccgg ccccgccgcg cccggtgcgc ccggtgacgc agatctgagt
6240cgaaacctag acttgtccat cttctggatt ggccaactta attaatgtat gaaataaaag
6300gatgcacaca tagtgacatg ctaatcacta taatgtgggc atcaaagttg tgtgttatgt
6360gtaattacta gttatctgaa taaaagagaa agagatcatc catatttctt atcctaaatg
6420aatgtcacgt gtctttataa ttctttgatg aaccagatgc atttcattaa ccaaatccat
6480atacatataa atattaatca tatataatta atatcaattg ggttagcaaa acaaatctag
6540tctaggtgtg ttttgcgaat tgcggccgcc accgcggtgg agctcgaatt cattccgatt
6600aatcgtggcc tcttgctctt caggatgaag agctatgttt aaacgtgcaa gcgctactag
6660acaattcagt acattaaaaa cgtccgcaat gtgttattaa gttgtctaag cgtcaatttg
6720tttacaccac aatatatcct gccaccagcc agccaacagc tccccgaccg gcagctcggc
6780acaaaatcac cactcgatac aggcagccca tcagtccggg acggcgtcag cgggagagcc
6840gttgtaaggc ggcagacttt gctcatgtta ccgatgctat tcggaagaac ggcaactaag
6900ctgccgggtt tgaaacacgg atgatctcgc ggagggtagc atgttgattg taacgatgac
6960agagcgttgc tgcctgtgat caaatatcat ctccctcgca gagatccgaa ttatcagcct
7020tcttattcat ttctcgctta accgtgacag gctgtcgatc ttgagaacta tgccgacata
7080ataggaaatc gctggataaa gccgctgagg aagctgagtg gcgctatttc tttagaagtg
7140aacgttgacg atcgtcgacc gtaccccgat gaattaattc ggacgtacgt tctgaacaca
7200gctggatact tacttgggcg attgtcatac atgacatcaa caatgtaccc gtttgtgtaa
7260ccgtctcttg gaggttcgta tgacactagt ggttcccctc agcttgcgac tagatgttga
7320ggcctaacat tttattagag agcaggctag ttgcttagat acatgatctt caggccgtta
7380tctgtcaggg caagcgaaaa ttggccattt atgacgacca atgccccgca gaagctccca
7440tctttgccgc catagacgcc gcgcccccct tttggggtgt agaacatcct tttgccagat
7500gtggaaaaga agttcgttgt cccattgttg gcaatgacgt agtagccggc gaaagtgcga
7560gacccatttg cgctatatat aagcctacga tttccgttgc gactattgtc gtaattggat
7620gaactattat cgtagttgct ctcagagttg tcgtaatttg atggactatt gtcgtaattg
7680cttatggagt tgtcgtagtt gcttggagaa atgtcgtagt tggatgggga gtagtcatag
7740ggaagacgag cttcatccac taaaacaatt ggcaggtcag caagtgcctg ccccgatgcc
7800atcgcaagta cgaggcttag aaccaccttc aacagatcgc gcatagtctt ccccagctct
7860ctaacgcttg agttaagccg cgccgcgaag cggcgtcggc ttgaacgaat tgttagacat
7920tatttgccga ctaccttggt gatctcgcct ttcacgtagt gaacaaattc ttccaactga
7980tctgcgcgcg aggccaagcg atcttcttgt ccaagataag cctgcctagc ttcaagtatg
8040acgggctgat actgggccgg caggcgctcc attgcccagt cggcagcgac atccttcggc
8100gcgattttgc cggttactgc gctgtaccaa atgcgggaca acgtaagcac tacatttcgc
8160tcatcgccag cccagtcggg cggcgagttc catagcgtta aggtttcatt tagcgcctca
8220aatagatcct gttcaggaac cggatcaaag agttcctccg ccgctggacc taccaaggca
8280acgctatgtt ctcttgcttt tgtcagcaag atagccagat caatgtcgat cgtggctggc
8340tcgaagatac ctgcaagaat gtcattgcgc tgccattctc caaattgcag ttcgcgctta
8400gctggataac gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg
8460agaatctcgc tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc
8520cgcgttgttt catcaagcct tacagtcacc gtaaccagca aatcaatatc actgtgtggc
8580ttcaggccgc catccactgc ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga
8640tggcgctcga tgacgccaac tacctctgat agttgagtcg atacttcggc gatcaccgct
8700tccctcatga tgtttaactc ctgaattaag ccgcgccgcg aagcggtgtc ggcttgaatg
8760aattgttagg cgtcatcctg tgctcccgag aaccagtacc agtacatcgc tgtttcgttc
8820gagacttgag gtctagtttt atacgtgaac aggtcaatgc cgccgagagt aaagccacat
8880tttgcgtaca aattgcaggc aggtacattg ttcgtttgtg tctctaatcg tatgccaagg
8940agctgtctgc ttagtgccca ctttttcgca aattcgatga gactgtgcgc gactcctttg
9000cctcggtgcg tgtgcgacac aacaatgtgt tcgatagagg ctagatcgtt ccatgttgag
9060ttgagttcaa tcttcccgac aagctcttgg tcgatgaatg cgccatagca agcagagtct
9120tcatcagagt catcatccga gatgtaatcc ttccggtagg ggctcacact tctggtagat
9180agttcaaagc cttggtcgga taggtgcaca tcgaacactt cacgaacaat gaaatggttc
9240tcagcatcca atgtttccgc cacctgctca gggatcaccg aaatcttcat atgacgccta
9300acgcctggca cagcggatcg caaacctggc gcggcttttg gcacaaaagg cgtgacaggt
9360ttgcgaatcc gttgctgcca cttgttaacc cttttgccag atttggtaac tataatttat
9420gttagaggcg aagtcttggg taaaaactgg cctaaaattg ctggggattt caggaaagta
9480aacatcacct tccggctcga tgtctattgt agatatatgt agtgtatcta cttgatcggg
9540ggatctgctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
9600cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
9660cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
9720gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
9780gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
9840ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
9900ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
9960agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
10020taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
10080cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
10140tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
10200gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
10260gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
10320tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
10380gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
10440cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
10500aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
10560tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
10620ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
10680attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
10740ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
10800tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
10860aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
10920acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
10980aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
11040agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg cagggggggg
11100gggggggggg gacttccatt gttcattcca cggacaaaaa cagagaaagg aaacgacaga
11160ggccaaaaag cctcgctttc agcacctgtc gtttcctttc ttttcagagg gtattttaaa
11220taaaaacatt aagttatgac gaagaagaac ggaaacgcct taaaccggaa aattttcata
11280aatagcgaaa acccgcgagg tcgccgcccc gtaacctgtc ggatcaccgg aaaggacccg
11340taaagtgata atgattatca tctacatatc acaacgtgcg tggaggccat caaaccacgt
11400caaataatca attatgacgc aggtatcgta ttaattgatc tgcatcaact taacgtaaaa
11460acaacttcag acaatacaaa tcagcgacac tgaatacggg gcaacctcat gtcccccccc
11520cccccccccc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
11580ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
11640gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
11700ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
11760ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
11820gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
11880ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
11940cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
12000ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
12060aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
12120gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
12180gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
12240cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattggtc gacgatcttg
12300ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggattgaagg cgagatccag
12360caactcgcgc cagatcatcc tgtgacggaa ctttggcgcg tgatgactgg ccaggacgtc
12420ggccgaaaga gcgacaagca gatcacgctt ttcgacagcg tcggatttgc gatcgaggat
12480ttttcggcgc tgcgctacgt ccgcgaccgc gttgagggat caagccacag cagcccactc
12540gaccttctag ccgacccaga cgagccaagg gatctttttg gaatgctgct ccgtcgtcag
12600gctttccgac gtttgggtgg ttgaacagaa gtcattatcg tacggaatgc caagcactcc
12660cgaggggaac cctgtggttg gcatgcacat acaaatggac gaacggataa accttttcac
12720gcccttttaa atatccgtta ttctaataaa cgctcttttc tcttaggttt acccgccaat
12780atatcctgtc aaacactgat agtttaaact gaaggcggga aacgacaatc tgatcatgag
12840cggagaatta agggagtcac gttatgaccc ccgccgatga cgcgggacaa gccgttttac
12900gtttggaact gacagaaccg caacgttgaa ggagccactc agcaagctgg tacgattgta
12960atacgactca ctatagggcg aattgagcgc tgtttaaacg ctcttcaact ggaagagcg
1301992991DNAartificial sequencevector 9ctttcctgcg ttatcccctg attctgtgga
taaccgtatt accgcctttg agtgagctga 60taccgctcgc cgcagccgaa cgaccgagcg
cagcgagtca gtgagcgagg aagcggaaga 120gcgcccaata cgcaaaccgc ctctccccgc
gcgttggccg attcattaat gcagctggca 180cgacaggttt cccgactgga aagcgggcag
tgagcgcaac gcaattaata cgcgtaccgc 240tagccaggaa gagtttgtag aaacgcaaaa
aggccatccg tcaggatggc cttctgctta 300gtttgatgcc tggcagttta tggcgggcgt
cctgcccgcc accctccggg ccgttgcttc 360acaacgttca aatccgctcc cggcggattt
gtcctactca ggagagcgtt caccgacaaa 420caacagataa aacgaaaggc ccagtcttcc
gactgagcct ttcgttttat ttgatgcctg 480gcagttccct actctcgcgt taacgctagc
atggatgttt tcccagtcac gacgttgtaa 540aacgacggcc agtcttaagc tcgggccctg
cagctctaga gctcgaattc tacaggtcac 600taataccatc taagtagttg gttcatagtg
actgcatatg ttgtgtttta cagtattatg 660tagtctgttt tttatgcaaa atctaattta
atatattgat atttatatca ttttacgttt 720ctcgttcaac tttcttgtac aaagtggccg
ttaacggatc cagacttgtc catcttctgg 780attggccaac ttaattaatg tatgaaataa
aaggatgcac acatagtgac atgctaatca 840ctataatgtg ggcatcaaag ttgtgtgtta
tgtgtaatta ctagttatct gaataaaaga 900gaaagagatc atccatattt cttatcctaa
atgaatgtca cgtgtcttta taattctttg 960atgaaccaga tgcatttcat taaccaaatc
catatacata taaatattaa tcatatataa 1020ttaatatcaa ttgggttagc aaaacaaatc
tagtctaggt gtgttttgcg aattgcggca 1080agcttgcggc cgccccgggc aactttatta
tacaaagttg gcattataaa aaagcattgc 1140ttatcaattt gttgcaacga acaggtcact
atcagtcaaa ataaaatcat tatttggagc 1200tccatggtag cgttaacgcg gccgcgatat
cccctatagt gagtcgtatt acatggtcat 1260agctgtttcc tggcagctct ggcccgtgtc
tcaaaatctc tgatgttaca ttgcacaaga 1320taaaaatata tcatcatgaa caataaaact
gtctgcttac ataaacagta atacaagggg 1380tgttatgagc catattcaac gggaaacgtc
gaggccgcga ttaaattcca acatggatgc 1440tgatttatat gggtataaat gggctcgcga
taatgtcggg caatcaggtg cgacaatcta 1500tcgcttgtat gggaagcccg atgcgccaga
gttgtttctg aaacatggca aaggtagcgt 1560tgccaatgat gttacagatg agatggtcag
actaaactgg ctgacggaat ttatgcctct 1620tccgaccatc aagcatttta tccgtactcc
tgatgatgca tggttactca ccactgcgat 1680ccccggaaaa acagcattcc aggtattaga
agaatatcct gattcaggtg aaaatattgt 1740tgatgcgctg gcagtgttcc tgcgccggtt
gcattcgatt cctgtttgta attgtccttt 1800taacagcgat cgcgtatttc gtctcgctca
ggcgcaatca cgaatgaata acggtttggt 1860tgatgcgagt gattttgatg acgagcgtaa
tggctggcct gttgaacaag tctggaaaga 1920aatgcataaa cttttgccat tctcaccgga
ttcagtcgtc actcatggtg atttctcact 1980tgataacctt atttttgacg aggggaaatt
aataggttgt attgatgttg gacgagtcgg 2040aatcgcagac cgataccagg atcttgccat
cctatggaac tgcctcggtg agttttctcc 2100ttcattacag aaacggcttt ttcaaaaata
tggtattgat aatcctgata tgaataaatt 2160gcagtttcat ttgatgctcg atgagttttt
ctaatcagaa ttggttaatt ggttgtaaca 2220ctggcagagc attacgctga cttgacggga
cggcgcaagc tcatgaccaa aatcccttaa 2280cgtgagttac gcgtcgttcc actgagcgtc
agaccccgta gaaaagatca aaggatcttc 2340ttgagatcct ttttttctgc gcgtaatctg
ctgcttgcaa acaaaaaaac caccgctacc 2400agcggtggtt tgtttgccgg atcaagagct
accaactctt tttccgaagg taactggctt 2460cagcagagcg cagataccaa atactgtcct
tctagtgtag ccgtagttag gccaccactt 2520caagaactct gtagcaccgc ctacatacct
cgctctgcta atcctgttac cagtggctgc 2580tgccagtggc gataagtcgt gtcttaccgg
gttggactca agacgatagt taccggataa 2640ggcgcagcgg tcgggctgaa cggggggttc
gtgcacacag cccagcttgg agcgaacgac 2700ctacaccgaa ctgagatacc tacagcgtga
gcattgagaa agcgccacgc ttcccgaagg 2760gagaaaggcg gacaggtatc cggtaagcgg
cagggtcgga acaggagagc gcacgaggga 2820gcttccaggg ggaaacgcct ggtatcttta
tagtcctgtc gggtttcgcc acctctgact 2880tgagcgtcga tttttgtgat gctcgtcagg
ggggcggagc ctatggaaaa acgccagcaa 2940cgcggccttt ttacggttcc tggccttttg
ctggcctttt gctcacatgt t 29911013807DNAartificial
sequencevector 10aagctggtac gattgtaata cgactcacta tagggcgaat tgagcgctgt
ttaaacgctc 60ttcaactgga agagcggtta ccagagctgg tcacctttgt ccaccaagat
ggaactgcgg 120ccgctcatta attaagtcag gcgcgcctct agttgaagac acgttcatgt
cttcatcgta 180agaagacact cagtagtctt cggccagaat ggccgtaggt gaattaagag
gagagaggag 240gtaaacattt tcttctattt tttcatattt tcaggataaa ttattgtaaa
agtttacaag 300atttccattt gactagtgta aatgaggaat attctctagt aagatcatta
tttcatctac 360ttcttttatc ttctaccagt agaggaataa acaatattta gctcctttgt
aaatacaaat 420taattttcgt tcttgacatc attcaatttt aattttacgt ataaaataaa
agatcatacc 480tattagaacg attaaggaga aatacaattc gaatgagaag gatgtgccgt
ttgttataat 540aaacagccac acgacgtaaa cgtaaaatga ccacatgatg ggccaataga
catggaccga 600ctactaataa tagtaagtta cattttagga tggaataaat atcataccga
catcagtttg 660aaagaaaagg gaaaaaaaga aaaaataaat aaaagatata ctaccgacat
gagttccaaa 720aagcaaaaaa aaagatcaag ccgacacaga cacgcgtaga gagcaaaatg
actttgacgt 780cacaccacga aaacagacgc ttcatacgtg tccctttatc tctctcagtc
tctctataaa 840cttagtgaga ccctcctctg ttttactcag gatccccggg taccgagctc
gaattcaccg 900gtcgccacca tggcccacag caagcacggc ctgaaggagg agatgaccat
gaagtaccac 960atggagggct gcgtgaacgg ccacaagttc gtgatcaccg gcgagggcat
cggctacccc 1020ttcaagggca agcagaccat caacctgtgc gtgatcgagg gcggccccct
gcccttcagc 1080gaggacatcc tgagcgccgg cttcaagtac ggcgaccgga tcttcaccga
gtacccccag 1140gacatcgtgg actacttcaa gaacagctgc cccgccggct acacctgggg
ccggagcttc 1200ctgttcgagg acggcgccgt gtgcatctgt aacgtggaca tcaccgtgag
cgtgaaggag 1260aactgcatct accacaagag catcttcaac ggcgtgaact tccccgccga
cggccccgtg 1320atgaagaaga tgaccaccaa ctgggaggcc agctgcgaga agatcatgcc
cgtgcctaag 1380cagggcatcc tgaagggcga cgtgagcatg tacctgctgc tgaaggacgg
cggccggtac 1440cggtgccagt tcgacaccgt gtacaaggcc aagagcgtgc ccagcaagat
gcccgagtgg 1500cacttcatcc agcacaagct gctgcgggag gaccggagcg acgccaagaa
ccagaagtgg 1560cagctgaccg agcacgccat cgccttcccc agcgccctgg cctgaagcgg
cccatggata 1620ttcgaacgcg taggtaccac atggttaacc tagacttgtc catcttctgg
attggccaac 1680ttaattaatg tatgaaataa aaggatgcac acatagtgac atgctaatca
ctataatgtg 1740ggcatcaaag ttgtgtgtta tgtgtaatta ctagttatct gaataaaaga
gaaagagatc 1800atccatattt cttatcctaa atgaatgtca cgtgtcttta taattctttg
atgaaccaga 1860tgcatttcat taaccaaatc catatacata taaatattaa tcatatataa
ttaatatcaa 1920ttgggttagc aaaacaaatc tagtctaggt gtgttttgcg aatgcggcca
ttggcctaga 1980aggccattta aatcctgagg atctggtctt cctaaggacc cgggatatcg
ctatcaactt 2040tgtatagaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata
ttaaattaga 2100ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
tcactatggt 2160cgacctgcag actggctgtg tataagggag cctgacattt atattcccca
gaacatcagg 2220ttaatggcgt ttttgatgtc attttcgcgg tggctgagat cagccacttc
ttccccgata 2280acggagaccg gcacactggc catatcggtg gtcatcatgc gccagctttc
atccccgata 2340tgcaccaccg ggtaaagttc acgggggact ttatctgaca gcagacgtgc
actggccagg 2400gggatcacca tccgtcgccc gggcgtgtca ataatatcac tctgtacatc
cacaaacaga 2460cgataacggc tctctctttt ataggtgtaa accttaaact gcatttcacc
agcccctgtt 2520ctcgtcggca aaagagccgt tcatttcaat aaaccgggcg acctcagcca
tcccttcctg 2580attttccgct ttccagcgtt cggcacgcag acgacgggct tcattctgca
tggttgtgct 2640taccgaaccg gagatattga catcatatat gccttgagca actgatagct
gtcgctgtca 2700actgtcactg taatacgctg cttcatagca tacctctttt tgacatactt
cgggtataca 2760tatcagtata tattcttata ccgcaaaaat cagcgcgcaa atacgcatac
tgttatctgg 2820cttttagtaa gccggatcct ctagattacg ccccgcctgc cactcatcgc
agtactgttg 2880taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
gaacctgaat 2940cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
tgaaaacggg 3000ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
tcacccaggg 3060attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
ccaggttttc 3120accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
cgtcgtggta 3180ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
aacaagggtg 3240aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt
ccggatgagc 3300attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
tatttttctt 3360tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
tacattgagc 3420aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
caacggtggt 3480atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
tcgacggatc 3540ctaactcaaa atccacacat tatacgagcc ggaagcataa agtgtaaagc
ctggggtgcc 3600ctaatgcggc cgccatagtg actggatatg ttgtgtttta cagtattatg
tagtctgttt 3660tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
ctcgttcaac 3720tttattatac aaagttgata gatatcggac cgattaaact ttaattcggt
ccgaagcttg 3780catgcctgca gtgcagcgtg acccggtcgt gcccctctct agagataatg
agcattgcat 3840gtctaagtta taaaaaatta ccacatattt tttttgtcac acttgtttga
agtgcagttt 3900atctatcttt atacatatat ttaaacttta ctctacgaat aatataatct
atagtactac 3960aataatatca gtgttttaga gaatcatata aatgaacagt tagacatggt
ctaaaggaca 4020attgagtatt ttgacaacag gactctacag ttttatcttt ttagtgtgca
tgtgttctcc 4080tttttttttg caaatagctt cacctatata atacttcatc cattttatta
gtacatccat 4140ttagggttta gggttaatgg tttttataga ctaatttttt tagtacatct
attttattct 4200attttagcct ctaaattaag aaaactaaaa ctctatttta gtttttttat
ttaataattt 4260agatataaaa tagaataaaa taaagtgact aaaaattaaa caaataccct
ttaagaaatt 4320aaaaaaacta aggaaacatt tttcttgttt cgagtagata atgccagcct
gttaaacgcc 4380gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg
gccaagcgaa 4440gcagacggca cggcatctct gtcgctgcct ctggacccct ctcgagagtt
ccgctccacc 4500gttggacttg ctccgctgtc ggcatccaga aattgcgtgg cggagcggca
gacgtgagcc 4560ggcacggcag gcggcctcct cctcctctca cggcaccggc agctacgggg
gattcctttc 4620ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata aatagacacc
ccctccacac 4680cctctttccc caacctcgtg ttgttcggag cgcacacaca cacaaccaga
tctcccccaa 4740atccacccgt cggcacctcc gcttcaaggt acgccgctcg tcctcccccc
cccccctctc 4800taccttctct agatcggcgt tccggtccat gcatggttag ggcccggtag
ttctacttct 4860gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt gctgctagcg
ttcgtacacg 4920gatgcgacct gtacgtcaga cacgttctga ttgctaactt gccagtgttt
ctctttgggg 4980aatcctggga tggctctagc cgttccgcag acgggatcga tttcatgatt
ttttttgttt 5040cgttgcatag ggtttggttt gcccttttcc tttatttcaa tatatgccgt
gcacttgttt 5100gtcgggtcat cttttcatgc ttttttttgt cttggttgtg atgatgtggt
ctggttgggc 5160ggtcgttcta gatcggagta gaattctgtt tcaaactacc tggtggattt
attaattttg 5220gatctgtatg tgtgtgccat acatattcat agttacgaat tgaagatgat
ggatggaaat 5280atcgatctag gataggtata catgttgatg cgggttttac tgatgcatat
acagagatgc 5340tttttgttcg cttggttgtg atgatgtggt gtggttgggc ggtcgttcat
tcgttctaga 5400tcggagtaga atactgtttc aaactacctg gtgtatttat taattttgga
actgtatgtg 5460tgtgtcatac atcttcatag ttacgagttt aagatggatg gaaatatcga
tctaggatag 5520gtatacatgt tgatgtgggt tttactgatg catatacatg atggcatatg
cagcatctat 5580tcatatgctc taaccttgag tacctatcta ttataataaa caagtatgtt
ttataattat 5640tttgatcttg atatacttgg atgatggcat atgcagcagc tatatgtgga
tttttttagc 5700cctgccttca tacgctattt atttgcttgg tactgtttct tttgtcgatg
ctcaccctgt 5760tgtttggtgt tacttctgca ggtcgacttt aacttagcct aggatccaca
cgacaccatg 5820tcccccgagc gccgccccgt cgagatccgc ccggccaccg ccgccgacat
ggccgccgtg 5880tgcgacatcg tgaaccacta catcgagacc tccaccgtga acttccgcac
cgagccgcag 5940accccgcagg agtggatcga cgacctggag cgcctccagg accgctaccc
gtggctcgtg 6000gccgaggtgg agggcgtggt ggccggcatc gcctacgccg gcccgtggaa
ggcccgcaac 6060gcctacgact ggaccgtgga gtccaccgtg tacgtgtccc accgccacca
gcgcctcggc 6120ctcggctcca ccctctacac ccacctcctc aagagcatgg aggcccaggg
cttcaagtcc 6180gtggtggccg tgatcggcct cccgaacgac ccgtccgtgc gcctccacga
ggccctcggc 6240tacaccgccc gcggcaccct ccgcgccgcc ggctacaagc acggcggctg
gcacgacgtc 6300ggcttctggc agcgcgactt cgagctgccg gccccgccgc gcccggtgcg
cccggtgacg 6360cagatctccg gtggaggcgg cagcggtggc ggaggctccg gaggcggtgg
ctccatggcc 6420tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga
gggctccgtg 6480aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
gggcacccag 6540accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
catcctgtcc 6600ccccagttcc agtacggctc caaggtgtac gtgaagcacc ccgccgacat
ccccgactac 6660aagaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
cgaggacggc 6720ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gctccttcat
ctacaaggtg 6780aagttcatcg gcgtgaactt cccctccgac ggccccgtaa tgcagaagaa
gactatgggc 6840tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg tgctgaaggg
cgagatccac 6900aaggccctga agctgaagga cggcggccac tacctggtgg agttcaagtc
catctacatg 6960gccaagaagc ccgtgcagct gcccggctac tactacgtgg actccaagct
ggacatcacc 7020tcccacaacg aggactacac catcgtggag cagtacgagc gcgccgaggg
ccgccaccac 7080ctgttcctgt agtcaggatc tgagtcgaaa cctagacttg tccatcttct
ggattggcca 7140acttaattaa tgtatgaaat aaaaggatgc acacatagtg acatgctaat
cactataatg 7200tgggcatcaa agttgtgtgt tatgtgtaat tactagttat ctgaataaaa
gagaaagaga 7260tcatccatat ttcttatcct aaatgaatgt cacgtgtctt tataattctt
tgatgaacca 7320gatgcatttc attaaccaaa tccatataca tataaatatt aatcatatat
aattaatatc 7380aattgggtta gcaaaacaaa tctagtctag gtgtgttttg cgaatgcggc
cgccaccgcg 7440gtggagctcg aattcattcc gattaatcgt ggcctcttgc tcttcaggat
gaagagctat 7500gtttaaacgt gcaagcgcta ctagacaatt cagtacatta aaaacgtccg
caatgtgtta 7560ttaagttgtc taagcgtcaa tttgtttaca ccacaatata tcctgccacc
agccagccaa 7620cagctccccg accggcagct cggcacaaaa tcaccactcg atacaggcag
cccatcagtc 7680cgggacggcg tcagcgggag agccgttgta aggcggcaga ctttgctcat
gttaccgatg 7740ctattcggaa gaacggcaac taagctgccg ggtttgaaac acggatgatc
tcgcggaggg 7800tagcatgttg attgtaacga tgacagagcg ttgctgcctg tgatcaaata
tcatctccct 7860cgcagagatc cgaattatca gccttcttat tcatttctcg cttaaccgtg
acaggctgtc 7920gatcttgaga actatgccga cataatagga aatcgctgga taaagccgct
gaggaagctg 7980agtggcgcta tttctttaga agtgaacgtt gacgatcgtc gaccgtaccc
cgatgaatta 8040attcggacgt acgttctgaa cacagctgga tacttacttg ggcgattgtc
atacatgaca 8100tcaacaatgt acccgtttgt gtaaccgtct cttggaggtt cgtatgacac
tagtggttcc 8160cctcagcttg cgactagatg ttgaggccta acattttatt agagagcagg
ctagttgctt 8220agatacatga tcttcaggcc gttatctgtc agggcaagcg aaaattggcc
atttatgacg 8280accaatgccc cgcagaagct cccatctttg ccgccataga cgccgcgccc
cccttttggg 8340gtgtagaaca tccttttgcc agatgtggaa aagaagttcg ttgtcccatt
gttggcaatg 8400acgtagtagc cggcgaaagt gcgagaccca tttgcgctat atataagcct
acgatttccg 8460ttgcgactat tgtcgtaatt ggatgaacta ttatcgtagt tgctctcaga
gttgtcgtaa 8520tttgatggac tattgtcgta attgcttatg gagttgtcgt agttgcttgg
agaaatgtcg 8580tagttggatg gggagtagtc atagggaaga cgagcttcat ccactaaaac
aattggcagg 8640tcagcaagtg cctgccccga tgccatcgca agtacgaggc ttagaaccac
cttcaacaga 8700tcgcgcatag tcttccccag ctctctaacg cttgagttaa gccgcgccgc
gaagcggcgt 8760cggcttgaac gaattgttag acattatttg ccgactacct tggtgatctc
gcctttcacg 8820tagtgaacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc
ttgtccaaga 8880taagcctgcc tagcttcaag tatgacgggc tgatactggg ccggcaggcg
ctccattgcc 8940cagtcggcag cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta
ccaaatgcgg 9000gacaacgtaa gcactacatt tcgctcatcg ccagcccagt cgggcggcga
gttccatagc 9060gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
aaagagttcc 9120tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
caagatagcc 9180agatcaatgt cgatcgtggc tggctcgaag atacctgcaa gaatgtcatt
gcgctgccat 9240tctccaaatt gcagttcgcg cttagctgga taacgccacg gaatgatgtc
gtcgtgcaca 9300acaatggtga cttctacagc gcggagaatc tcgctctctc caggggaagc
cgaagtttcc 9360aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacagt
caccgtaacc 9420agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
gtacaaatgt 9480acggccagca acgtcggttc gagatggcgc tcgatgacgc caactacctc
tgatagttga 9540gtcgatactt cggcgatcac cgcttccctc atgatgttta actcctgaat
taagccgcgc 9600cgcgaagcgg tgtcggcttg aatgaattgt taggcgtcat cctgtgctcc
cgagaaccag 9660taccagtaca tcgctgtttc gttcgagact tgaggtctag ttttatacgt
gaacaggtca 9720atgccgccga gagtaaagcc acattttgcg tacaaattgc aggcaggtac
attgttcgtt 9780tgtgtctcta atcgtatgcc aaggagctgt ctgcttagtg cccacttttt
cgcaaattcg 9840atgagactgt gcgcgactcc tttgcctcgg tgcgtgtgcg acacaacaat
gtgttcgata 9900gaggctagat cgttccatgt tgagttgagt tcaatcttcc cgacaagctc
ttggtcgatg 9960aatgcgccat agcaagcaga gtcttcatca gagtcatcat ccgagatgta
atccttccgg 10020taggggctca cacttctggt agatagttca aagccttggt cggataggtg
cacatcgaac 10080acttcacgaa caatgaaatg gttctcagca tccaatgttt ccgccacctg
ctcagggatc 10140accgaaatct tcatatgacg cctaacgcct ggcacagcgg atcgcaaacc
tggcgcggct 10200tttggcacaa aaggcgtgac aggtttgcga atccgttgct gccacttgtt
aacccttttg 10260ccagatttgg taactataat ttatgttaga ggcgaagtct tgggtaaaaa
ctggcctaaa 10320attgctgggg atttcaggaa agtaaacatc accttccggc tcgatgtcta
ttgtagatat 10380atgtagtgta tctacttgat cgggggatct gctgcctcgc gcgtttcggt
gatgacggtg 10440aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
gcggatgccg 10500ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
ggcgcagcca 10560tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
catcagagca 10620gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
taaggagaaa 10680ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
cggtcgttcg 10740gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
cagaatcagg 10800ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa 10860ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
acaaaaatcg 10920acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc 10980tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
acctgtccgc 11040ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
atctcagttc 11100ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 11160ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
acttatcgcc 11220actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga 11280gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
gtatctgcgc 11340tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
gcaaacaaac 11400caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 11460atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
acgaaaactc 11520acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
tccttttaaa 11580ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
ctgacagtta 11640ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
catccatagt 11700tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
ctggccccag 11760tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
caataaacca 11820gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
ccatccagtc 11880tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
tgcgcaacgt 11940tgttgccatt gctgcagggg gggggggggg gggggacttc cattgttcat
tccacggaca 12000aaaacagaga aaggaaacga cagaggccaa aaagcctcgc tttcagcacc
tgtcgtttcc 12060tttcttttca gagggtattt taaataaaaa cattaagtta tgacgaagaa
gaacggaaac 12120gccttaaacc ggaaaatttt cataaatagc gaaaacccgc gaggtcgccg
ccccgtaacc 12180tgtcggatca ccggaaagga cccgtaaagt gataatgatt atcatctaca
tatcacaacg 12240tgcgtggagg ccatcaaacc acgtcaaata atcaattatg acgcaggtat
cgtattaatt 12300gatctgcatc aacttaacgt aaaaacaact tcagacaata caaatcagcg
acactgaata 12360cggggcaacc tcatgtcccc cccccccccc cccctgcagg catcgtggtg
tcacgctcgt 12420cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
acatgatccc 12480ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
agaagtaagt 12540tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
actgtcatgc 12600catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
tgagaatagt 12660gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg ggataatacc
gcgccacata 12720gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
ctctcaagga 12780tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
tgatcttcag 12840catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
aatgccgcaa 12900aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
tttcaatatt 12960attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
tgtatttaga 13020aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
gacgtctaag 13080aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
ccctttcgtc 13140ttcaagaatt ggtcgacgat cttgctgcgt tcggatattt tcgtggagtt
cccgccacag 13200acccggattg aaggcgagat ccagcaactc gcgccagatc atcctgtgac
ggaactttgg 13260cgcgtgatga ctggccagga cgtcggccga aagagcgaca agcagatcac
gcttttcgac 13320agcgtcggat ttgcgatcga ggatttttcg gcgctgcgct acgtccgcga
ccgcgttgag 13380ggatcaagcc acagcagccc actcgacctt ctagccgacc cagacgagcc
aagggatctt 13440tttggaatgc tgctccgtcg tcaggctttc cgacgtttgg gtggttgaac
agaagtcatt 13500atcgtacgga atgccaagca ctcccgaggg gaaccctgtg gttggcatgc
acatacaaat 13560ggacgaacgg ataaaccttt tcacgccctt ttaaatatcc gttattctaa
taaacgctct 13620tttctcttag gtttacccgc caatatatcc tgtcaaacac tgatagttta
aactgaaggc 13680gggaaacgac aatctgatca tgagcggaga attaagggag tcacgttatg
acccccgccg 13740atgacgcggg acaagccgtt ttacgtttgg aactgacaga accgcaacgt
tgaaggagcc 13800actcagc
13807114678DNAartificial sequencevector 11gaaaggccca gtcttccgac
tgagcctttc gttttatttg atgcctggca gttccctact 60ctcgcgttaa cgctagcatg
gatgttttcc cagtcacgac gttgtaaaac gacggccagt 120cttaagctcg ggcccgcgtt
aacgctacca tggagctcca aataatgatt ttattttgac 180tgatagtgac ctgttcgttg
caacaaattg ataagcaatg cttttttata atgccaactt 240tgtatagaaa agttgggccg
aattcgagct cggtacggcc agaatggccc ggaccgggtt 300accgaattcg agctcggtac
cctgggatcc ctggtaatta ttggctgtag gattctaaac 360agagcctaaa tagctggaat
agctctagcc ctcaatccaa actaatgata tctatactta 420tgcaactcta aatttttatt
ctaaaagtaa tatttcattt ttgtcaacga gattctctac 480tctattccac aatcttttga
agcaatattt accttaaatc tgtactctat accaataatc 540atatattcta ttatttattt
ttatctctct cctaaggagc atccccctat gtctgcatgg 600cccccgcctc gggtcccaat
ctcttgctct gctagtagca cagaagaaaa cactagaaat 660gacttgcttg acttagagta
tcagataaac atcatgttta cttaacttta atttgtatcg 720gtttctacta tttttataat
atttttgtct ctatagatac tacgtgcaac agtataatca 780acctagttta atccagagcg
aaggattttt tactaagtac gtgactccat atgcacagcg 840ttccttttat ggttcctcac
tgggcacagc ataaacgaac cctgtccaat gttttcagcg 900cgaacaaaca gaaattccat
cagcgaacaa acaacataca tgcgagatga aaataaataa 960taaaaaaagc tccgtctcga
taggccggca cgaatcgaga gcctccatag ccagtttttt 1020ccatcggaac ggcggttcgc
gcacctaatt atatgcacca cacgcctata aagccaacca 1080acccgtcgga ggggcgcaag
ccagacagaa gacagcccgt cagcccctct cgtttttcat 1140ccgccttcgc ctccaaccgc
gtgcgctcca cgcctcctcc aggaaagcga ggatctcccc 1200caaatccacc cgtcggcacc
tccgcttcaa ggtacgccgc tcgtcctccc cccccccccc 1260tctctacctt ctctagatcg
gcgttccggt ccatggttag ggcccggtag ttctacttct 1320gttcatgttt gtgttagatc
cgtgtttgtg ttagatccgt gctgctagcg ttcgtacacg 1380gatgcgacct gtacgtcaga
cacgttctga ttgctaactt gccagtgttt ctctttgggg 1440aatcctggga tggctctagc
cgttccgcag acgggatcga tttcatgatt ttttttgttt 1500cgttgcatag ggtttggttt
gcccttttcc tttatttcaa tatatgccgt gcacttgttt 1560gtcgggtcat cttttcatgc
ttttttttgt cttggttgtg atgatgtggt ctggttgggc 1620ggtcgttcta gatcggagta
gaattctgtt tcaaactacc tggtggattt attaattttg 1680gatctgtatg tgtgtgccat
acatattcat agttacgaat tgaagatgat ggatggaaat 1740atcgatctag gataggtata
catgttgatg cgggttttac tgatgcatat acagagatgc 1800tttttgttcg cttggttgtg
atgatgtggt gtggttgggc ggtcgttcat tcgttctaga 1860tcggagtaga atactgtttc
aaactacctg gtgtatttat taattttgga actgtatgtg 1920tgtgtcatac atcttcatag
ttacgagttt aagatggatg gaaatatcga tctaggatag 1980gtatacatgt tgatgtgggt
tttactgatg catatacatg atggcatatg cagcatctat 2040tcatatgctc taaccttgag
tacctatcta ttataataaa caagtatgtt ttataattat 2100tttgatcttg atatacttgg
atgatggcat atgcagcagc tatatgtgga tttttttagc 2160cctgccttca tacgctattt
atttgcttgg tactgtttct tttgtcgatg ctcaccctgt 2220tgtttggtgt tacttctgca
ggtcgactct agaagcttgg tcacccggtc cgggcctaga 2280aggccagctt caagtttgta
caaaaaagtt gaacgagaaa cgtaaaatga tataaatatc 2340aatatattaa attagatttt
gcataaaaaa cagactacat aatactgtaa aacacaacat 2400atgcagtcac tatgaatcaa
ctacttagat ggtattagtg acctgtagaa ttcgagctct 2460agagctgcag ggcggccgcg
atatccccta tagtgagtcg tattacatgg tcatagctgt 2520ttcctggcag ctctggcccg
tgtctcaaaa tctctgatgt tacattgcac aagataaaaa 2580tatatcatca tgaacaataa
aactgtctgc ttacataaac agtaatacaa ggggtgttat 2640gagccatatt caacgggaaa
cgtcgaggcc gcgattaaat tccaacatgg atgctgattt 2700atatgggtat aaatgggctc
gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt 2760gtatgggaag cccgatgcgc
cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa 2820tgatgttaca gatgagatgg
tcagactaaa ctggctgacg gaatttatgc ctcttccgac 2880catcaagcat tttatccgta
ctcctgatga tgcatggtta ctcaccactg cgatccccgg 2940aaaaacagca ttccaggtat
tagaagaata tcctgattca ggtgaaaata ttgttgatgc 3000gctggcagtg ttcctgcgcc
ggttgcattc gattcctgtt tgtaattgtc cttttaacag 3060cgatcgcgta tttcgtctcg
ctcaggcgca atcacgaatg aataacggtt tggttgatgc 3120gagtgatttt gatgacgagc
gtaatggctg gcctgttgaa caagtctgga aagaaatgca 3180taaacttttg ccattctcac
cggattcagt cgtcactcat ggtgatttct cacttgataa 3240ccttattttt gacgagggga
aattaatagg ttgtattgat gttggacgag tcggaatcgc 3300agaccgatac caggatcttg
ccatcctatg gaactgcctc ggtgagtttt ctccttcatt 3360acagaaacgg ctttttcaaa
aatatggtat tgataatcct gatatgaata aattgcagtt 3420tcatttgatg ctcgatgagt
ttttctaatc agaattggtt aattggttgt aacactggca 3480gagcattacg ctgacttgac
gggacggcgc aagctcatga ccaaaatccc ttaacgtgag 3540ttacgcgtcg ttccactgag
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 3600tccttttttt ctgcgcgtaa
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 3660ggtttgtttg ccggatcaag
agctaccaac tctttttccg aaggtaactg gcttcagcag 3720agcgcagata ccaaatactg
tccttctagt gtagccgtag ttaggccacc acttcaagaa 3780ctctgtagca ccgcctacat
acctcgctct gctaatcctg ttaccagtgg ctgctgccag 3840tggcgataag tcgtgtctta
ccgggttgga ctcaagacga tagttaccgg ataaggcgca 3900gcggtcgggc tgaacggggg
gttcgtgcac acagcccagc ttggagcgaa cgacctacac 3960cgaactgaga tacctacagc
gtgagcattg agaaagcgcc acgcttcccg aagggagaaa 4020ggcggacagg tatccggtaa
gcggcagggt cggaacagga gagcgcacga gggagcttcc 4080agggggaaac gcctggtatc
tttatagtcc tgtcgggttt cgccacctct gacttgagcg 4140tcgatttttg tgatgctcgt
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 4200ctttttacgg ttcctggcct
tttgctggcc ttttgctcac atgttctttc ctgcgttatc 4260ccctgattct gtggataacc
gtattaccgc ctttgagtga gctgataccg ctcgccgcag 4320ccgaacgacc gagcgcagcg
agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 4380accgcctctc cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga 4440ctggaaagcg ggcagtgagc
gcaacgcaat taatacgcgt accgctagcc aggaagagtt 4500tgtagaaacg caaaaaggcc
atccgtcagg atggccttct gcttagtttg atgcctggca 4560gtttatggcg ggcgtcctgc
ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc 4620gctcccggcg gatttgtcct
actcaggaga gcgttcaccg acaaacaaca gataaaac 4678123505DNAartificial
sequencevector 12gatccccggg taccgagctc gaattcggcc caagtttgta caaaaaagtt
gaacgagaaa 60cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
cagactacat 120aatactgtaa aacacaacat atgcagtcac tatgaatcaa ctacttagat
ggtattagtg 180acctgtagaa ttcgagctct agagctgcag ggcggccgcg atatccccta
tagtgagtcg 240tattacatgg tcatagctgt ttcctggcag ctctggcccg tgtctcaaaa
tctctgatgt 300tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc
ttacataaac 360agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc
gcgattaaat 420tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
cgggcaatca 480ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt
tctgaaacat 540ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
ctggctgacg 600gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
tgcatggtta 660ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata
tcctgattca 720ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
gattcctgtt 780tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
atcacgaatg 840aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
gcctgttgaa 900caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt
cgtcactcat 960ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
ttgtattgat 1020gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
gaactgcctc 1080ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
tgataatcct 1140gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
agaattggtt 1200aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc
aagctcatga 1260ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc
cgtagaaaag 1320atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
gcaaacaaaa 1380aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
tctttttccg 1440aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
gtagccgtag 1500ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
gctaatcctg 1560ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
ctcaagacga 1620tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
acagcccagc 1680ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagcattg
agaaagcgcc 1740acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
cggaacagga 1800gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
tgtcgggttt 1860cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
gagcctatgg 1920aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
ttttgctcac 1980atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
ctttgagtga 2040gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
cgaggaagcg 2100gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
ttaatgcagc 2160tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
taatacgcgt 2220accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg
atggccttct 2280gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct
ccgggccgtt 2340gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga
gcgttcaccg 2400acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt
tttatttgat 2460gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca
gtcacgacgt 2520tgtaaaacga cggccagtct taagctcggg cccgcgttaa cgctaccatg
gagctccaaa 2580taatgatttt attttgactg atagtgacct gttcgttgca acaaattgat
aagcaatgct 2640tttttataat gccaactttg tatagaaaag ttgaagctta aatccttaca
gaattgctgt 2700agtttcatag tgctagatgt ggacagcaaa gcgccgctgt atgcttctgc
ttttcttttt 2760tggtgtgtgt agccacatcc tttgttcctg cccggcgcca tcccacttgg
ttgttttttt 2820ttatgattga aagccttcat gcttcctcgg tcaatcaccg gtgcgcactg
ggagcatcgc 2880cggaaaaaaa attcttcggc taagagtaac ttctttctcc ttttcttctc
tgatctcgcg 2940agcagtgctg ataacgtgtt gtaatctact tagcggtaac gagattgaga
gagacaaaat 3000gacagaacta ttgtctttat tgcagagtgt catgtattta tacaggggat
acaaagtctc 3060ccaaggggtg tgtcccttgg gagtaactgc cagttgatca caggacaata
ttttgtaaca 3120aaacgtacac atcgtcaaaa tagcgaggca tgaaactggc cttggccatg
gacgcgtgaa 3180gcgcgccatg cgttggatat gtggtcaata agtatataca atacaatgtt
taacagagct 3240gatagtactg ctttggcaca tttttgtcca cgcttcatga gagataaaac
acctgcacgt 3300aaattcacat gctgcactga aggcccgatc actgaggagc gaactgccgt
aactcccttc 3360tatatatacc cccagtccct gtttcagttt tcgtcaagct agcagcacca
agttgtcgat 3420cacttgcctg ctcttgagct cgattaagct atcatcagct acagcatccg
atcccaaact 3480gcaactgtag cagcgacaac tgccg
35051349765DNAartificial sequencevector 13gggggggggg
ggggggggtt ccattgttca ttccacggac aaaaacagag aaaggaaacg 60acagaggcca
aaaagctcgc tttcagcacc tgtcgtttcc tttcttttca gagggtattt 120taaataaaaa
cattaagtta tgacgaagaa gaacggaaac gccttaaacc ggaaaatttt 180cataaatagc
gaaaacccgc gaggtcgccg ccccgtaacc tgtcggatca ccggaaagga 240cccgtaaagt
gataatgatt atcatctaca tatcacaacg tgcgtggagg ccatcaaacc 300acgtcaaata
atcaattatg acgcaggtat cgtattaatt gatctgcatc aacttaacgt 360aaaaacaact
tcagacaata caaatcagcg acactgaata cggggcaacc tcatgtcccc 420cccccccccc
cccctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 480agctccggtt
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 540gttagctcct
tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 600atggttatgg
cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 660gtgactggtg
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 720tcttgcccgg
cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 780atcattggaa
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 840agttcgatgt
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 900gtttctgggt
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 960cggaaatgtt
gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 1020tattgtctca
tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 1080ccgcgcacat
ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 1140ttaacctata
aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt cggagctttt 1200gccattctca
ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt 1260tgacgagggg
aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata 1320ccaggatctt
gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg 1380gctttttcaa
aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat 1440gctcgatgag
tttttctaat cagaattggt taattggttg taacactggc agagcattac 1500gctgacttga
cgggacggcg gctttgttga ataaatcgaa cttttgctga gttgaaggat 1560cagatcacgc
atcttcccga caacgcagac cgttccgtgg caaagcaaaa gttcaaaatc 1620accaactggt
ccacctacaa caaagctctc atcaaccgtg gctccctcac tttctggctg 1680gatgatgggg
cgattcaggc ctggtatgag tcagcaacac cttcttcacg aggcagacct 1740cagcgccaga
aggccgccag agaggccgag cgcggccgtg aggcttggac gctagggcag 1800ggcatgaaaa
agcccgtagc gggctgctac gggcgtctga cgcggtggaa agggggaggg 1860gatgttgtct
acatggctct gctgtagtga gtgggttgcg ctccggcagc ggtcctgatc 1920aatcgtcacc
ctttctcggt ccttcaacgt tcctgacaac gagcctcctt ttcgccaatc 1980catcgacaat
caccgcgagt ccctgctcga acgctgcgtc cggaccggct tcgtcgaagg 2040cgtctatcgc
ggcccgcaac agcggcgaga gcggagcctg ttcaacggtg ccgccgcgct 2100cgccggcatc
gctgtcgccg gcctgctcct caagcacggc cccaacagtg aagtagctga 2160ttgtcatcag
cgcattgacg gcgtccccgg ccgaaaaacc cgcctcgcag aggaagcgaa 2220gctgcgcgtc
ggccgtttcc atctgcggtg cgcccggtcg cgtgccggca tggatgcgcg 2280cgccatcgcg
gtaggcgagc agcgcctgcc tgaagctgcg ggcattcccg atcagaaatg 2340agcgccagtc
gtcgtcggct ctcggcaccg aatgcgtatg attctccgcc agcatggctt 2400cggccagtgc
gtcgagcagc gcccgcttgt tcctgaagtg ccagtaaagc gccggctgct 2460gaacccccaa
ccgttccgcc agtttgcgtg tcgtcagacc gtctacgccg acctcgttca 2520acaggtccag
ggcggcacgg atcactgtat tcggctgcaa ctttgtcatg cttgacactt 2580tatcactgat
aaacataata tgtccaccaa cttatcagtg ataaagaatc cgcgcgttca 2640atcggaccag
cggaggctgg tccggaggcc agacgtgaaa cccaacatac ccctgatcgt 2700aattctgagc
actgtcgcgc tcgacgctgt cggcatcggc ctgattatgc cggtgctgcc 2760gggcctcctg
cgcgatctgg ttcactcgaa cgacgtcacc gcccactatg gcattctgct 2820ggcgctgtat
gcgttggtgc aatttgcctg cgcacctgtg ctgggcgcgc tgtcggatcg 2880tttcgggcgg
cggccaatct tgctcgtctc gctggccggc gccactgtcg actacgccat 2940catggcgaca
gcgcctttcc tttgggttct ctatatcggg cggatcgtgg ccggcatcac 3000cggggcgact
ggggcggtag ccggcgctta tattgccgat atcactgatg gcgatgagcg 3060cgcgcggcac
ttcggcttca tgagcgcctg tttcgggttc gggatggtcg cgggacctgt 3120gctcggtggg
ctgatgggcg gtttctcccc ccacgctccg ttcttcgccg cggcagcctt 3180gaacggcctc
aatttcctga cgggctgttt ccttttgccg gagtcgcaca aaggcgaacg 3240ccggccgtta
cgccgggagg ctctcaaccc gctcgcttcg ttccggtggg cccggggcat 3300gaccgtcgtc
gccgccctga tggcggtctt cttcatcatg caacttgtcg gacaggtgcc 3360ggccgcgctt
tgggtcattt tcggcgagga tcgctttcac tgggacgcga ccacgatcgg 3420catttcgctt
gccgcatttg gcattctgca ttcactcgcc caggcaatga tcaccggccc 3480tgtagccgcc
cggctcggcg aaaggcgggc actcatgctc ggaatgattg ccgacggcac 3540aggctacatc
ctgcttgcct tcgcgacacg gggatggatg gcgttcccga tcatggtcct 3600gcttgcttcg
ggtggcatcg gaatgccggc gctgcaagca atgttgtcca ggcaggtgga 3660tgaggaacgt
caggggcagc tgcaaggctc actggcggcg ctcaccagcc tgacctcgat 3720cgtcggaccc
ctcctcttca cggcgatcta tgcggcttct ataacaacgt ggaacgggtg 3780ggcatggatt
gcaggcgctg ccctctactt gctctgcctg ccggcgctgc gtcgcgggct 3840ttggagcggc
gcagggcaac gagccgatcg ctgatcgtgg aaacgatagg cctatgccat 3900gcgggtcaag
gcgacttccg gcaagctata cgcgccctag gagtgcggtt ggaacgttgg 3960cccagccaga
tactcccgat cacgagcagg acgccgatga tttgaagcgc actcagcgtc 4020tgatccaaga
acaaccatcc tagcaacacg gcggtccccg ggctgagaaa gcccagtaag 4080gaaacaactg
taggttcgag tcgcgagatc ccccggaacc aaaggaagta ggttaaaccc 4140gctccgatca
ggccgagcca cgccaggccg agaacattgg ttcctgtagg catcgggatt 4200ggcggatcaa
acactaaagc tactggaacg agcagaagtc ctccggccgc cagttgccag 4260gcggtaaagg
tgagcagagg cacgggaggt tgccacttgc gggtcagcac ggttccgaac 4320gccatggaaa
ccgcccccgc caggcccgct gcgacgccga caggatctag cgctgcgttt 4380ggtgtcaaca
ccaacagcgc cacgcccgca gttccgcaaa tagcccccag gaccgccatc 4440aatcgtatcg
ggctacctag cagagcggca gagatgaaca cgaccatcag cggctgcaca 4500gcgcctaccg
tcgccgcgac cccgcccggc aggcggtaga ccgaaataaa caacaagctc 4560cagaatagcg
aaatattaag tgcgccgagg atgaagatgc gcatccacca gattcccgtt 4620ggaatctgtc
ggacgatcat cacgagcaat aaacccgccg gcaacgcccg cagcagcata 4680ccggcgaccc
ctcggcctcg ctgttcgggc tccacgaaaa cgccggacag atgcgccttg 4740tgagcgtcct
tggggccgtc ctcctgtttg aagaccgaca gcccaatgat ctcgccgtcg 4800atgtaggcgc
cgaatgccac ggcatctcgc aaccgttcag cgaacgcctc catgggcttt 4860ttctcctcgt
gctcgtaaac ggacccgaac atctctggag ctttcttcag ggccgacaat 4920cggatctcgc
ggaaatcctg cacgtcggcc gctccaagcc gtcgaatctg agccttaatc 4980acaattgtca
attttaatcc tctgtttatc ggcagttcgt agagcgcgcc gtgcgtcccg 5040agcgatactg
agcgaagcaa gtgcgtcgag cagtgcccgc ttgttcctga aatgccagta 5100aagcgctggc
tgctgaaccc ccagccggaa ctgaccccac aaggccctag cgtttgcaat 5160gcaccaggtc
atcattgacc caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc 5220aggcttcgcc
gacctgctcg cgccacttct tcacgcgggt ggaatccgat ccgcacatga 5280ggcggaaggt
ttccagcttg agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca 5340tccgtcgggc
cgtcggcgac agcttgcggt acttctccca tatgaatttc gtgtagtggt 5400cgccagcaaa
cagcacgacg atttcctcgt cgatcaggac ctggcaacgg gacgttttct 5460tgccacggtc
caggacgcgg aagcggtgca gcagcgacac cgattccagg tgcccaacgc 5520ggtcggacgt
gaagcccatc gccgtcgcct gtaggcgcga caggcattcc tcggccttcg 5580tgtaataccg
gccattgatc gaccagccca ggtcctggca aagctcgtag aacgtgaagg 5640tgatcggctc
gccgataggg gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt 5700cgtcatcgtc
ggcccgcagc tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt 5760ggaaaatgac
cttgttttgc agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca 5820gggcagagcg
ggccgtgtcg tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat 5880cgaacaagga
aagctgcatt tccttgatct gctgcttcgt gtgtttcagc aacgcggcct 5940gcttggcctc
gctgacctgt tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg 6000tcatagttcc
tcgcgtgtcg atggtcatcg acttcgccaa acctgccgcc tcctgttcga 6060gacgacgcga
acgctccacg gcggccgatg gcgcgggcag ggcaggggga gccagttgca 6120cgctgtcgcg
ctcgatcttg gccgtagctt gctggaccat cgagccgacg gactggaagg 6180tttcgcgggg
cgcacgcatg acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa 6240accccgcgtc
gatcagttct tgcctgtatg ccttccggtc aaacgtccga ttcattcacc 6300ctccttgcgg
gattgccccg actcacgccg gggcaatgtg cccttattcc tgatttgacc 6360cgcctggtgc
cttggtgtcc agataatcca ccttatcggc aatgaagtcg gtcccgtaga 6420ccgtctggcc
gtccttctcg tacttggtat tccgaatctt gccctgcacg aataccagcg 6480accccttgcc
caaatacttg ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga 6540agaagtcggt
gcgctcctgc ttgtcgccgg catcgttgcg ccactcttca ttaaccgcta 6600tatcgaaaat
tgcttgcggc ttgttagaat tgccatgacg tacctcggtg tcacgggtaa 6660gattaccgat
aaactggaac tgattatggc tcatatcgaa agtctccttg agaaaggaga 6720ctctagttta
gctaaacatt ggttccgctg tcaagaactt tagcggctaa aattttgcgg 6780gccgcgacca
aaggtgcgag gggcggcttc cgctgtgtac aaccagatat ttttcaccaa 6840catccttcgt
ctgctcgatg agcggggcat gacgaaacat gagctgtcgg agagggcagg 6900ggtttcaatt
tcgtttttat cagacttaac caacggtaag gccaacccct cgttgaaggt 6960gatggaggcc
attgccgacg ccctggaaac tcccctacct cttctcctgg agtccaccga 7020ccttgaccgc
gaggcactcg cggagattgc gggtcatcct ttcaagagca gcgtgccgcc 7080cggatacgaa
cgcatcagtg tggttttgcc gtcacataag gcgtttatcg taaagaaatg 7140gggcgacgac
acccgaaaaa agctgcgtgg aaggctctga cgccaagggt tagggcttgc 7200acttccttct
ttagccgcta aaacggcccc ttctctgcgg gccgtcggct cgcgcatcat 7260atcgacatcc
tcaacggaag ccgtgccgcg aatggcatcg ggcgggtgcg ctttgacagt 7320tgttttctat
cagaacccct acgtcgtgcg gttcgattag ctgtttgtct tgcaggctaa 7380acactttcgg
tatatcgttt gcctgtgcga taatgttgct aatgatttgt tgcgtagggg 7440ttactgaaaa
gtgagcggga aagaagagtt tcagaccatc aaggagcggg ccaagcgcaa 7500gctggaacgc
gacatgggtg cggacctgtt ggccgcgctc aacgacccga aaaccgttga 7560agtcatgctc
aacgcggacg gcaaggtgtg gcacgaacgc cttggcgagc cgatgcggta 7620catctgcgac
atgcggccca gccagtcgca ggcgattata gaaacggtgg ccggattcca 7680cggcaaagag
gtcacgcggc attcgcccat cctggaaggc gagttcccct tggatggcag 7740ccgctttgcc
ggccaattgc cgccggtcgt ggccgcgcca acctttgcga tccgcaagcg 7800cgcggtcgcc
atcttcacgc tggaacagta cgtcgaggcg ggcatcatga cccgcgagca 7860atacgaggtc
attaaaagcg ccgtcgcggc gcatcgaaac atcctcgtca ttggcggtac 7920tggctcgggc
aagaccacgc tcgtcaacgc gatcatcaat gaaatggtcg ccttcaaccc 7980gtctgagcgc
gtcgtcatca tcgaggacac cggcgaaatc cagtgcgccg cagagaacgc 8040cgtccaatac
cacaccagca tcgacgtctc gatgacgctg ctgctcaaga caacgctgcg 8100tatgcgcccc
gaccgcatcc tggtcggtga ggtacgtggc cccgaagccc ttgatctgtt 8160gatggcctgg
aacaccgggc atgaaggagg tgccgccacc ctgcacgcaa acaaccccaa 8220agcgggcctg
agccggctcg ccatgcttat cagcatgcac ccggattcac cgaaacccat 8280tgagccgctg
attggcgagg cggttcatgt ggtcgtccat atcgccagga cccctagcgg 8340ccgtcgagtg
caagaaattc tcgaagttct tggttacgag aacggccagt acatcaccaa 8400aaccctgtaa
ggagtatttc caatgacaac ggctgttccg ttccgtctga ccatgaatcg 8460cggcattttg
ttctaccttg ccgtgttctt cgttctcgct ctcgcgttat ccgcgcatcc 8520ggcgatggcc
tcggaaggca ccggcggcag cttgccatat gagagctggc tgacgaacct 8580gcgcaactcc
gtaaccggcc cggtggcctt cgcgctgtcc atcatcggca tcgtcgtcgc 8640cggcggcgtg
ctgatcttcg gcggcgaact caacgccttc ttccgaaccc tgatcttcct 8700ggttctggtg
atggcgctgc tggtcggcgc gcagaacgtg atgagcacct tcttcggtcg 8760tggtgccgaa
atcgcggccc tcggcaacgg ggcgctgcac caggtgcaag tcgcggcggc 8820ggatgccgtg
cgtgcggtag cggctggacg gctcgcctaa tcatggctct gcgcacgatc 8880cccatccgtc
gcgcaggcaa ccgagaaaac ctgttcatgg gtggtgatcg tgaactggtg 8940atgttctcgg
gcctgatggc gtttgcgctg attttcagcg cccaagagct gcgggccacc 9000gtggtcggtc
tgatcctgtg gttcggggcg ctctatgcgt tccgaatcat ggcgaaggcc 9060gatccgaaga
tgcggttcgt gtacctgcgt caccgccggt acaagccgta ttacccggcc 9120cgctcgaccc
cgttccgcga gaacaccaat agccaaggga agcaataccg atgatccaag 9180caattgcgat
tgcaatcgcg ggcctcggcg cgcttctgtt gttcatcctc tttgcccgca 9240tccgcgcggt
cgatgccgaa ctgaaactga aaaagcatcg ttccaaggac gccggcctgg 9300ccgatctgct
caactacgcc gctgtcgtcg atgacggcgt aatcgtgggc aagaacggca 9360gctttatggc
tgcctggctg tacaagggcg atgacaacgc aagcagcacc gaccagcagc 9420gcgaagtagt
gtccgcccgc atcaaccagg ccctcgcggg cctgggaagt gggtggatga 9480tccatgtgga
cgccgtgcgg cgtcctgctc cgaactacgc ggagcggggc ctgtcggcgt 9540tccctgaccg
tctgacggca gcgattgaag aagagcgctc ggtcttgcct tgctcgtcgg 9600tgatgtactt
caccagctcc gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct 9660tggcaatcac
gcgcaccccc cggccgtttt agcggctaaa aaagtcatgg ctctgccctc 9720gggcggacca
cgcccatcat gaccttgcca agctcgtcct gcttctcttc gatcttcgcc 9780agcagggcga
ggatcgtggc atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag 9840agtttcagca
ggccgcccag gcggcccagg tcgccattga tgcgggccag ctcgcggacg 9900tgctcatagt
ccacgacgcc cgtgattttg tagccctggc cgacggccag caggtaggcc 9960gacaggctca
tgccggccgc cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg 10020cagtacacct
tgataggtgg gctgcccttc ctggttggct tggtttcatc agccatccgc 10080ttgccctcat
ctgttacgcc ggcggtagcc ggccagcctc gcagagcagg attcccgttg 10140agcaccgcca
ggtgcgaata agggacagtg aagaaggaac acccgctcgc gggtgggcct 10200acttcaccta
tcctgcccgg ctgacgccgt tggatacacc aaggaaagtc tacacgaacc 10260ctttggcaaa
atcctgtata tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata 10320atgaccccga
agcagggtta tgcagcggaa aagcgctgct tccctgctgt tttgtggaat 10380atctaccgac
tggaaacagg caaatgcagg aaattactga actgagggga caggcgagag 10440acgatgccaa
agagctacac cgacgagctg gccgagtggg ttgaatcccg cgcggccaag 10500aagcgccggc
gtgatgaggc tgcggttgcg ttcctggcgg tgagggcgga tgtcgaggcg 10560gcgttagcgt
ccggctatgc gctcgtcacc atttgggagc acatgcggga aacggggaag 10620gtcaagttct
cctacgagac gttccgctcg cacgccaggc ggcacatcaa ggccaagccc 10680gccgatgtgc
ccgcaccgca ggccaaggct gcggaacccg cgccggcacc caagacgccg 10740gagccacggc
ggccgaagca ggggggcaag gctgaaaagc cggcccccgc tgcggccccg 10800accggcttca
ccttcaaccc aacaccggac aaaaaggatc tactgtaatg gcgaaaattc 10860acatggtttt
gcagggcaag ggcggggtcg gcaagtcggc catcgccgcg atcattgcgc 10920agtacaagat
ggacaagggg cagacaccct tgtgcatcga caccgacccg gtgaacgcga 10980cgttcgaggg
ctacaaggcc ctgaacgtcc gccggctgaa catcatggcc ggcgacgaaa 11040ttaactcgcg
caacttcgac accctggtcg agctgattgc gccgaccaag gatgacgtgg 11100tgatcgacaa
cggtgccagc tcgttcgtgc ctctgtcgca ttacctcatc agcaaccagg 11160tgccggctct
gctgcaagaa atggggcatg agctggtcat ccataccgtc gtcaccggcg 11220gccaggctct
cctggacacg gtgagcggct tcgcccagct cgccagccag ttcccggccg 11280aagcgctttt
cgtggtctgg ctgaacccgt attgggggcc tatcgagcat gagggcaaga 11340gctttgagca
gatgaaggcg tacacggcca acaaggcccg cgtgtcgtcc atcatccaga 11400ttccggccct
caaggaagaa acctacggcc gcgatttcag cgacatgctg caagagcggc 11460tgacgttcga
ccaggcgctg gccgatgaat cgctcacgat catgacgcgg caacgcctca 11520agatcgtgcg
gcgcggcctg tttgaacagc tcgacgcggc ggccgtgcta tgagcgacca 11580gattgaagag
ctgatccggg agattgcggc caagcacggc atcgccgtcg gccgcgacga 11640cccggtgctg
atcctgcata ccatcaacgc ccggctcatg gccgacagtg cggccaagca 11700agaggaaatc
cttgccgcgt tcaaggaaga gctggaaggg atcgcccatc gttggggcga 11760ggacgccaag
gccaaagcgg agcggatgct gaacgcggcc ctggcggcca gcaaggacgc 11820aatggcgaag
gtaatgaagg acagcgccgc gcaggcggcc gaagcgatcc gcagggaaat 11880cgacgacggc
cttggccgcc agctcgcggc caaggtcgcg gacgcgcggc gcgtggcgat 11940gatgaacatg
atcgccggcg gcatggtgtt gttcgcggcc gccctggtgg tgtgggcctc 12000gttatgaatc
gcagaggcgc agatgaaaaa gcccggcgtt gccgggcttt gtttttgcgt 12060tagctgggct
tgtttgacag gcccaagctc tgactgcgcc cgcgctcgcg ctcctgggcc 12120tgtttcttct
cctgctcctg cttgcgcatc agggcctggt gccgtcgggc tgcttcacgc 12180atcgaatccc
agtcgccggc cagctcggga tgctccgcgc gcatcttgcg cgtcgccagt 12240tcctcgatct
tgggcgcgtg aatgcccatg ccttccttga tttcgcgcac catgtccagc 12300cgcgtgtgca
gggtctgcaa gcgggcttgc tgttgggcct gctgctgctg ccaggcggcc 12360tttgtacgcg
gcagggacag caagccgggg gcattggact gtagctgctg caaacgcgcc 12420tgctgacggt
ctacgagctg ttctaggcgg tcctcgatgc gctccacctg gtcatgcttt 12480gcctgcacgt
agagcgcaag ggtctgctgg taggtctgct cgatgggcgc ggattctaag 12540agggcctgct
gttccgtctc ggcctcctgg gccgcctgta gcaaatcctc gccgctgttg 12600ccgctggact
gctttactgc cggggactgc tgttgccctg ctcgcgccgt cgtcgcagtt 12660cggcttgccc
ccactcgatt gactgcttca tttcgagccg cagcgatgcg atctcggatt 12720gcgtcaacgg
acggggcagc gcggaggtgt ccggcttctc cttgggtgag tcggtcgatg 12780ccatagccaa
aggtttcctt ccaaaatgcg tccattgctg gaccgtgttt ctcattgatg 12840cccgcaagca
tcttcggctt gaccgccagg tcaagcgcgc cttcatgggc ggtcatgacg 12900gacgccgcca
tgaccttgcc gccgttgttc tcgatgtagc cgcgtaatga ggcaatggtg 12960ccgcccatcg
tcagcgtgtc atcgacaacg atgtacttct ggccggggat cacctccccc 13020tcgaaagtcg
ggttgaacgc caggcgatga tctgaaccgg ctccggttcg ggcgaccttc 13080tcccgctgca
caatgtccgt ttcgacctca aggccaaggc ggtcggccag aacgaccgcc 13140atcatggccg
gaatcttgtt gttccccgcc gcctcgacgg cgaggactgg aacgatgcgg 13200ggcttgtcgt
cgccgatcag cgtcttgagc tgggcaacag tgtcgtccga aatcaggcgc 13260tcgaccaaat
taagcgccgc ttccgcgtcg ccctgcttcg cagcctggta ttcaggctcg 13320ttggtcaaag
aaccaaggtc gccgttgcga accaccttcg ggaagtctcc ccacggtgcg 13380cgctcggctc
tgctgtagct gctcaagacg cctccctttt tagccgctaa aactctaacg 13440agtgcgcccg
cgactcaact tgacgctttc ggcacttacc tgtgccttgc cacttgcgtc 13500ataggtgatg
cttttcgcac tcccgatttc aggtacttta tcgaaatctg accgggcgtg 13560cattacaaag
ttcttcccca cctgttggta aatgctgccg ctatctgcgt ggacgatgct 13620gccgtcgtgg
cgctgcgact tatcggcctt ttgggccata tagatgttgt aaatgccagg 13680tttcagggcc
ccggctttat ctaccttctg gttcgtccat gcgccttggt tctcggtctg 13740gacaattctt
tgcccattca tgaccaggag gcggtgtttc attgggtgac tcctgacggt 13800tgcctctggt
gttaaacgtg tcctggtcgc ttgccggcta aaaaaaagcc gacctcggca 13860gttcgaggcc
ggctttccct agagccgggc gcgtcaaggt tgttccatct attttagtga 13920actgcgttcg
atttatcagt tactttcctc ccgctttgtg tttcctccca ctcgtttccg 13980cgtctagccg
acccctcaac atagcggcct cttcttgggc tgcctttgcc tcttgccgcg 14040cttcgtcacg
ctcggcttgc accgtcgtaa agcgctcggc ctgcctggcc gcctcttgcg 14100ccgccaactt
cctttgctcc tggtgggcct cggcgtcggc ctgcgccttc gctttcaccg 14160ctgccaactc
cgtgcgcaaa ctctccgctt cgcgcctggt ggcgtcgcgc tcgccgcgaa 14220gcgcctgcat
ttcctggttg gccgcgtcca gggtcttgcg gctctcttct ttgaatgcgc 14280gggcgtcctg
gtgagcgtag tccagctcgg cgcgcagctc ctgcgctcga cgctccacct 14340cgtcggcccg
ctgcgtcgcc agcgcggccc gctgctcggc tcctgccagg gcggtgcgtg 14400cttcggccag
ggcttgccgc tggcgtgcgg ccagctcggc cgcctcggcg gcctgctgct 14460ctagcaatgt
aacgcgcgcc tgggcttctt ccagctcgcg ggcctgcgcc tcgaaggcgt 14520cggccagctc
cccgcgcacg gcttccaact cgttgcgctc acgatcccag ccggcttgcg 14580ctgcctgcaa
cgattcattg gcaagggcct gggcggcttg ccagagggcg gccacggcct 14640ggttgccggc
ctgctgcacc gcgtccggca cctggactgc cagcggggcg gcctgcgccg 14700tgcgctggcg
tcgccattcg cgcatgccgg cgctggcgtc gttcatgttg acgcgggcgg 14760ccttacgcac
tgcatccacg gtcgggaagt tctcccggtc gccttgctcg aacagctcgt 14820ccgcagccgc
aaaaatgcgg tcgcgcgtct ctttgttcag ttccatgttg gctccggtaa 14880ttggtaagaa
taataatact cttacctacc ttatcagcgc aagagtttag ctgaacagtt 14940ctcgacttaa
cggcaggttt tttagcggct gaagggcagg caaaaaaagc cccgcacggt 15000cggcgggggc
aaagggtcag cgggaagggg attagcgggc gtcgggcttc ttcatgcgtc 15060ggggccgcgc
ttcttgggat ggagcacgac gaagcgcgca cgcgcatcgt cctcggccct 15120atcggcccgc
gtcgcggtca ggaacttgtc gcgcgctagg tcctccctgg tgggcaccag 15180gggcatgaac
tcggcctgct cgatgtaggt ccactccatg accgcatcgc agtcgaggcc 15240gcgttccttc
accgtctctt gcaggtcgcg gtacgcccgc tcgttgagcg gctggtaacg 15300ggccaattgg
tcgtaaatgg ctgtcggcca tgagcggcct ttcctgttga gccagcagcc 15360gacgacgaag
ccggcaatgc aggcccctgg cacaaccagg ccgacgccgg gggcagggga 15420tggcagcagc
tcgccaacca ggaaccccgc cgcgatgatg ccgatgccgg tcaaccagcc 15480cttgaaacta
tccggccccg aaacacccct gcgcattgcc tggatgctgc gccggatagc 15540ttgcaacatc
aggagccgtt tcttttgttc gtcagtcatg gtccgccctc accagttgtt 15600cgtatcggtg
tcggacgaac tgaaatcgca agagctgccg gtatcggtcc agccgctgtc 15660cgtgtcgctg
ctgccgaagc acggcgaggg gtccgcgaac gccgcagacg gcgtatccgg 15720ccgcagcgca
tcgcccagca tggccccggt cagcgagccg ccggccaggt agcccagcat 15780ggtgctgttg
gtcgccccgg ccaccagggc cgacgtgacg aaatcgccgt cattccctct 15840ggattgttcg
ctgctcggcg gggcagtgcg ccgcgccggc ggcgtcgtgg atggctcggg 15900ttggctggcc
tgcgacggcc ggcgaaaggt gcgcagcagc tcgttatcga ccggctgcgg 15960cgtcggggcc
gccgccttgc gctgcggtcg gtgttccttc ttcggctcgc gcagcttgaa 16020cagcatgatc
gcggaaacca gcagcaacgc cgcgcctacg cctcccgcga tgtagaacag 16080catcggattc
attcttcggt cctccttgta gcggaaccgt tgtctgtgcg gcgcgggtgg 16140cccgcgccgc
tgtctttggg gatcagccct cgatgagcgc gaccagtttc acgtcggcaa 16200ggttcgcctc
gaactcctgg ccgtcgtcct cgtacttcaa ccaggcatag ccttccgccg 16260gcggccgacg
gttgaggata aggcgggcag ggcgctcgtc gtgctcgacc tggacgatgg 16320cctttttcag
cttgtccggg tccggctcct tcgcgccctt ttccttggcg tccttaccgt 16380cctggtcgcc
gtcctcgccg tcctggccgt cgccggcctc cgcgtcacgc tcggcatcag 16440tctggccgtt
gaaggcatcg acggtgttgg gatcgcggcc cttctcgtcc aggaactcgc 16500gcagcagctt
gaccgtgccg cgcgtgattt cctgggtgtc gtcgtcaagc cacgcctcga 16560cttcctccgg
gcgcttcttg aaggccgtca ccagctcgtt caccacggtc acgtcgcgca 16620cgcggccggt
gttgaacgca tcggcgatct tctccggcag gtccagcagc gtgacgtgct 16680gggtgatgaa
cgccggcgac ttgccgattt ccttggcgat atcgcctttc ttcttgccct 16740tcgccagctc
gcggccaatg aagtcggcaa tttcgcgcgg ggtcagctcg ttgcgttgca 16800ggttctcgat
aacctggtcg gcttcgttgt agtcgttgtc gatgaacgcc gggatggact 16860tcttgccggc
ccacttcgag ccacggtagc ggcgggcgcc gtgattgatg atatagcggc 16920ccggctgctc
ctggttctcg cgcaccgaaa tgggtgactt caccccgcgc tctttgatcg 16980tggcaccgat
ttccgcgatg ctctccgggg aaaagccggg gttgtcggcc gtccgcggct 17040gatgcggatc
ttcgtcgatc aggtccaggt ccagctcgat agggccggaa ccgccctgag 17100acgccgcagg
agcgtccagg aggctcgaca ggtcgccgat gctatccaac cccaggccgg 17160acggctgcgc
cgcgcctgcg gcttcctgag cggccgcagc ggtgtttttc ttggtggtct 17220tggcttgagc
cgcagtcatt gggaaatctc catcttcgtg aacacgtaat cagccagggc 17280gcgaacctct
ttcgatgcct tgcgcgcggc cgttttcttg atcttccaga ccggcacacc 17340ggatgcgagg
gcatcggcga tgctgctgcg caggccaacg gtggccggaa tcatcatctt 17400ggggtacgcg
gccagcagct cggcttggtg gcgcgcgtgg cgcggattcc gcgcatcgac 17460cttgctgggc
accatgccaa ggaattgcag cttggcgttc ttctggcgca cgttcgcaat 17520ggtcgtgacc
atcttcttga tgccctggat gctgtacgcc tcaagctcga tgggggacag 17580cacatagtcg
gccgcgaaga gggcggccgc caggccgacg ccaagggtcg gggccgtgtc 17640gatcaggcac
acgtcgaagc cttggttcgc cagggccttg atgttcgccc cgaacagctc 17700gcgggcgtcg
tccagcgaca gccgttcggc gttcgccagt accgggttgg actcgatgag 17760ggcgaggcgc
gcggcctggc cgtcgccggc tgcgggtgcg gtttcggtcc agccgccggc 17820agggacagcg
ccgaacagct tgcttgcatg caggccggta gcaaagtcct tgagcgtgta 17880ggacgcattg
ccctgggggt ccaggtcgat cacggcaacc cgcaagccgc gctcgaaaaa 17940gtcgaaggca
agatgcacaa gggtcgaagt cttgccgacg ccgcctttct ggttggccgt 18000gaccaaagtt
ttcatcgttt ggtttcctgt tttttcttgg cgtccgcttc ccacttccgg 18060acgatgtacg
cctgatgttc cggcagaacc gccgttaccc gcgcgtaccc ctcgggcaag 18120ttcttgtcct
cgaacgcggc ccacacgcga tgcaccgctt gcgacactgc gcccctggtc 18180agtcccagcg
acgttgcgaa cgtcgcctgt ggcttcccat cgactaagac gccccgcgct 18240atctcgatgg
tctgctgccc cacttccagc ccctggatcg cctcctggaa ctggctttcg 18300gtaagccgtt
tcttcatgga taacacccat aatttgctcc gcgccttggt tgaacatagc 18360ggtgacagcc
gccagcacat gagagaagtt tagctaaaca tttctcgcac gtcaacacct 18420ttagccgcta
aaactcgtcc ttggcgtaac aaaacaaaag cccggaaacc gggctttcgt 18480ctcttgccgc
ttatggctct gcacccggct ccatcaccaa caggtcgcgc acgcgcttca 18540ctcggttgcg
gatcgacact gccagcccaa caaagccggt tgccgccgcc gccaggatcg 18600cgccgatgat
gccggccaca ccggccatcg cccaccaggt cgccgccttc cggttccatt 18660cctgctggta
ctgcttcgca atgctggacc tcggctcacc ataggctgac cgctcgatgg 18720cgtatgccgc
ttctcccctt ggcgtaaaac ccagcgccgc aggcggcatt gccatgctgc 18780ccgccgcttt
cccgaccacg acgcgcgcac caggcttgcg gtccagacct tcggccacgg 18840cgagctgcgc
aaggacataa tcagccgccg acttggctcc acgcgcctcg atcagctctt 18900gcactcgcgc
gaaatccttg gcctccacgg ccgccatgaa tcgcgcacgc ggcgaaggct 18960ccgcagggcc
ggcgtcgtga tcgccgccga gaatgccctt caccaagttc gacgacacga 19020aaatcatgct
gacggctatc accatcatgc agacggatcg cacgaacccg ctgaattgaa 19080cacgagcacg
gcacccgcga ccactatgcc aagaatgccc aaggtaaaaa ttgccggccc 19140cgccatgaag
tccgtgaatg ccccgacggc cgaagtgaag ggcaggccgc cacccaggcc 19200gccgccctca
ctgcccggca cctggtcgct gaatgtcgat gccagcacct gcggcacgtc 19260aatgcttccg
ggcgtcgcgc tcgggctgat cgcccatccc gttactgccc cgatcccggc 19320aatggcaagg
actgccagcg ctgccatttt tggggtgagg ccgttcgcgg ccgaggggcg 19380cagcccctgg
ggggatggga ggcccgcgtt agcgggccgg gagggttcga gaaggggggg 19440cacccccctt
cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta aaaacaaggt 19500ttataaatat
tggtttaaaa gcaggttaaa agacaggtta gcggtggccg aaaaacgggc 19560ggaaaccctt
gcaaatgctg gattttctgc ctgtggacag cccctcaaat gtcaataggt 19620gcgcccctca
tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc cctcatctgt 19680cagtagtcgc
gcccctcaag tgtcaatacc gcagggcact tatccccagg cttgtccaca 19740tcatctgtgg
gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag gctggccagc 19800tccacgtcgc
cggccgaaat cgagcctgcc cctcatctgt caacgccgcg ccgggtgagt 19860cggcccctca
agtgtcaacg tccgcccctc atctgtcagt gagggccaag ttttccgcga 19920ggtatccaca
acgccggcgg ccgcggtgtc tcgcacacgg cttcgacggc gtttctggcg 19980cgtttgcagg
gccatagacg gccgccagcc cagcggcgag ggcaaccagc ccggtgagcg 20040tcggaaaggc
gctggaagcc ccgtagcgac gcggagaggg gcgagacaag ccaagggcgc 20100aggctcgatg
cgcagcacga catagccggt tctcgcaagg acgagaattt ccctgcggtg 20160cccctcaagt
gtcaatgaaa gtttccaacg cgagccattc gcgagagcct tgagtccacg 20220ctagatgaga
gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac 20280ggaacggtct
gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg 20340atttattcaa
caaagccacg ttgtgtctca aaatctctga tgttacattg cacaagataa 20400aaatatatca
tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt 20460tatgagccat
attcaacggg aaacgtcttg ctcgactcta gagctcgttc ctcgaggcct 20520cgaggcctcg
aggaacggta cctgcgggga agcttacaat aatgtgtgtt gttaagtctt 20580gttgcctgtc
atcgtctgac tgactttcgt cataaatccc ggcctccgta acccagcttt 20640gggcaagctc
acggatttga tccggcggaa cgggaatatc gagatgccgg gctgaacgct 20700gcagttccag
ctttcccttt cgggacaggt actccagctg attgattatc tgctgaaggg 20760tcttggttcc
acctcctggc acaatgcgaa tgattacttg agcgcgatcg ggcatccaat 20820tttctcccgt
caggtgcgtg gtcaagtgct acaaggcacc tttcagtaac gagcgaccgt 20880cgatccgtcg
ccgggatacg gacaaaatgg agcgcagtag tccatcgagg gcggcgaaag 20940cctcgccaaa
agcaatacgt tcatctcgca cagcctccag atccgatcga gggtcttcgg 21000cgtaggcaga
tagaagcatg gatacattgc ttgagagtat tccgatggac tgaagtatgg 21060cttccatctt
ttctcgtgtg tctgcatcta tttcgagaaa gcccccgatg cggcgcaccg 21120caacgcgaat
tgccatacta tccgaaagtc ccagcaggcg cgcttgatag gaaaaggttt 21180catactcggc
cgatcgcaga cgggcactca cgaccttgaa cccttcaact ttcagggatc 21240gatgctggtt
gatggtagtc tcactcgacg tggctctggt gtgttttgac atagcttcct 21300ccaaagaaag
cggaaggtct ggatactcca gcacgaaatg tgcccgggta gacggatgga 21360agtctagccc
tgctcaatat gaaatcaaca gtacatttac agtcaatact gaatatactt 21420gctacatttg
caattgtctt ataacgaatg tgaaataaaa atagtgtaac aacgctttta 21480ctcatcgata
atcacaaaaa catttatacg aacaaaaata caaatgcact ccggtttcac 21540aggataggcg
ggatcagaat atgcaacttt tgacgttttg ttctttcaaa gggggtgctg 21600gcaaaaccac
cgcactcatg ggcctttgcg ctgctttggc aaatgacggt aaacgagtgg 21660ccctctttga
tgccgacgaa aaccggcctc tgacgcgatg gagagaaaac gccttacaaa 21720gcagtactgg
gatcctcgct gtgaagtcta ttccgccgac gaaatgcccc ttcttgaagc 21780agcctatgaa
aatgccgagc tcgaaggatt tgattatgcg ttggccgata cgcgtggcgg 21840ctcgagcgag
ctcaacaaca caatcatcgc tagctcaaac ctgcttctga tccccaccat 21900gctaacgccg
ctcgacatcg atgaggcact atctacctac cgctacgtca tcgagctgct 21960gttgagtgaa
aatttggcaa ttcctacagc tgttttgcgc caacgcgtcc cggtcggccg 22020attgacaaca
tcgcaacgca ggatgtcaga gacgctagag agccttccag ttgtaccgtc 22080tcccatgcat
gaaagagatg catttgccgc gatgaaagaa cgcggcatgt tgcatcttac 22140attactaaac
acgggaactg atccgacgat gcgcctcata gagaggaatc ttcggattgc 22200gatggaggaa
gtcgtggtca tttcgaaact gatcagcaaa atcttggagg cttgaagatg 22260gcaattcgca
agcccgcatt gtcggtcggc gaagcacggc ggcttgctgg tgctcgaccc 22320gagatccacc
atcccaaccc gacacttgtt ccccagaagc tggacctcca gcacttgcct 22380gaaaaagccg
acgagaaaga ccagcaacgt gagcctctcg tcgccgatca catttacagt 22440cccgatcgac
aacttaagct aactgtggat gcccttagtc cacctccgtc cccgaaaaag 22500ctccaggttt
ttctttcagc gcgaccgccc gcgcctcaag tgtcgaaaac atatgacaac 22560ctcgttcggc
aatacagtcc ctcgaagtcg ctacaaatga ttttaaggcg cgcgttggac 22620gatttcgaaa
gcatgctggc agatggatca tttcgcgtgg ccccgaaaag ttatccgatc 22680ccttcaacta
cagaaaaatc cgttctcgtt cagacctcac gcatgttccc ggttgcgttg 22740ctcgaggtcg
ctcgaagtca ttttgatccg ttggggttgg agaccgctcg agctttcggc 22800cacaagctgg
ctaccgccgc gctcgcgtca ttctttgctg gagagaagcc atcgagcaat 22860tggtgaagag
ggacctatcg gaacccctca ccaaatattg agtgtaggtt tgaggccgct 22920ggccgcgtcc
tcagtcacct tttgagccag ataattaaga gccaaatgca attggctcag 22980gctgccatcg
tccccccgtg cgaaacctgc acgtccgcgt caaagaaata accggcacct 23040cttgctgttt
ttatcagttg agggcttgac ggatccgcct caagtttgcg gcgcagccgc 23100aaaatgagaa
catctatact cctgtcgtaa acctcctcgt cgcgtactcg actggcaatg 23160agaagttgct
cgcgcgatag aacgtcgcgg ggtttctcta aaaacgcgag gagaagattg 23220aactcacctg
ccgtaagttt cacctcaccg ccagcttcgg acatcaagcg acgttgcctg 23280agattaagtg
tccagtcagt aaaacaaaaa gaccgtcggt ctttggagcg gacaacgttg 23340gggcgcacgc
gcaaggcaac ccgaatgcgt gcaagaaact ctctcgtact aaacggctta 23400gcgataaaat
cacttgctcc tagctcgagt gcaacaactt tatccgtctc ctcaaggcgg 23460tcgccactga
taattatgat tggaatatca gactttgccg ccagatttcg aacgatctca 23520agcccatctt
cacgacctaa atttagatca acaaccacga catcgaccgt cgcggaagag 23580agtactctag
tgaactgggt gctgtcggct accgcggtca ctttgaaggc gtggatcgta 23640aggtattcga
taataagatg ccgcatagcg acatcgtcat cgataagaag aacgtgtttc 23700aacggctcac
ctttcaatct aaaatctgaa cccttgttca cagcgcttga gaaattttca 23760cgtgaaggat
gtacaatcat ctccagctaa atgggcagtt cgtcagaatt gcggctgacc 23820gcggatgacg
aaaatgcgaa ccaagtattt caattttatg acaaaagttc tcaatcgttg 23880ttacaagtga
aacgcttcga ggttacagct actattgatt aaggagatcg cctatggtct 23940cgccccggcg
tcgtgcgtcc gccgcgagcc agatctcgcc tacttcataa acgtcctcat 24000aggcacggaa
tggaatgatg acatcgatcg ccgtagagag catgtcaatc agtgtgcgat 24060cttccaagct
agcaccttgg gcgctacttt tgacaaggga aaacagtttc ttgaatcctt 24120ggattggatt
cgcgccgtgt attgttgaaa tcgatcccgg atgtcccgag acgacttcac 24180tcagataagc
ccatgctgca tcgtcgcgca tctcgccaag caatatccgg tccggccgca 24240tacgcagact
tgcttggagc aagtgctcgg cgctcacagc acccagccca gcaccgttct 24300tggagtagag
tagtctaaca tgattatcgt gtggaatgac gagttcgagc gtatcttcta 24360tggtgattag
cctttcctgg ggggggatgg cgctgatcaa ggtcttgctc attgttgtct 24420tgccgcttcc
ggtagggcca catagcaaca tcgtcagtcg gctgacgacg catgcgtgca 24480gaaacgcttc
caaatccccg ttgtcaaaat gctgaaggat agcttcatca tcctgatttt 24540ggcgtttcct
tcgtgtctgc cactggttcc acctcgaagc atcataacgg gaggagactt 24600ctttaagacc
agaaacacgc gagcttggcc gtcgaatggt caagctgacg gtgcccgagg 24660gaacggtcgg
cggcagacag atttgtagtc gttcaccacc aggaagttca gtggcgcaga 24720gggggttacg
tggtccgaca tcctgctttc tcagcgcgcc cgctaaaata gcgatatctt 24780caagatcatc
ataagagacg ggcaaaggca tcttggtaaa aatgccggct tggcgcacaa 24840atgcctctcc
aggtcgattg atcgcaattt cttcagtctt cgggtcatcg agccattcca 24900aaatcggctt
cagaagaaag cgtagttgcg gatccacttc catttacaat gtatcctatc 24960tctaagcgga
aatttgaatt cattaagagc ggcggttcct cccccgcgtg gcgccgccag 25020tcaggcggag
ctggtaaaca ccaaagaaat cgaggtcccg tgctacgaaa atggaaacgg 25080tgtcaccctg
attcttcttc agggttggcg gtatgttgat ggttgcctta agggctgtct 25140cagttgtctg
ctcaccgtta ttttgaaagc tgttgaagct catcccgcca cccgagctgc 25200cggcgtaggt
gctagctgcc tggaaggcgc cttgaacaac actcaagagc atagctccgc 25260taaaacgctg
ccagaagtgg ctgtcgaccg agcccggcaa tcctgagcga ccgagttcgt 25320ccgcgcttgg
cgatgttaac gagatcatcg catggtcagg tgtctcggcg cgatcccaca 25380acacaaaaac
gcgcccatct ccctgttgca agccacgctg tatttcgcca acaacggtgg 25440tgccacgatc
aagaagcacg atattgttcg ttgttccacg aatatcctga ggcaagacac 25500actttacata
gcctgccaaa tttgtgtcga ttgcggtttg caagatgcac ggaattattg 25560tcccttgcgt
taccataaaa tcggggtgcg gcaagagcgt ggcgctgctg ggctgcagct 25620cggtgggttt
catacgtatc gacaaatcgt tctcgccgga cacttcgcca ttcggcaagg 25680agttgtcgtc
acgcttgcct tcttgtcttc ggcccgtgtc gccctgaatg gcgcgtttgc 25740tgaccccttg
atcgccgctg ctatatgcaa aaatcggtgt ttcttccggc cgtggctcat 25800gccgctccgg
ttcgcccctc ggcggtagag gagcagcagg ctgaacagcc tcttgaaccg 25860ctggaggatc
cggcggcacc tcaatcggag ctggatgaaa tggcttggtg tttgttgcga 25920tcaaagttga
cggcgatgcg ttctcattca ccttcttttg gcgcccacct agccaaatga 25980ggcttaatga
taacgcgaga acgacacctc cgacgatcaa tttctgagac cccgaaagac 26040gccggcgatg
tttgtcggag accagggatc cagatgcatc aacctcatgt gccgcttgct 26100gactatcgtt
attcatccct tcgccccctt caggacgcgt ttcacatcgg gcctcaccgt 26160gcccgtttgc
ggcctttggc caacgggatc gtaagcggtg ttccagatac atagtactgt 26220gtggccatcc
ctcagacgcc aacctcggga aaccgaagaa atctcgacat cgctcccttt 26280aactgaatag
ttggcaacag cttccttgcc atcaggattg atggtgtaga tggagggtat 26340gcgtacattg
cccggaaagt ggaataccgt cgtaaatcca ttgtcgaaga cttcgagtgg 26400caacagcgaa
cgatcgcctt gggcgacgta gtgccaatta ctgtccgccg caccaagggc 26460tgtgacaggc
tgatccaata aattctcagc tttccgttga tattgtgctt ccgcgtgtag 26520tctgtccaca
acagccttct gttgtgcctc ccttcgccga gccgccgcat cgtcggcggg 26580gtaggcgaat
tggacgctgt aatagagatc gggctgctct ttatcgaggt gggacagagt 26640cttggaactt
atactgaaaa cataacggcg catcccggag tcgcttgcgg ttagcacgat 26700tactggctga
ggcgtgagga cctggcttgc cttgaaaaat agataatttc cccgcggtag 26760ggctgctaga
tctttgctat ttgaaacggc aaccgctgtc accgtttcgt tcgtggcgaa 26820tgttacgacc
aaagtagctc caaccgccgt cgagaggcgc accacttgat cgggattgta 26880agccaaataa
cgcatgcgcg gatctagctt gcccgccatt ggagtgtctt cagcctccgc 26940accagtcgca
gcggcaaata aacatgctaa aatgaaaagt gcttttctga tcatggttcg 27000ctgtggccta
cgtttgaaac ggtatcttcc gatgtctgat aggaggtgac aaccagacct 27060gccgggttgg
ttagtctcaa tctgccgggc aagctggtca ccttttcgta gcgaactgtc 27120gcggtccacg
tactcaccac aggcattttg ccgtcaacga cgagggtcct tttatagcga 27180atttgctgcg
tgcttggagt tacatcattt gaagcgatgt gctcgacctc caccctgccg 27240cgtttgccaa
gaatgacttg aggcgaactg ggattgggat agttgaagaa ttgctggtaa 27300tcctggcgca
ctgttggggc actgaagttc gataccaggt cgtaggcgta ctgagcggtg 27360tcggcatcat
aactctcgcg caggcgaacg tactcccaca atgaggcgtt aacgacggcc 27420tcctcttgag
ttgcaggcaa tcgcgagaca gacacctcgc tgtcaacggt gccgtccggc 27480cgtatccata
gatatacggg cacaagcctg ctcaacggca ccattgtggc tatagcgaac 27540gcttgagcaa
catttcccaa aatcgcgata gctgcgacag ctgcaatgag tttggagaga 27600cgtcgcgccg
atttcgctcg cgcggtttga aaggcttcta cttccttata gtgctcggca 27660aggctttcgc
gcgccactag catggcatat tcaggccccg tcatagcgtc cacccgaatt 27720gccgagctga
agatctgacg gagtaggctg ccatcgcccc acattcagcg ggaagatcgg 27780gcctttgcag
ctcgctaatg tgtcgtttgt ctggcagccg ctcaaagcga caactaggca 27840cagcaggcaa
tacttcatag aattctccat tgaggcgaat ttttgcgcga cctagcctcg 27900ctcaacctga
gcgaagcgac ggtacaagct gctggcagat tgggttgcgc cgctccagta 27960actgcctcca
atgttgccgg cgatcgccgg caaagcgaca atgagcgcat cccctgtcag 28020aaaaaacata
tcgagttcgt aaagaccaat gatcttggcc gcggtcgtac cggcgaaggt 28080gattacacca
agcataaggg tgagcgcagt cgcttcggtt aggatgacga tcgttgccac 28140gaggtttaag
aggagaagca agagaccgta ggtgataagt tgcccgatcc acttagctgc 28200gatgtcccgc
gtgcgatcaa aaatatatcc gacgaggatc agaggcccga tcgcgagaag 28260cactttcgtg
agaattccaa cggcgtcgta aactccgaag gcagaccaga gcgtgccgta 28320aaggacccac
tgtgcccctt ggaaagcaag gatgtcctgg tcgttcatcg gaccgatttc 28380ggatgcgatt
ttctgaaaaa cggcctgggt cacggcgaac attgtatcca actgtgccgg 28440aacagtctgc
agaggcaagc cggttacact aaactgctga acaaagtttg ggaccgtctt 28500ttcgaagatg
gaaaccacat agtcttggta gttagcctgc ccaacaatta gagcaacaac 28560gatggtgacc
gtgatcaccc gagtgatacc gctacgggta tcgacttcgc cgcgtatgac 28620taaaataccc
tgaacaataa tccaaagagt gacacaggcg atcaatggcg cactcaccgc 28680ctcctggata
gtctcaagca tcgagtccaa gcctgtcgtg aaggctacat cgaagatcgt 28740atgaatggcc
gtaaacggcg ccggaatcgt gaaattcatc gattggacct gaacttgact 28800ggtttgtcgc
ataatgttgg ataaaatgag ctcgcattcg gcgaggatgc gggcggatga 28860acaaatcgcc
cagccttagg ggagggcacc aaagatgaca gcggtctttt gatgctcctt 28920gcgttgagcg
gccgcctctt ccgcctcgtg aaggccggcc tgcgcggtag tcatcgttaa 28980taggcttgtc
gcctgtacat tttgaatcat tgcgtcatgg atctgcttga gaagcaaacc 29040attggtcacg
gttgcctgca tgatattgcg agatcgggaa agctgagcag acgtatcagc 29100attcgccgtc
aagcgtttgt ccatcgtttc cagattgtca gccgcaatgc cagcgctgtt 29160tgcggaaccg
gtgatctgcg atcgcaacag gtccgcttca gcatcactac ccacgactgc 29220acgatctgta
tcgctggtga tcgcacgtgc cgtggtcgac attggcattc gcggcgaaaa 29280catttcattg
tctaggtcct tcgtcgaagg atactgattt ttctggttga gcgaagtcag 29340tagtccagta
acgccgtagg ccgacgtcaa catcgtaacc atcgctatag tctgagtgag 29400attctccgca
gtcgcgagcg cagtcgcgag cgtctcagcc tccgttgccg ggtcgctaac 29460aacaaactgc
gcccgcgcgg gctgaatata tagaaagctg caggtcaaaa ctgttgcaat 29520aagttgcgtc
gtcttcatcg tttcctacct tatcaatctt ctgcctcgtg gtgacgggcc 29580atgaattcgc
tgagccagcc agatgagttg ccttcttgtg cctcgcgtag tcgagttgca 29640aagcgcaccg
tgttggcacg ccccgaaagc acggcgacat attcacgcat atcccgcaga 29700tcaaattcgc
agatgacgct tccactttct cgtttaagaa gaaacttacg gctgccgacc 29760gtcatgtctt
cacggatcgc ctgaaattcc ttttcggtac atttcagtcc atcgacataa 29820gccgatcgat
ctgcggttgg tgatggatag aaaatcttcg tcatacattg cgcaaccaag 29880ctggctccta
gcggcgattc cagaacatgc tctggttgct gcgttgccag tattagcatc 29940ccgttgtttt
ttcgaacggt caggaggaat ttgtcgacga cagtcgaaaa tttagggttt 30000aacaaatagg
cgcgaaactc atcgcagctc atcacaaaac ggcggccgtc gatcatggct 30060ccaatccgat
gcaggagata tgctgcagcg ggagcgcata cttcctcgta ttcgagaaga 30120tgcgtcatgt
cgaagccggt aatcgacgga tctaacttta cttcgtcaac ttcgccgtca 30180aatgcccagc
caagcgcatg gccccggcac cagcgttgga gccgcgctcc tgcgccttcg 30240gcgggcccat
gcaacaaaaa ttcacgtaac cccgcgattg aacgcatttg tggatcaaac 30300gagagctgac
gatggatacc acggaccaga cggcggttct cttccggaga aatcccaccc 30360cgaccatcac
tctcgatgag agccacgatc cattcgcgca gaaaatcgtg tgaggctgct 30420gtgttttcta
ggccacgcaa cggcgccaac ccgctgggtg tgcctctgtg aagtgccaaa 30480tatgttcctc
ctgtggcgcg aaccagcaat tcgccacccc ggtccttgtc aaagaacacg 30540accgtacctg
cacggtcgac catgctctgt tcgagcatgg ctagaacaaa catcatgagc 30600gtcgtcttac
ccctcccgat aggcccgaat attgccgtca tgccaacatc gtgctcatgc 30660gggatatagt
cgaaaggcgt tccgccattg gtacgaaatc gggcaatcgc gttgccccag 30720tggcctgagc
tggcgccctc tggaaagttt tcgaaagaga caaaccctgc gaaattgcgt 30780gaagtgattg
cgccagggcg tgtgcgccac ttaaaattcc ccggcaattg ggaccaatag 30840gccgcttcca
taccaatacc ttcttggaca accacggcac ctgcatccgc cattcgtgtc 30900cgagcccgcg
cgcccctgtc cccaagacta ttgagatcgt ctgcatagac gcaaaggctc 30960aaatgatgtg
agcccataac gaattcgttg ctcgcaagtg cgtcctcagc ctcggataat 31020ttgccgattt
gagtcacggc tttatcgccg gaactcagca tctggctcga tttgaggcta 31080agtttcgcgt
gcgcttgcgg gcgagtcagg aacgaaaaac tctgcgtgag aacaagtgga 31140aaatcgaggg
atagcagcgc gttgagcatg cccggccgtg tttttgcagg gtattcgcga 31200aacgaataga
tggatccaac gtaactgtct tttggcgttc tgatctcgag tcctcgcttg 31260ccgcaaatga
ctctgtcggt ataaatcgaa gcgccgagtg agccgctgac gaccggaacc 31320ggtgtgaacc
gaccagtcat gatcaaccgt agcgcttcgc caatttcggt gaagagcaca 31380ccctgcttct
cgcggatgcc aagacgatgc aggccatacg ctttaagaga gccagcgaca 31440acatgccaaa
gatcttccat gttcctgatc tggcccgtga gatcgttttc cctttttccg 31500cttagcttgg
tgaacctcct ctttaccttc cctaaagccg cctgtgggta gacaatcaac 31560gtaaggaagt
gttcattgcg gaggagttgg ccggagagca cgcgctgttc aaaagcttcg 31620ttcaggctag
cggcgaaaac actacggaag tgtcgcggcg ccgatgatgg cacgtcggca 31680tgacgtacga
ggtgagcata tattgacaca tgatcatcag cgatattgcg caacagcgtg 31740ttgaacgcac
gacaacgcgc attgcgcatt tcagtttcct caagctcgaa tgcaacgcca 31800tcaattctcg
caatggtcat gatcgatccg tcttcaagaa ggacgatatg gtcgctgagg 31860tggccaatat
aagggagata gatctcaccg gatctttcgg tcgttccact cgcgccgagc 31920atcacaccat
tcctctccct cgtgggggaa ccctaattgg atttgggcta acagtagcgc 31980ccccccaaac
tgcactatca atgcttcttc ccgcggtccg caaaaatagc aggacgacgc 32040tcgccgcatt
gtagtctcgc tccacgatga gccgggctgc aaaccataac ggcacgagaa 32100cgacttcgta
gagcgggttc tgaacgataa cgatgacaaa gccggcgaac atcatgaata 32160accctgccaa
tgtcagtggc accccaagaa acaatgcggg ccgtgtggct gcgaggtaaa 32220gggtcgattc
ttccaaacga tcagccatca actaccgcca gtgagcgttt ggccgaggaa 32280gctcgcccca
aacatgataa caatgccgcc gacgacgccg gcaaccagcc caagcgaagc 32340ccgcccgaac
atccaggaga tcccgatagc gacaatgccg agaacagcga gtgactggcc 32400gaacggacca
aggataaacg tgcatatatt gttaaccatt gtggcggggt cagtgccgcc 32460acccgcagat
tgcgctgcgg cgggtccgga tgaggaaatg ctccatgcaa ttgcaccgca 32520caagcttggg
gcgcagctcg atatcacgcg catcatcgca ttcgagagcg agaggcgatt 32580tagatgtaaa
cggtatctct caaagcatcg catcaatgcg cacctcctta gtataagtcg 32640aataagactt
gattgtcgtc tgcggatttg ccgttgtcct ggtgtggcgg tggcggagcg 32700attaaaccgc
cagcgccatc ctcctgcgag cggcgctgat atgaccccca aacatcccac 32760gtctcttcgg
attttagcgc ctcgtgatcg tcttttggag gctcgattaa cgcgggcacc 32820agcgattgag
cagctgtttc aacttttcgc acgtagccgt ttgcaaaacc gccgatgaaa 32880ttaccggtgt
tgtaagcgga gatcgcccga cgaagcgcaa attgcttctc gtcaatcgtt 32940tcgccgcctg
cataacgact tttcagcatg tttgcagcgg cagataatga tgtgcacgcc 33000tggagcgcac
cgtcaggtgt cagaccgagc atagaaaaat ttcgagagtt tatttgcatg 33060aggccaacat
ccagcgaatg ccgtgcatcg agacggtgcc tgacgacttg ggttgcttgg 33120ctgtgatctt
gccagtgaag cgtttcgccg gtcgtgttgt catgaatcgc taaaggatca 33180aagcgactct
ccaccttagc tatcgccgca agcgtagatg tcgcaactga tggggcacac 33240ttgcgagcaa
catggtcaaa ctcagcagat gagagtggcg tggcaaggct cgacgaacag 33300aaggagacca
tcaaggcaag agaaagcgac cccgatctct taagcatacc ttatctcctt 33360agctcgcaac
taacaccgcc tctcccgttg gaagaagtgc gttgttttat gttgaagatt 33420atcgggaggg
tcggttactc gaaaattttc aattgcttct ttatgatttc aattgaagcg 33480agaaacctcg
cccggcgtct tggaacgcaa catggaccga gaaccgcgca tccatgacta 33540agcaaccgga
tcgacctatt caggccgcag ttggtcaggt caggctcaga acgaaaatgc 33600tcggcgaggt
tacgctgtct gtaaacccat tcgatgaacg ggaagcttcc ttccgattgc 33660tcttggcagg
aatattggcc catgcctgct tgcgctttgc aaatgctctt atcgcgttgg 33720tatcatatgc
cttgtccgcc agcagaaacg cactctaagc gattatttgt aaaaatgttt 33780cggtcatgcg
gcggtcatgg gcttgacccg ctgtcagcgc aagacggatc ggtcaaccgt 33840cggcatcgac
aacagcgtga atcttggtgg tcaaaccgcc acgggaacgt cccatacagc 33900catcgtcttg
atcccgctgt ttcccgtcgc cgcatgttgg tggacgcgga cacaggaact 33960gtcaatcatg
acgacattct atcgaaagcc ttggaaatca cactcagaat atgatcccag 34020acgtctgcct
cacgccatcg tacaaagcga ttgtagcagg ttgtacagga accgtatcga 34080tcaggaacgt
ctgcccaggg cgggcccgtc cggaagcgcc acaagatgac attgatcacc 34140cgcgtcaacg
cgcggcacgc gacgcggctt atttgggaac aaaggactga acaacagtcc 34200attcgaaatc
ggtgacatca aagcggggac gggttatcag tggcctccaa gtcaagcctc 34260aatgaatcaa
aatcagaccg atttgcaaac ctgatttatg agtgtgcggc ctaaatgatg 34320aaatcgtcct
tctagatcgc ctccgtggtg tagcaacacc tcgcagtatc gccgtgctga 34380ccttggccag
ggaattgact ggcaagggtg ctttcacatg accgctcttt tggccgcgat 34440agatgatttc
gttgctgctt tgggcacgta gaaggagaga agtcatatcg gagaaattcc 34500tcctggcgcg
agagcctgct ctatcgcgac ggcatcccac tgtcgggaac agaccggatc 34560attcacgagg
cgaaagtcgt caacacatgc gttataggca tcttcccttg aaggatgatc 34620ttgttgctgc
caatctggag gtgcggcagc cgcaggcaga tgcgatctca gcgcaacttg 34680cggcaaaaca
tctcactcac ctgaaaacca ctagcgagtc tcgcgatcag acgaaggcct 34740tttacttaac
gacacaatat ccgatgtctg catcacaggc gtcgctatcc cagtcaatac 34800taaagcggtg
caggaactaa agattactga tgacttaggc gtgccacgag gcctgagacg 34860acgcgcgtag
acagtttttt gaaatcatta tcaaagtgat ggcctccgct gaagcctatc 34920acctctgcgc
cggtctgtcg gagagatggg caagcattat tacggtcttc gcgcccgtac 34980atgcattgga
cgattgcagg gtcaatggat ctgagatcat ccagaggatt gccgccctta 35040ccttccgttt
cgagttggag ccagccccta aatgagacga catagtcgac ttgatgtgac 35100aatgccaaga
gagagatttg cttaacccga tttttttgct caagcgtaag cctattgaag 35160cttgccggca
tgacgtccgc gccgaaagaa tatcctacaa gtaaaacatt ctgcacaccg 35220aaatgcttgg
tgtagacatc gattatgtga ccaagatcct tagcagtttc gcttggggac 35280cgctccgacc
agaaataccg aagtgaactg acgccaatga caggaatccc ttccgtctgc 35340agataggtac
catcgataga tctgctgcct cgcgcgtttc ggtgatgacg gtgaaaacct 35400ctgacacatg
cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 35460acaagcccgt
cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca 35520gtcacgtagc
gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta 35580ctgagagtgc
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 35640atcaggcgct
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 35700cgagcggtat
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 35760gcaggaaaga
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 35820ttgctggcgt
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 35880agtcagaggt
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 35940tccctcgtgc
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 36000ccttcgggaa
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 36060gtcgttcgct
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 36120ttatccggta
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 36180gcagccactg
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 36240aagtggtggc
ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 36300aagccagtta
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 36360ggtagcggtg
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 36420gaagatcctt
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 36480gggattttgg
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 36540tgaagtttta
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 36600ttaatcagtg
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 36660ctccccgtcg
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 36720atgataccgc
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 36780ggaagggccg
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 36840tgttgccggg
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 36900attgctgcag
gggggggggg ggggggggac ttccattgtt cattccacgg acaaaaacag 36960agaaaggaaa
cgacagaggc caaaaagcct cgctttcagc acctgtcgtt tcctttcttt 37020tcagagggta
ttttaaataa aaacattaag ttatgacgaa gaagaacgga aacgccttaa 37080accggaaaat
tttcataaat agcgaaaacc cgcgaggtcg ccgccccgta acctgtcgga 37140tcaccggaaa
ggacccgtaa agtgataatg attatcatct acatatcaca acgtgcgtgg 37200aggccatcaa
accacgtcaa ataatcaatt atgacgcagg tatcgtatta attgatctgc 37260atcaacttaa
cgtaaaaaca acttcagaca atacaaatca gcgacactga atacggggca 37320acctcatgtc
cccccccccc ccccccctgc aggcatcgtg gtgtcacgct cgtcgtttgg 37380tatggcttca
ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 37440gtgcaaaaaa
gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 37500agtgttatca
ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 37560aagatgcttt
tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 37620gcgaccgagt
tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac 37680tttaaaagtg
ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 37740gctgttgaga
tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 37800tactttcacc
agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 37860aataagggcg
acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 37920catttatcag
ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 37980acaaataggg
gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 38040tattatcatg
acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga 38100attggtcgac
gatcttgctg cgttcggata ttttcgtgga gttcccgcca cagacccgga 38160ttgaaggcga
gatccagcaa ctcgcgccag atcatcctgt gacggaactt tggcgcgtga 38220tgactggcca
ggacgtcggc cgaaagagcg acaagcagat cacgcttttc gacagcgtcg 38280gatttgcgat
cgaggatttt tcggcgctgc gctacgtccg cgaccgcgtt gagggatcaa 38340gccacagcag
cccactcgac cttctagccg acccagacga gccaagggat ctttttggaa 38400tgctgctccg
tcgtcaggct ttccgacgtt tgggtggttg aacagaagtc attatcgtac 38460ggaatgccaa
gcactcccga ggggaaccct gtggttggca tgcacataca aatggacgaa 38520cggataaacc
ttttcacgcc cttttaaata tccgttattc taataaacgc tcttttctct 38580taggtttacc
cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac 38640gacaatctga
tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc 38700gggacaagcc
gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc 38760aagctggtac
gattgtaata cgactcacta tagggcgaat tgagcgctgt ttaaacgctc 38820ttcaactgga
agagcggtta cccggaccga agcttgaagt tcctattccg aagttcctat 38880tctctagaaa
gtataggaac ttcagatctc gatgctcacc ctgttgtttg gtgttacttc 38940tgcaggtcga
ctctagagga tccaccatga gcccagaacg acgcccggcc gacatccgcc 39000gtgccaccga
ggcggacatg ccggcggtct gcaccatcgt caaccactac atcgagacaa 39060gcacggtcaa
cttccgtacc gagccgcagg aaccgcagga ctggacggac gacctcgtcc 39120gtctgcggga
gcgctatccc tggctcgtcg ccgaggtgga cggcgaggtc gccggcatcg 39180cctacgcggg
cccctggaag gcacgcaacg cctacgactg gacggccgag tcgaccgtgt 39240acgtctcccc
ccgccaccag cggacgggac tgggctccac gctctacacc cacctgctga 39300agtccctgga
ggcacagggc ttcaagagcg tggtcgctgt catcgggctg cccaacgacc 39360cgagcgtgcg
catgcacgag gcgctcggat atgccccccg cggcatgctg cgggcggccg 39420gcttcaagca
cgggaactgg catgacgtgg gtttctggca gctggacttc agcctgccgg 39480taccgccccg
tccggtcctg cccgtcaccg agatctgatc cgtcgaccaa cctagacttg 39540tccatcttct
ggattggcca acttaattaa tgtatgaaat aaaaggatgc acacatagtg 39600acatgctaat
cactataatg tgggcatcaa agttgtgtgt tatgtgtaat tactagttat 39660ctgaataaaa
gagaaagaga tcatccatat ttcttatcct aaatgaatgt cacgtgtctt 39720tataattctt
tgatgaacca gatgcatttc attaaccaaa tccatataca tataaatatt 39780aatcatatat
aattaatatc aattgggtta gcaaaacaaa tctagtctag gtgtgttttg 39840cgaattgcgg
ccgcgatctg gggaattccc atggacaccg gtaattccca tgatcttctc 39900tccttcatca
atggatgcca tgtttcataa caataacacc aaatgtttga tgagctacca 39960acaattgcgc
aaagactatg gctaagctcg agctcgctcg ctacaagttg ttgactttca 40020aatacaagtt
tgtttttgga acaccaaata ttctacatga tctttcacta agttgcgcac 40080cactatcaaa
agattatcta ggccattatt caagtaaaga gtgaacacgt ctaagaccca 40140caaccacacc
aaatagaata cgcatacatg caacatattg tgcaagaagt atccaactgg 40200actcccatgt
attctaaaac tattttcgta gagttaaagt tatgacaaac ttatcaaata 40260aaaatttgaa
cgctggacca aaactttcat ctttcaaatc caccatcgtc tatcctcata 40320aattgttttg
attataacac atctacgtaa atcatttgtt ttgaacaata ctaatttaat 40380tttattaagt
caaataacct gcttagaaaa taatccctcc acctcattta acaatttctt 40440gtcaaacaca
caccaagaaa aaaattaatg aaagagaaaa gaaatgaaaa ggacatggag 40500ttgaatacta
gcaaaattga ttgaaggaag attcacaatt gaaattgaaa ccatttaatt 40560tattttcggg
tccataataa taaattggta agaataaaaa cccgatcaag tccggtacag 40620tacaattcca
ctccaccaac tccttactta aacccctatt tatacccact ctcatcctca 40680ctcttccttc
acctctcaca ctctcttctc tctctcaaaa ccctcacaca aacgctgcgt 40740ttagtgtaag
aaattcaatc cggcgccttg gcgcgccgat catccacaag tttgtacaaa 40800aaagctgaac
gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat 40860aaaaaacaga
ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat 40920taggcacccc
aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt 40980aggatttaaa
tacgcgttga tccggcttac taaaagccag ataacagtat gcgtatttgc 41040gcgctgattt
ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa 41100gaggtatgct
atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct 41160caaggcatat
atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc 41220cgtcgtctgc
gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc 41280cggtttattg
aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt 41340taaggtttac
acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga 41400tatcattgac
acgcccggtc gacggatggt gatccccctg gccagtgcac gtctgctgtc 41460agataaagtc
tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat 41520gatgaccacc
gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct 41580cagccaccgc
gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat 41640gtcaggctcc
cttatacaca gccagtctgc aggtcgacca tagtgactgg atatgttgtg 41700ttttacagta
ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta 41760tatcatttta
cgtttctcgt tcagctttct tgtacaaagt ggtgttaacc tagacttgtc 41820catcttctgg
attggccaac ttaattaatg tatgaaataa aaggatgcac acatagtgac 41880atgctaatca
ctataatgtg ggcatcaaag ttgtgtgtta tgtgtaatta ctagttatct 41940gaataaaaga
gaaagagatc atccatattt cttatcctaa atgaatgtca cgtgtcttta 42000taattctttg
atgaaccaga tgcatttcat taaccaaatc catatacata taaatattaa 42060tcatatataa
ttaatatcaa ttgggttagc aaaacaaatc tagtctaggt gtgttttgcg 42120aattgcggcc
gccaccgcgg tggagctcga attccggtcc gggtcacctt tgtccaccaa 42180gatggaactg
cggccgctca ttaattaagt caggcgcgcc tctagttgaa gacacgttca 42240tgtcttcatc
gtaagaagac actcagtagt cttcggccag aatggccatc tggattcagc 42300aggcctagaa
ggccatttaa atcctgagga tctggtcttc ctaaggaccc gggatatcgg 42360accgattaaa
ctttaattcg gtccgaagct tgaagttcct attccgaagt tcctattctc 42420cagaaagtat
aggaacttcg catgcctgca gtgcagcgtg acccggtcgt gcccctctct 42480agagataatg
agcattgcat gtctaagtta taaaaaatta ccacatattt tttttgtcac 42540acttgtttga
agtgcagttt atctatcttt atacatatat ttaaacttta ctctacgaat 42600aatataatct
atagtactac aataatatca gtgttttaga gaatcatata aatgaacagt 42660tagacatggt
ctaaaggaca attgagtatt ttgacaacag gactctacag ttttatcttt 42720ttagtgtgca
tgtgttctcc tttttttttg caaatagctt cacctatata atacttcatc 42780cattttatta
gtacatccat ttagggttta gggttaatgg tttttataga ctaatttttt 42840tagtacatct
attttattct attttagcct ctaaattaag aaaactaaaa ctctatttta 42900gtttttttat
ttaataattt agatataaaa tagaataaaa taaagtgact aaaaattaaa 42960caaataccct
ttaagaaatt aaaaaaacta aggaaacatt tttcttgttt cgagtagata 43020atgccagcct
gttaaacgcc gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc 43080gtcgcgtcgg
gccaagcgaa gcagacggca cggcatctct gtcgctgcct ctggacccct 43140ctcgagagtt
ccgctccacc gttggacttg ctccgctgtc ggcatccaga aattgcgtgg 43200cggagcggca
gacgtgagcc ggcacggcag gcggcctcct cctcctctca cggcaccggc 43260agctacgggg
gattcctttc ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata 43320aatagacacc
ccctccacac cctctttccc caacctcgtg ttgttcggag cgcacacaca 43380cacaaccaga
tctcccccaa atccacccgt cggcacctcc gcttcaaggt acgccgctcg 43440tcctcccccc
cccccctctc taccttctct agatcggcgt tccggtccat gcatggttag 43500ggcccggtag
ttctacttct gttcatgttt gtgttagatc cgtgtttgtg ttagatccgt 43560gctgctagcg
ttcgtacacg gatgcgacct gtacgtcaga cacgttctga ttgctaactt 43620gccagtgttt
ctctttgggg aatcctggga tggctctagc cgttccgcag acgggatcga 43680tttcatgatt
ttttttgttt cgttgcatag ggtttggttt gcccttttcc tttatttcaa 43740tatatgccgt
gcacttgttt gtcgggtcat cttttcatgc ttttttttgt cttggttgtg 43800atgatgtggt
ctggttgggc ggtcgttcta gatcggagta gaattctgtt tcaaactacc 43860tggtggattt
attaattttg gatctgtatg tgtgtgccat acatattcat agttacgaat 43920tgaagatgat
ggatggaaat atcgatctag gataggtata catgttgatg cgggttttac 43980tgatgcatat
acagagatgc tttttgttcg cttggttgtg atgatgtggt gtggttgggc 44040ggtcgttcat
tcgttctaga tcggagtaga atactgtttc aaactacctg gtgtatttat 44100taattttgga
actgtatgtg tgtgtcatac atcttcatag ttacgagttt aagatggatg 44160gaaatatcga
tctaggatag gtatacatgt tgatgtgggt tttactgatg catatacatg 44220atggcatatg
cagcatctat tcatatgctc taaccttgag tacctatcta ttataataaa 44280caagtatgtt
ttataattat tttgatcttg atatacttgg atgatggcat atgcagcagc 44340tatatgtgga
tttttttagc cctgccttca tacgctattt atttgcttgg tactgtttct 44400tttgtcgatg
ctcaccctgt tgtttggtgt tacttctgca ggtcgacttt aacttagcct 44460aggatccaca
cgacaccatg atagaggtga aaccgattaa cgcagaggat acctatgaac 44520taaggcatag
aatactcaga ccaaaccagc cgatagaagc gtgtatgttt gaaagcgatt 44580tacttcgtgg
tgcatttcac ttaggcggct attacggggg caaactgatt tccatagctt 44640cattccacca
ggccgagcac tcagaactcc aaggccagaa acagtaccag ctccgaggta 44700tggctacctt
ggaaggttat cgtgagcaga aggcgggatc gagtctaatt aaacacgctg 44760aagaaattct
tcgtaagagg ggggcggact tgctttggtg taatgcgcgg acatccgcct 44820caggctacta
caaaaagtta ggcttcagcg agcagggaga ggtattcgac acgccgccag 44880taggacctca
catcctgatg tataaaagga tcacataact agctagtcag ttaacctaga 44940cttgtccatc
ttctggattg gccaacttaa ttaatgtatg aaataaaagg atgcacacat 45000agtgacatgc
taatcactat aatgtgggca tcaaagttgt gtgttatgtg taattactag 45060ttatctgaat
aaaagagaaa gagatcatcc atatttctta tcctaaatga atgtcacgtg 45120tctttataat
tctttgatga accagatgca tttcattaac caaatccata tacatataaa 45180tattaatcat
atataattaa tatcaattgg gttagcaaaa caaatctagt ctaggtgtgt 45240tttgcgaatt
cagagctcga attcattccg attaatcgtg gcctcttgct cttcaggatg 45300aagagctatg
tttaaacgtg caagcgctac tagacaattc agtacattaa aaacgtccgc 45360aatgtgttat
taagttgtct aagcgtcaat ttgtttacac cacaatatat cctgccacca 45420gccagccaac
agctccccga ccggcagctc ggcacaaaat caccactcga tacaggcagc 45480ccatcagtcc
gggacggcgt cagcgggaga gccgttgtaa ggcggcagac tttgctcatg 45540ttaccgatgc
tattcggaag aacggcaact aagctgccgg gtttgaaaca cggatgatct 45600cgcggagggt
agcatgttga ttgtaacgat gacagagcgt tgctgcctgt gatcaaatat 45660catctccctc
gcagagatcc gaattatcag ccttcttatt catttctcgc ttaaccgtga 45720caggctgtcg
atcttgagaa ctatgccgac ataataggaa atcgctggat aaagccgctg 45780aggaagctga
gtggcgctat ttctttagaa gtgaacgttg acgatcgtcg accgtacccc 45840gatgaattaa
ttcggacgta cgttctgaac acagctggat acttacttgg gcgattgtca 45900tacatgacat
caacaatgta cccgtttgtg taaccgtctc ttggaggttc gtatgacact 45960agtggttccc
ctcagcttgc gactagatgt tgaggcctaa cattttatta gagagcaggc 46020tagttgctta
gatacatgat cttcaggccg ttatctgtca gggcaagcga aaattggcca 46080tttatgacga
ccaatgcccc gcagaagctc ccatctttgc cgccatagac gccgcgcccc 46140ccttttgggg
tgtagaacat ccttttgcca gatgtggaaa agaagttcgt tgtcccattg 46200ttggcaatga
cgtagtagcc ggcgaaagtg cgagacccat ttgcgctata tataagccta 46260cgatttccgt
tgcgactatt gtcgtaattg gatgaactat tatcgtagtt gctctcagag 46320ttgtcgtaat
ttgatggact attgtcgtaa ttgcttatgg agttgtcgta gttgcttgga 46380gaaatgtcgt
agttggatgg ggagtagtca tagggaagac gagcttcatc cactaaaaca 46440attggcaggt
cagcaagtgc ctgccccgat gccatcgcaa gtacgaggct tagaaccacc 46500ttcaacagat
cgcgcatagt cttccccagc tctctaacgc ttgagttaag ccgcgccgcg 46560aagcggcgtc
ggcttgaacg aattgttaga cattatttgc cgactacctt ggtgatctcg 46620cctttcacgt
agtgaacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct 46680tgtccaagat
aagcctgcct agcttcaagt atgacgggct gatactgggc cggcaggcgc 46740tccattgccc
agtcggcagc gacatccttc ggcgcgattt tgccggttac tgcgctgtac 46800caaatgcggg
acaacgtaag cactacattt cgctcatcgc cagcccagtc gggcggcgag 46860ttccatagcg
ttaaggtttc atttagcgcc tcaaatagat cctgttcagg aaccggatca 46920aagagttcct
ccgccgctgg acctaccaag gcaacgctat gttctcttgc ttttgtcagc 46980aagatagcca
gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag aatgtcattg 47040cgctgccatt
ctccaaattg cagttcgcgc ttagctggat aacgccacgg aatgatgtcg 47100tcgtgcacaa
caatggtgac ttctacagcg cggagaatct cgctctctcc aggggaagcc 47160gaagtttcca
aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag ccttacagtc 47220accgtaacca
gcaaatcaat atcactgtgt ggcttcaggc cgccatccac tgcggagccg 47280tacaaatgta
cggccagcaa cgtcggttcg agatggcgct cgatgacgcc aactacctct 47340gatagttgag
tcgatacttc ggcgatcacc gcttccctca tgatgtttaa ctcctgaatt 47400aagccgcgcc
gcgaagcggt gtcggcttga atgaattgtt aggcgtcatc ctgtgctccc 47460gagaaccagt
accagtacat cgctgtttcg ttcgagactt gaggtctagt tttatacgtg 47520aacaggtcaa
tgccgccgag agtaaagcca cattttgcgt acaaattgca ggcaggtaca 47580ttgttcgttt
gtgtctctaa tcgtatgcca aggagctgtc tgcttagtgc ccactttttc 47640gcaaattcga
tgagactgtg cgcgactcct ttgcctcggt gcgtgtgcga cacaacaatg 47700tgttcgatag
aggctagatc gttccatgtt gagttgagtt caatcttccc gacaagctct 47760tggtcgatga
atgcgccata gcaagcagag tcttcatcag agtcatcatc cgagatgtaa 47820tccttccggt
aggggctcac acttctggta gatagttcaa agccttggtc ggataggtgc 47880acatcgaaca
cttcacgaac aatgaaatgg ttctcagcat ccaatgtttc cgccacctgc 47940tcagggatca
ccgaaatctt catatgacgc ctaacgcctg gcacagcgga tcgcaaacct 48000ggcgcggctt
ttggcacaaa aggcgtgaca ggtttgcgaa tccgttgctg ccacttgtta 48060acccttttgc
cagatttggt aactataatt tatgttagag gcgaagtctt gggtaaaaac 48120tggcctaaaa
ttgctgggga tttcaggaaa gtaaacatca ccttccggct cgatgtctat 48180tgtagatata
tgtagtgtat ctacttgatc gggggatctg ctgcctcgcg cgtttcggtg 48240atgacggtga
aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 48300cggatgccgg
gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 48360gcgcagccat
gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc 48420atcagagcag
attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt 48480aaggagaaaa
taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 48540ggtcgttcgg
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 48600agaatcaggg
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 48660ccgtaaaaag
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 48720caaaaatcga
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 48780gtttccccct
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 48840cctgtccgcc
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 48900tctcagttcg
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 48960gcccgaccgc
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 49020cttatcgcca
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 49080tgctacagag
ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 49140tatctgcgct
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 49200caaacaaacc
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 49260aaaaaaagga
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 49320cgaaaactca
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 49380ccttttaaat
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 49440tgacagttac
caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 49500atccatagtt
gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 49560tggccccagt
gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 49620aataaaccag
ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 49680catccagtct
attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 49740gcgcaacgtt
gttgccattg ctgca 49765141670DNAZea
Maysmisc_feature(1660)..(1660)n is a, c, g, or t 14atggcgcccc gcggccgcac
cctcctcccg ctcgccgcgg ccaccgtcct cgtcgcttcc 60accatcttcc tcttcgccgc
cgccggtgcg cgctggcggc ccgccgacac cgacctcccc 120gtcccgccac acgccttccc
cacggccgtc cccgcggccg tgaccgcttc ttcttactcc 180aacgccaccg ccggcaagga
gctctccttt ctcgacgaga acggccgccc cgatgacccc 240tcctccgcct cagcatccag
ctccacctcc ggcgccaccc cggccgctgg tgtcgtcaga 300tgtgaccccc gcgacgccgt
cagggtattc atgtacgaca tgccgcccga gttccacttc 360ggcctcctcg gctggtcgcc
gccgtcccct gactccgtct ggccagacgt caccgccgcc 420tccccgccgc cgcgctaccc
cggggggctc aaccagcagc acagcgtgga gtactggctc 480acgctcgacc tcctctcctc
ctcgcccccc tgcggccgtc actccgcagt gcgggtctcc 540gattcccgcg atgccgacct
cgtcttcgtc cccttcttcg cgtccctcag ctacaaycgc 600cactaccggc ccgtgccgcc
cgagaagggc agcagggaca gggccatcca ggagaagctg 660gtgcgggacc tcgcggcgcg
gccggagtgg aggaggtacg gtggtgccga ccacgtcatc 720gtcgcgcacc accccaacag
cttgctgcac gcccgggcgg tgctgcaccc cgccgtgttc 780gtgctgtcag acttcgggag
gtacccaccg agggtggcca gcttggagaa ggatgtcatt 840gcgccataca agcacatggc
caagacgttc gtcaatgact cggccgggtt cgatgaccgg 900ccgaccctgt tatacttccg
gggagcaatt tacaggaagg agggagggag cattcgacag 960gagctatatt atatgctsaa
agaagaaaag gatgtttact tttcctttgg aagtgtccag 1020gaccatgggg ccagcaaagc
tagccaagga atgcactcat caaaattttg cctaaatatt 1080gctggggaca ccccttcttc
caatcgtctg tttgatgcga tagttaccca ctgtgtccct 1140gttatcatca gtgacgacat
tgagctacct tatgaggatg tgttggatta ttcaaaattc 1200tccatctttg tccgttcgtc
tgatgctgtt aagaaaggtt acctgatgag actgctcagt 1260ggtgtaagca agcaacaatg
gacaaagatg tgggataggc tcaaagaggt ggataaacat 1320tttgagtatc agtatccatc
acagaaggat gatgcagtcc agatgatctg gcaagcattg 1380tctagaaagg tgccatcaat
taagctgaag gttcacagat ctaatagatt ttcaagatct 1440aacagaggaa aataaacaga
aaggggtgtg tctatcttgt ctctattggc taatctaatg 1500taacacattt cactgacaca
ggctctcagc ctttcccagt tgcacaaaat agatagattg 1560taatactcag gttatcttta
ggaaaggttt gtaccttaag atttgttggt tcagttgaag 1620catatactgt caagtcaaag
gttgttttgt aaggtatgtn aaatgtaatc 167015484PRTZea Mays 15Met
Ala Pro Arg Gly Arg Thr Leu Leu Pro Leu Ala Ala Ala Thr Val1
5 10 15Leu Val Ala Ser Thr Ile Phe
Leu Phe Ala Ala Ala Gly Ala Arg Trp 20 25
30Arg Pro Ala Asp Thr Asp Leu Pro Val Pro Pro His Ala Phe
Pro Thr 35 40 45Ala Val Pro Ala
Ala Val Thr Ala Ser Ser Tyr Ser Asn Ala Thr Ala 50 55
60Gly Lys Glu Leu Ser Phe Leu Asp Glu Asn Gly Arg Pro
Asp Asp Pro65 70 75
80Ser Ser Ala Ser Ala Ser Ser Ser Thr Ser Gly Ala Thr Pro Ala Ala
85 90 95Gly Val Val Arg Cys Asp
Pro Arg Asp Ala Val Arg Val Phe Met Tyr 100
105 110Asp Met Pro Pro Glu Phe His Phe Gly Leu Leu Gly
Trp Ser Pro Pro 115 120 125Ser Pro
Asp Ser Val Trp Pro Asp Val Thr Ala Ala Ser Pro Pro Pro 130
135 140Arg Tyr Pro Gly Gly Leu Asn Gln Gln His Ser
Val Glu Tyr Trp Leu145 150 155
160Thr Leu Asp Leu Leu Ser Ser Ser Pro Pro Cys Gly Arg His Ser Ala
165 170 175Val Arg Val Ser
Asp Ser Arg Asp Ala Asp Leu Val Phe Val Pro Phe 180
185 190Phe Ala Ser Leu Ser Tyr Asn Arg His Tyr Arg
Pro Val Pro Pro Glu 195 200 205Lys
Gly Ser Arg Asp Arg Ala Ile Gln Glu Lys Leu Val Arg Asp Leu 210
215 220Ala Ala Arg Pro Glu Trp Arg Arg Tyr Gly
Gly Ala Asp His Val Ile225 230 235
240Val Ala His His Pro Asn Ser Leu Leu His Ala Arg Ala Val Leu
His 245 250 255Pro Ala Val
Phe Val Leu Ser Asp Phe Gly Arg Tyr Pro Pro Arg Val 260
265 270Ala Ser Leu Glu Lys Asp Val Ile Ala Pro
Tyr Lys His Met Ala Lys 275 280
285Thr Phe Val Asn Asp Ser Ala Gly Phe Asp Asp Arg Pro Thr Leu Leu 290
295 300Tyr Phe Arg Gly Ala Ile Tyr Arg
Lys Glu Gly Gly Ser Ile Arg Gln305 310
315 320Glu Leu Tyr Tyr Met Leu Lys Glu Glu Lys Asp Val
Tyr Phe Ser Phe 325 330
335Gly Ser Val Gln Asp His Gly Ala Ser Lys Ala Ser Gln Gly Met His
340 345 350Ser Ser Lys Phe Cys Leu
Asn Ile Ala Gly Asp Thr Pro Ser Ser Asn 355 360
365Arg Leu Phe Asp Ala Ile Val Thr His Cys Val Pro Val Ile
Ile Ser 370 375 380Asp Asp Ile Glu Leu
Pro Tyr Glu Asp Val Leu Asp Tyr Ser Lys Phe385 390
395 400Ser Ile Phe Val Arg Ser Ser Asp Ala Val
Lys Lys Gly Tyr Leu Met 405 410
415Arg Leu Leu Ser Gly Val Ser Lys Gln Gln Trp Thr Lys Met Trp Asp
420 425 430Arg Leu Lys Glu Val
Asp Lys His Phe Glu Tyr Gln Tyr Pro Ser Gln 435
440 445Lys Asp Asp Ala Val Gln Met Ile Trp Gln Ala Leu
Ser Arg Lys Val 450 455 460Pro Ser Ile
Lys Leu Lys Val His Arg Ser Asn Arg Phe Ser Arg Ser465
470 475 480Asn Arg Gly Lys161776DNAZea
Maysmisc_feature(764)..(924)n is a, c, g, or t 16ccccctccca gctagccatg
gccgcgcccc gctctggtcg ccgcccgcac tgccactgct 60agccatggcc gccgccgccg
cctgccggag cccgctggtc tggctcttcg cgctcgtcac 120cgcactcttc ttcttctcct
ggtacctcct cctcgactcc gccgcgggtc cagccgccgc 180ccgccgcccc aaccagtggc
tccgcctcgg cggcggcggg cggcgctccg gtcccggtag 240gaaatgcgac cccgcggagg
cgctgctgcg agtgttcatg tacgacctgc cccccgagtt 300ccacttcgga ctgctcgact
ggaagccccc cggcttcggc ggcggcgtgt ggcccgacat 360cagggacggc gtgcctgact
acccgggggg cctcaacctg cagcacagca tcgagtattg 420gctcaccctc gacctcctgg
cctccgagca gggcgcgccc acgccctgcg cagtggcgcg 480ggtgcgccac gcggcggacg
ccgacgtcgt cttcgtgccc ttcttcgcct cgctcagctt 540caaccgccac tcccgggtgg
taccgcccgc gcgggacagc gaggaccgcg cgctgcagcg 600gaggctcctc gagttcctcg
ccgcgcggcc cgagtggcgg aggactggcg ggcgggacca 660cgtcgtgctc gcgcatcacc
ccaacgggat gctcgacgcg cgctacaggt tctggccctg 720cgtcttcgtg ctctgcgact
tcgggaggta cccgcccagc gtcnnnnnnn nnnnnnnnnn 780nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900nnnnnnnnnn nnnnnnnnnn
nnnngaaaga cgagaaagat gtgcatttct catttggaag 960cgtagccggt aatgggatcg
agcaagcaac acaagggatg cggtcatcca agttctgcct 1020caacattgca ggtgacactc
catcctccaa ccgcctcttt gactccatag tcagtcactg 1080tgttcccgtc acgatcagcg
atgagattga gctcccgttt gaggatgtcc tcgactactc 1140gaagttcagt gtcatagtac
gtggcgcaga cgcagtcaag aaggggtttc taatgaacct 1200gatcaaaggg atcagccgag
aagagtggac acgcatgtgg aacaggctaa aggaagtgga 1260aaagcacttt gagtaccaat
acccatctca gaccgatgat gccgtgcaga tgatatggaa 1320ggccattgct cggaaggtgc
cgtctatccg gctgaagatt aacagactgc aaagattttc 1380tctgtttgag actaacagga
cagatgagac tctaccccca tcttcttctt ggctacagaa 1440tcaggctcct tgattttgga
ctagcaagct caggctttcg ccatgttttc aatctcgtag 1500aatcaagagt tgatactgaa
gaccaaatct taatcgcttg actgggggca gatgttagta 1560tgttacagct gcagacatta
gcggatagta aagttgaaac actttagcat agcagaatta 1620catatccagt ggcacatcat
tttcttcatt tttttttctt ttgcttgcga ttcatccaaa 1680gtgtcctcgg ctgcagacta
aggacaatga attttgtatg tgaagctgta tattctaggc 1740ggcaatatag ttactgatca
gttacagttg ctgggc 177617462PRTZea
maysmisc_feature(234)..(287)Xaa can be any naturally occurring amino acid
17Met Ala Ala Ala Ala Ala Cys Arg Ser Pro Leu Val Trp Leu Phe Ala1
5 10 15Leu Val Thr Ala Leu Phe
Phe Phe Ser Trp Tyr Leu Leu Leu Asp Ser 20 25
30Ala Ala Gly Pro Ala Ala Ala Arg Arg Pro Asn Gln Trp
Leu Arg Leu 35 40 45Gly Gly Gly
Gly Arg Arg Ser Gly Pro Gly Arg Lys Cys Asp Pro Ala 50
55 60Glu Ala Leu Leu Arg Val Phe Met Tyr Asp Leu Pro
Pro Glu Phe His65 70 75
80Phe Gly Leu Leu Asp Trp Lys Pro Pro Gly Phe Gly Gly Gly Val Trp
85 90 95Pro Asp Ile Arg Asp Gly
Val Pro Asp Tyr Pro Gly Gly Leu Asn Leu 100
105 110Gln His Ser Ile Glu Tyr Trp Leu Thr Leu Asp Leu
Leu Ala Ser Glu 115 120 125Gln Gly
Ala Pro Thr Pro Cys Ala Val Ala Arg Val Arg His Ala Ala 130
135 140Asp Ala Asp Val Val Phe Val Pro Phe Phe Ala
Ser Leu Ser Phe Asn145 150 155
160Arg His Ser Arg Val Val Pro Pro Ala Arg Asp Ser Glu Asp Arg Ala
165 170 175Leu Gln Arg Arg
Leu Leu Glu Phe Leu Ala Ala Arg Pro Glu Trp Arg 180
185 190Arg Thr Gly Gly Arg Asp His Val Val Leu Ala
His His Pro Asn Gly 195 200 205Met
Leu Asp Ala Arg Tyr Arg Phe Trp Pro Cys Val Phe Val Leu Cys 210
215 220Asp Phe Gly Arg Tyr Pro Pro Ser Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa225 230 235
240Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 245 250 255Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 260
265 270Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Lys 275 280
285Asp Glu Lys Asp Val His Phe Ser Phe Gly Ser Val Ala Gly Asn Gly 290
295 300Ile Glu Gln Ala Thr Gln Gly Met
Arg Ser Ser Lys Phe Cys Leu Asn305 310
315 320Ile Ala Gly Asp Thr Pro Ser Ser Asn Arg Leu Phe
Asp Ser Ile Val 325 330
335Ser His Cys Val Pro Val Thr Ile Ser Asp Glu Ile Glu Leu Pro Phe
340 345 350Glu Asp Val Leu Asp Tyr
Ser Lys Phe Ser Val Ile Val Arg Gly Ala 355 360
365Asp Ala Val Lys Lys Gly Phe Leu Met Asn Leu Ile Lys Gly
Ile Ser 370 375 380Arg Glu Glu Trp Thr
Arg Met Trp Asn Arg Leu Lys Glu Val Glu Lys385 390
395 400His Phe Glu Tyr Gln Tyr Pro Ser Gln Thr
Asp Asp Ala Val Gln Met 405 410
415Ile Trp Lys Ala Ile Ala Arg Lys Val Pro Ser Ile Arg Leu Lys Ile
420 425 430Asn Arg Leu Gln Arg
Phe Ser Leu Phe Glu Thr Asn Arg Thr Asp Glu 435
440 445Thr Leu Pro Pro Ser Ser Ser Trp Leu Gln Asn Gln
Ala Pro 450 455 460181174DNAZea mays
18gcacgagtct tcgtgccctt cttctcctcg ctcagcttca acgtgcacgg tcgcaacatg
60accgaccctg acaccgaggc cgaccgcctc ctgcaggttg aacttgtgga tattctctgg
120aagtctaaat attggcaacg ttctgcgggc cgtgaccatg tcattcctat gcatcaccct
180aatgctttca gattcctgcg agcaatggtg aatgcatcta ttcttatagt ttcagacttt
240gggagataca caaaggaact ggcttccctg aggaaagatg ttgtggcacc atatgtgcat
300gttgtgggtt ccttccttga tgacgatcca cctgatccat ttgaggctcg ccatacactg
360cttttctttc gaggccgtac tgtcaggaaa gatgaaggga aaatccggtc aaaacttgag
420aagatattaa aaggcaagga aggggtgcgc tttgaggata gcattgccac gggcgacggc
480attaacatat ctacagaagg tatgcggtca tcaaagtttt gtctccaccc tgctggggac
540actccttcct catgccgcct gtttgatgcc atagtcagtc attgtgttcc tgtgatagtc
600agcagtcgaa tcgagctccc ttttgaagat gagattgatt acagtgagtt ctcccttttc
660ttctccgttg aagaggctct aagacctgat tacttgctga acgagctcag acaggtcccc
720aaaaggaagt gggttgatat gtggttgaag cttaagaatg tctcccatca ttatgaattc
780cagtatcccc ccaggaaggg cgacgcggtg aacatgatat ggaggcaggt gaggcacaag
840atccccgcag ttaaccttgc tattcacagg aacagaagac tgaaaattcc agactggtgg
900ggataatgat tggtggtgaa tcgtgtacat attaccatat ccactgttag tcctggttat
960tttcggtgcg ttatgatgga aacattgctc accgtccttt gtgaaccaaa gtgttcatct
1020taagatccaa ggaccgagtc cacaactatt tgctgacagg aactgagatt atcacctctt
1080ttttggtcga tttttgtgac ggcttcctat ttccccccct gatgctacaa aatagagggg
1140acaaataaac ttacagtaac attatagagg agtc
117419282PRTZea mays 19Met Thr Asp Pro Asp Thr Glu Ala Asp Arg Leu Leu
Gln Val Glu Leu1 5 10
15Val Asp Ile Leu Trp Lys Ser Lys Tyr Trp Gln Arg Ser Ala Gly Arg
20 25 30Asp His Val Ile Pro Met His
His Pro Asn Ala Phe Arg Phe Leu Arg 35 40
45Ala Met Val Asn Ala Ser Ile Leu Ile Val Ser Asp Phe Gly Arg
Tyr 50 55 60Thr Lys Glu Leu Ala Ser
Leu Arg Lys Asp Val Val Ala Pro Tyr Val65 70
75 80His Val Val Gly Ser Phe Leu Asp Asp Asp Pro
Pro Asp Pro Phe Glu 85 90
95Ala Arg His Thr Leu Leu Phe Phe Arg Gly Arg Thr Val Arg Lys Asp
100 105 110Glu Gly Lys Ile Arg Ser
Lys Leu Glu Lys Ile Leu Lys Gly Lys Glu 115 120
125Gly Val Arg Phe Glu Asp Ser Ile Ala Thr Gly Asp Gly Ile
Asn Ile 130 135 140Ser Thr Glu Gly Met
Arg Ser Ser Lys Phe Cys Leu His Pro Ala Gly145 150
155 160Asp Thr Pro Ser Ser Cys Arg Leu Phe Asp
Ala Ile Val Ser His Cys 165 170
175Val Pro Val Ile Val Ser Ser Arg Ile Glu Leu Pro Phe Glu Asp Glu
180 185 190Ile Asp Tyr Ser Glu
Phe Ser Leu Phe Phe Ser Val Glu Glu Ala Leu 195
200 205Arg Pro Asp Tyr Leu Leu Asn Glu Leu Arg Gln Val
Pro Lys Arg Lys 210 215 220Trp Val Asp
Met Trp Leu Lys Leu Lys Asn Val Ser His His Tyr Glu225
230 235 240Phe Gln Tyr Pro Pro Arg Lys
Gly Asp Ala Val Asn Met Ile Trp Arg 245
250 255Gln Val Arg His Lys Ile Pro Ala Val Asn Leu Ala
Ile His Arg Asn 260 265 270Arg
Arg Leu Lys Ile Pro Asp Trp Trp Gly 275
280201834DNAZea mays 20ctccatagcg accaaccggc cgccgcgggt gaccaaccca
gccgcgcttt cccccgtcag 60atcccctcct agccagccat ggccgagccc cgttccgccc
tccgcccgca ttgccactgt 120tagtgccatg gccaccgcct gccggagccc gctcgtctgg
ctcttcgcgc tcgccgccgc 180gctattcttc ctctcctggt acctcctcct caactccgcc
gcgggcccaa ccgccgcccg 240ccgccccaac caggggctcc gcctcggcgg ccccggtagg
aaatgcgacc ccgcggaggc 300gctgctgcga gtgttcatgt acgacctgcc ccccgagttc
cacttcggac tgctcgactg 360gaagcccccg ggcttcggcg gcggcgtgtg gcccgacgtc
agggacggcg tgccggacta 420cccggggggg ctcaaccttc agcacagcat cgagtactgg
ctcaccctcg acctcttggc 480ctccgagcag ggcgcgccca cgccctgcgc agcggcgcgg
gtgcgccacg cggcggacgc 540cgacgtcgtc ttcgtgcctt tcttcgcctc gctcagcttc
aaccgccact cccgggtggt 600gccgcccgcg cggaacagcg aggaccgcgc gctgcaacgg
aggctcctcg agttcctcgc 660cgcgcggcct gagtggcgca ggaccggcgg gcgggaccac
gtcgtgctcg cgcatcaccc 720caacggtatg ctcgacgcgc gctacaggtt ctggccctgc
gtcttcgtgc tctgcgactt 780cgggaggtac ccgcccagcg tcgccaacct cgacaaggac
atcatcgcgc cctatcggca 840cctcgtcgcc aacttcgcta atgacaccgc cggatacgac
gaccggccga cgctgctcta 900cttccaaggc gccatctaca ggaaggatgg tggttccatt
cggcaagaac tgtattacct 960tctgaaagac gagaaagatg tgcatttctc atttggaagt
gtagctggta atgggatcga 1020gcaggcaaca caaggtatgc ggtcatccaa gttctgcctc
aacattgcag gtgacactcc 1080atcctccaac cgcctcttcg actccattgt cagtcactgt
gttcccgtca tcatcagcga 1140tgagattgag ctcccgtttg aggatgtcct cgactattca
aagttcagcg tcatagtacg 1200tggcgcagat gcagtcaaga aggggtttct aaagagcctg
atcaaaggga tcagccaaga 1260agagtggaca cgcatgtgga acaagctaaa ggaagtagaa
aagcacttcg agtaccaata 1320cccatctcag actgatgatg ccgtgcagat gatatggaag
gctattgctc ggaaggtgcc 1380ctctatccgt ctgaagatta acagactacg gagattttct
cggtttgata ctaataggac 1440agatgaaact ctacccagtc cttcttggct acagaatcag
gcttcttgat ttttggacta 1500gcaaactcca gctttcacca tgttttcaat cctgccgaaa
caagagttga tactgaagac 1560caaaccttaa tcgcatgact gggggcagat gttacagctg
cagacattaa cggagagtaa 1620agttgaaaca ctctagcata acagaattac atatccaatg
gcacatcatt tttcttcatt 1680tttcttactt aagattcatc caaaatgtcc ttggctgcag
actaaggaca atgaattttg 1740tatgaaaagc tgtacattct aggcggcaat ataattactg
accagttaca gttgctgaaa 1800aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaag
183421453PRTZea mays 21Met Ala Thr Ala Cys Arg Ser
Pro Leu Val Trp Leu Phe Ala Leu Ala1 5 10
15Ala Ala Leu Phe Phe Leu Ser Trp Tyr Leu Leu Leu Asn
Ser Ala Ala 20 25 30Gly Pro
Thr Ala Ala Arg Arg Pro Asn Gln Gly Leu Arg Leu Gly Gly 35
40 45Pro Gly Arg Lys Cys Asp Pro Ala Glu Ala
Leu Leu Arg Val Phe Met 50 55 60Tyr
Asp Leu Pro Pro Glu Phe His Phe Gly Leu Leu Asp Trp Lys Pro65
70 75 80Pro Gly Phe Gly Gly Gly
Val Trp Pro Asp Val Arg Asp Gly Val Pro 85
90 95Asp Tyr Pro Gly Gly Leu Asn Leu Gln His Ser Ile
Glu Tyr Trp Leu 100 105 110Thr
Leu Asp Leu Leu Ala Ser Glu Gln Gly Ala Pro Thr Pro Cys Ala 115
120 125Ala Ala Arg Val Arg His Ala Ala Asp
Ala Asp Val Val Phe Val Pro 130 135
140Phe Phe Ala Ser Leu Ser Phe Asn Arg His Ser Arg Val Val Pro Pro145
150 155 160Ala Arg Asn Ser
Glu Asp Arg Ala Leu Gln Arg Arg Leu Leu Glu Phe 165
170 175Leu Ala Ala Arg Pro Glu Trp Arg Arg Thr
Gly Gly Arg Asp His Val 180 185
190Val Leu Ala His His Pro Asn Gly Met Leu Asp Ala Arg Tyr Arg Phe
195 200 205Trp Pro Cys Val Phe Val Leu
Cys Asp Phe Gly Arg Tyr Pro Pro Ser 210 215
220Val Ala Asn Leu Asp Lys Asp Ile Ile Ala Pro Tyr Arg His Leu
Val225 230 235 240Ala Asn
Phe Ala Asn Asp Thr Ala Gly Tyr Asp Asp Arg Pro Thr Leu
245 250 255Leu Tyr Phe Gln Gly Ala Ile
Tyr Arg Lys Asp Gly Gly Ser Ile Arg 260 265
270Gln Glu Leu Tyr Tyr Leu Leu Lys Asp Glu Lys Asp Val His
Phe Ser 275 280 285Phe Gly Ser Val
Ala Gly Asn Gly Ile Glu Gln Ala Thr Gln Gly Met 290
295 300Arg Ser Ser Lys Phe Cys Leu Asn Ile Ala Gly Asp
Thr Pro Ser Ser305 310 315
320Asn Arg Leu Phe Asp Ser Ile Val Ser His Cys Val Pro Val Ile Ile
325 330 335Ser Asp Glu Ile Glu
Leu Pro Phe Glu Asp Val Leu Asp Tyr Ser Lys 340
345 350Phe Ser Val Ile Val Arg Gly Ala Asp Ala Val Lys
Lys Gly Phe Leu 355 360 365Lys Ser
Leu Ile Lys Gly Ile Ser Gln Glu Glu Trp Thr Arg Met Trp 370
375 380Asn Lys Leu Lys Glu Val Glu Lys His Phe Glu
Tyr Gln Tyr Pro Ser385 390 395
400Gln Thr Asp Asp Ala Val Gln Met Ile Trp Lys Ala Ile Ala Arg Lys
405 410 415Val Pro Ser Ile
Arg Leu Lys Ile Asn Arg Leu Arg Arg Phe Ser Arg 420
425 430Phe Asp Thr Asn Arg Thr Asp Glu Thr Leu Pro
Ser Pro Ser Trp Leu 435 440 445Gln
Asn Gln Ala Ser 450222080DNAOryza sativa 22ggcatagcgg catgcatcgc
agcataggcc ggtcaagaag acgatggccc tgacgaggcg 60cctcctcatc gatctctcgt
caagacgccg ccttttcaac gccggcaaat tctccaccac 120gcacaagaag aaaccagtgc
tccatgaagg gagggagcag gggtgttgtt gaaatggagc 180agcagctcgt ttctttgatt
tgatctttgg cagtaagtaa ttcggggttt gaatttatta 240tttttgtttt gggcgaattt
cattccagcg gtgagtttgg ctggattctt gcgctgctcg 300cgcgcgctgg tgtcgtggat
ggtggcggag aggaagatgc agccatcgcc ggcggcgccg 360ccggcggcgg agcaccggag
gcgggcgctg ctgcgctacg tggtgttcct cgcggtctcc 420ctcctcgcct tctcctgctg
ggctctcgtc agctcgcgga tcgacggcgc cgtcctcgcg 480gcgaccgccg gcggcgagca
tgacgaccac gatggcatta ttgttagaag cagcacccaa 540gcggagatgc cagcgagagg
cgggaacgcg acgtcgcgcg gcgccgtcga ggtgggtgtg 600ggtactccgg cggcgatgat
cacccggcag ccgtcgtcgg gagagacgac gacgacggcg 660gcgttggcgg cgacgtgcga
cgcggagagc gcgctgctga gggtgtacct ctacgacctc 720ccgccggagt tccacttcgg
catgctgggg tgggacggca aggcggccgg cgcggcgtgg 780ccggacgtgg ccggcgaccc
gcgcgccgtg ccgcgctacc cgggcggcct gaacctgcag 840cacagcgtcg agtactggct
caccctggac atcctctcct ccaccacctc cggcgaccac 900cgccgccgcc gtccgtgcac
cgccgtgagg gtgacgaacg cgagccttgc cgacgtgttc 960ctggtgccgt tcttcgcgtc
gctgagctac aaccggcagt cgaagtcgcc gcacggcggc 1020catgggagtg gcggccggag
cgacaggcag ctgcagggcg agctggtgag gtacctggcg 1080aggcgggagg agtggcggcg
gtggggcggc gcggaccacc tcgtcgtgcc gcaccacccg 1140aacagcatga tggacgcccg
gcggcggctc agcgccgcca tgttcgtcct ctccgacttc 1200ggcaggtacc cgccggacgt
cgccaacctg aggaaggacg tgatcgcgcc gtacaagcac 1260gtcgtcccct ccctcggcga
cggcgactcg ccggggttcg agcagcgccc cgtcctcgct 1320tacttccagg gcgccattca
taggaaaaat ggtggaaggg ttcgtcagag gctgtaccag 1380ctgatcaagg acgagaagga
cgtccacttc acctacggca gcgtccgtca gaacggcatc 1440aggcgcgcca ccaaggggat
ggcctcctcc aagttctgcc tcaacatcgc cggcgacacc 1500ccctcctcca accgcctctt
cgacgccatc gtcagccact gcgtccccgt gatcatcagc 1560gacgacatcg agctcccctt
cgaggacgtc ctcgactact ccgccttctg cgtgttcgtc 1620cgcgcctccg acgccgtcaa
gaggggcttc ctgctgcatc tcctcagggg gatctcccag 1680gaagaatgga cggcaatgtg
gaggaggctg aaggaggttg cacaccactt cgagtaccag 1740tacccttcgc agcctggtga
cgctgttcag atgatctggg gagctgtagc tcggaagatg 1800catttggtga agctgcaact
tcacaagcgt ggtagatatc agaggacatt ttctgaatca 1860taaaaggtta gggtagcaaa
gcatggagga gattcagagc tataggttgc agtttttgat 1920caataatttt catgtggctg
gtaaagttct atggtcattt tgcaatctaa gttgcaagga 1980ctatgtgcag ccagtctttg
tccaagaggt aattattaga cagttagtta taaattatat 2040tattcttatt aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 208023514PRTOryza sativa
23Met Val Ala Glu Arg Lys Met Gln Pro Ser Pro Ala Ala Pro Pro Ala1
5 10 15Ala Glu His Arg Arg Arg
Ala Leu Leu Arg Tyr Val Val Phe Leu Ala 20 25
30Val Ser Leu Leu Ala Phe Ser Cys Trp Ala Leu Val Ser
Ser Arg Ile 35 40 45Asp Gly Ala
Val Leu Ala Ala Thr Ala Gly Gly Glu His Asp Asp His 50
55 60Asp Gly Ile Ile Val Arg Ser Ser Thr Gln Ala Glu
Met Pro Ala Arg65 70 75
80Gly Gly Asn Ala Thr Ser Arg Gly Ala Val Glu Val Gly Val Gly Thr
85 90 95Pro Ala Ala Met Ile Thr
Arg Gln Pro Ser Ser Gly Glu Thr Thr Thr 100
105 110Thr Ala Ala Leu Ala Ala Thr Cys Asp Ala Glu Ser
Ala Leu Leu Arg 115 120 125Val Tyr
Leu Tyr Asp Leu Pro Pro Glu Phe His Phe Gly Met Leu Gly 130
135 140Trp Asp Gly Lys Ala Ala Gly Ala Ala Trp Pro
Asp Val Ala Gly Asp145 150 155
160Pro Arg Ala Val Pro Arg Tyr Pro Gly Gly Leu Asn Leu Gln His Ser
165 170 175Val Glu Tyr Trp
Leu Thr Leu Asp Ile Leu Ser Ser Thr Thr Ser Gly 180
185 190Asp His Arg Arg Arg Arg Pro Cys Thr Ala Val
Arg Val Thr Asn Ala 195 200 205Ser
Leu Ala Asp Val Phe Leu Val Pro Phe Phe Ala Ser Leu Ser Tyr 210
215 220Asn Arg Gln Ser Lys Ser Pro His Gly Gly
His Gly Ser Gly Gly Arg225 230 235
240Ser Asp Arg Gln Leu Gln Gly Glu Leu Val Arg Tyr Leu Ala Arg
Arg 245 250 255Glu Glu Trp
Arg Arg Trp Gly Gly Ala Asp His Leu Val Val Pro His 260
265 270His Pro Asn Ser Met Met Asp Ala Arg Arg
Arg Leu Ser Ala Ala Met 275 280
285Phe Val Leu Ser Asp Phe Gly Arg Tyr Pro Pro Asp Val Ala Asn Leu 290
295 300Arg Lys Asp Val Ile Ala Pro Tyr
Lys His Val Val Pro Ser Leu Gly305 310
315 320Asp Gly Asp Ser Pro Gly Phe Glu Gln Arg Pro Val
Leu Ala Tyr Phe 325 330
335Gln Gly Ala Ile His Arg Lys Asn Gly Gly Arg Val Arg Gln Arg Leu
340 345 350Tyr Gln Leu Ile Lys Asp
Glu Lys Asp Val His Phe Thr Tyr Gly Ser 355 360
365Val Arg Gln Asn Gly Ile Arg Arg Ala Thr Lys Gly Met Ala
Ser Ser 370 375 380Lys Phe Cys Leu Asn
Ile Ala Gly Asp Thr Pro Ser Ser Asn Arg Leu385 390
395 400Phe Asp Ala Ile Val Ser His Cys Val Pro
Val Ile Ile Ser Asp Asp 405 410
415Ile Glu Leu Pro Phe Glu Asp Val Leu Asp Tyr Ser Ala Phe Cys Val
420 425 430Phe Val Arg Ala Ser
Asp Ala Val Lys Arg Gly Phe Leu Leu His Leu 435
440 445Leu Arg Gly Ile Ser Gln Glu Glu Trp Thr Ala Met
Trp Arg Arg Leu 450 455 460Lys Glu Val
Ala His His Phe Glu Tyr Gln Tyr Pro Ser Gln Pro Gly465
470 475 480Asp Ala Val Gln Met Ile Trp
Gly Ala Val Ala Arg Lys Met His Leu 485
490 495Val Lys Leu Gln Leu His Lys Arg Gly Arg Tyr Gln
Arg Thr Phe Ser 500 505 510Glu
Ser241802DNAZea mays 24ccccctccca gctagccatg gccgcgcccc gctctggtcg
ccgcccgcac tgccactgct 60agccatggcc gccgccgccg cctgccggag cccgctggtc
tggctcttcg cgctcgtcac 120cgcactcttc ttcttctcct ggtacctcct cctcgactcc
gccgcgggtc cagccgccgc 180ccgccgcccc aaccagtggc tccgcctcgg cggcggcggg
cggcgctccg gtcccggtag 240gaaatgcgac cccgcggagg cgctgctgcg agtgttcatg
tacgacctgc cccccgagtt 300ccacttcgga ctgctcgact ggaagccccc cggcttcggc
ggcggcgtgt ggcccgacat 360cagggacggc gtgcctgact acccgggggg cctcaacctg
cagcacagca tcgagtattg 420gctcaccctc gacctcctgg cctccgagca gggcgcgccc
acgccctgcg cagtggcgcg 480ggtgcgccac gcggcggacg ccgacgtcgt cttcgtgccc
ttcttcgcct cgctcagctt 540caaccgccac tcccgggtgg taccgcccgc gcgggacagc
gaggaccgcg cgctgcagcg 600gaggctcctc gagttcctcg ccgcgcggcc cgagtggcgg
aggactggcg ggcgggacca 660cgtcgtgctc gcgcatcacc ccaacgggat gctcgacgcg
cgctacaggt tctggccctg 720cgtcttcgtg ctctgcgact tcgggaggta cccgcccagc
gtcgccaacc tcgacaagga 780cgtcatcgcg ccctaccggc acctcgtcgc caacttcgct
aatgacaccg ccggatacga 840cgaccggccg acattgctct acttccaagg cgccatctac
aggaaggacg gtggtttcat 900ccggcaagaa ctgtattacc ttctgaaaga cgagaaagat
gtgcatttct catttggaag 960cgtagccggt aatgggatcg agcaagcaac acaagggatg
cggtcatcca agttctgcct 1020caacattgca ggtgacactc catcctccaa ccgcctcttt
gactccatag tcagtcactg 1080tgttcccgtc acgatcagcg atgagattga gctcccgttt
gaggatgtcc tcgactactc 1140gaagttcagt gtcatagtac gtggcgcaga cgcagtcaag
aaggggtttc taatgaacct 1200gatcaaaggg atcagccgag aagagtggac acgcatgtgg
aacaggctaa aggaagtgga 1260aaagcacttt gagtaccaat acccatctca gaccgatgat
gccgtgcaga tgatatggaa 1320ggccattgct cggaaggtgc cgtctatccg gctgaagatt
aacagactgc aaagattttc 1380tctgtttgag actaacagga cagatgagac tctaccccca
tcttcttctt ggctacagaa 1440tcaggctcct tgattttgga ctagcaagct caggctttcg
ccatgttttc aatctcgtag 1500aatcaagagt tgatactgaa gaccaaatct taatcgcttg
actgggggca gatgttagta 1560tgttacagct gcagacatta gcggatagta aagttgaaac
actttagcat agcagaatta 1620catatccagt ggcacatcat tttcttcatt ttttttttct
tttgcttgcg attcatccaa 1680agtgtcctcg gctgcagact aaggacaatg aattttgtat
gtgaagctgt atattctagg 1740cggcaatata gttactgatc agttacagtt gctgagcaaa
aaaaaaaaaa aaaaaaaaaa 1800aa
180225462PRTZea mays 25Met Ala Ala Ala Ala Ala Cys
Arg Ser Pro Leu Val Trp Leu Phe Ala1 5 10
15Leu Val Thr Ala Leu Phe Phe Phe Ser Trp Tyr Leu Leu
Leu Asp Ser 20 25 30Ala Ala
Gly Pro Ala Ala Ala Arg Arg Pro Asn Gln Trp Leu Arg Leu 35
40 45Gly Gly Gly Gly Arg Arg Ser Gly Pro Gly
Arg Lys Cys Asp Pro Ala 50 55 60Glu
Ala Leu Leu Arg Val Phe Met Tyr Asp Leu Pro Pro Glu Phe His65
70 75 80Phe Gly Leu Leu Asp Trp
Lys Pro Pro Gly Phe Gly Gly Gly Val Trp 85
90 95Pro Asp Ile Arg Asp Gly Val Pro Asp Tyr Pro Gly
Gly Leu Asn Leu 100 105 110Gln
His Ser Ile Glu Tyr Trp Leu Thr Leu Asp Leu Leu Ala Ser Glu 115
120 125Gln Gly Ala Pro Thr Pro Cys Ala Val
Ala Arg Val Arg His Ala Ala 130 135
140Asp Ala Asp Val Val Phe Val Pro Phe Phe Ala Ser Leu Ser Phe Asn145
150 155 160Arg His Ser Arg
Val Val Pro Pro Ala Arg Asp Ser Glu Asp Arg Ala 165
170 175Leu Gln Arg Arg Leu Leu Glu Phe Leu Ala
Ala Arg Pro Glu Trp Arg 180 185
190Arg Thr Gly Gly Arg Asp His Val Val Leu Ala His His Pro Asn Gly
195 200 205Met Leu Asp Ala Arg Tyr Arg
Phe Trp Pro Cys Val Phe Val Leu Cys 210 215
220Asp Phe Gly Arg Tyr Pro Pro Ser Val Ala Asn Leu Asp Lys Asp
Val225 230 235 240Ile Ala
Pro Tyr Arg His Leu Val Ala Asn Phe Ala Asn Asp Thr Ala
245 250 255Gly Tyr Asp Asp Arg Pro Thr
Leu Leu Tyr Phe Gln Gly Ala Ile Tyr 260 265
270Arg Lys Asp Gly Gly Phe Ile Arg Gln Glu Leu Tyr Tyr Leu
Leu Lys 275 280 285Asp Glu Lys Asp
Val His Phe Ser Phe Gly Ser Val Ala Gly Asn Gly 290
295 300Ile Glu Gln Ala Thr Gln Gly Met Arg Ser Ser Lys
Phe Cys Leu Asn305 310 315
320Ile Ala Gly Asp Thr Pro Ser Ser Asn Arg Leu Phe Asp Ser Ile Val
325 330 335Ser His Cys Val Pro
Val Thr Ile Ser Asp Glu Ile Glu Leu Pro Phe 340
345 350Glu Asp Val Leu Asp Tyr Ser Lys Phe Ser Val Ile
Val Arg Gly Ala 355 360 365Asp Ala
Val Lys Lys Gly Phe Leu Met Asn Leu Ile Lys Gly Ile Ser 370
375 380Arg Glu Glu Trp Thr Arg Met Trp Asn Arg Leu
Lys Glu Val Glu Lys385 390 395
400His Phe Glu Tyr Gln Tyr Pro Ser Gln Thr Asp Asp Ala Val Gln Met
405 410 415Ile Trp Lys Ala
Ile Ala Arg Lys Val Pro Ser Ile Arg Leu Lys Ile 420
425 430Asn Arg Leu Gln Arg Phe Ser Leu Phe Glu Thr
Asn Arg Thr Asp Glu 435 440 445Thr
Leu Pro Pro Ser Ser Ser Trp Leu Gln Asn Gln Ala Pro 450
455 460261674DNAZea mays 26ctagcatctc ttttctcata
tgcagtacca catatatttc gttttacctc tattgagtat 60aaaatcttta aatcgtagag
tcttctaccg taactctatt tccctattta ctattgtacg 120acttttaaca actagcacta
catcgtcgac aaaaggtata caccaaggga tttcccctct 180acgccccctg tgaccttatt
catcacaaag gcgaaaaggt aagtgcttaa agttgatctt 240tgatgtagta ctgtcctaat
tgtgaagttg tatatgtcca tattacttgt tcgaacacta 300gtcacaacat tggtccttag
gagtatcttc atgaaggtgc ctcaaaataa tgctcccaaa 360attaaaatac tacaacataa
agtgtttagg gtactaaaaa aacaaatcga actccaccag 420ttaagcccta tatcatgtat
tttagaaaat aaagtatatt attagaaaac aaaaggttca 480gtatattgta gaaaaaatag
gacactaatc tagggtgtag tatatctgga tcactttatt 540gtctaaccta tgtttttttg
ttaaaaaatt tataaaacag gattacctgt ttttatgagt 600tgaacccaat atcttaaaac
aacacaatga ttaaaggctt tattggagat attctaatga 660gcccaatgta tttcattaaa
atttatgttt gtccaaatcc atttcataac attttttggt 720actttatcat aaaccttaat
caaatcaata aaaacatgtg tagatcatcc ttttgttctc 780tatactggtc tatcacttgt
cttattaaaa atgactttca tggttgatct tttaggtatg 840aaaccgattt ggttagaaga
gatcctcgct attccattga gggagggagc attcgacagg 900agctatatta tatgctcaaa
gaagaaaagg atgtttactt ttcctttgga agtgtccagg 960accatggggc cagcaaagct
agccaaggaa tgcactcatc aaaattttgc ctaaatattg 1020ctggggacac cccttcttcc
aatcgtctgt ttgatgcgat agttacccac tgtgtccctg 1080ttatcatcag tgacgacatt
gagctacctt atgaggatgt gttggattat tcaaaattct 1140ccatctttgt ccgttcgtct
gatgctgtta agaaaggtta cctgatgaga ctgctcagtg 1200gtgtaagcaa gcaacaatgg
acaaagatgt gggataggct caaagaggtg gataaacatt 1260ttgagtatca gtatccatca
cagaaggatg atgcagtcca gatgatctgg caagcattgt 1320ctagaaaggt gccatcaatt
aagctgaagg ttcacagatc taatagattt tcaagatcta 1380acagaggaaa ataaacagaa
aggggtgtgt ctatcttgtc tctattggct aatctaatgt 1440aacacatttc actgacacag
gctctcagcc tttcccagtt gcacaaaata gatagattgt 1500aatactcagg ttatctttag
gaaaggtttg taccttaaga tttgttggtt cagttgaagc 1560atatactgtc aagtcaaagg
ttgttttgta aggtatgtta aatgtaatca tgaaagaaag 1620acttggtttt gctgtttcaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1674271674PRTZea mays 27Cys
Thr Ala Gly Cys Ala Thr Cys Thr Cys Thr Thr Thr Thr Cys Thr1
5 10 15Cys Ala Thr Ala Thr Gly Cys
Ala Gly Thr Ala Cys Cys Ala Cys Ala 20 25
30Thr Ala Thr Ala Thr Thr Thr Cys Gly Thr Thr Thr Thr Ala
Cys Cys 35 40 45Thr Cys Thr Ala
Thr Thr Gly Ala Gly Thr Ala Thr Ala Ala Ala Ala 50 55
60Thr Cys Thr Thr Thr Ala Ala Ala Thr Cys Gly Thr Ala
Gly Ala Gly65 70 75
80Thr Cys Thr Thr Cys Thr Ala Cys Cys Gly Thr Ala Ala Cys Thr Cys
85 90 95Thr Ala Thr Thr Thr Cys
Cys Cys Thr Ala Thr Thr Thr Ala Cys Thr 100
105 110Ala Thr Thr Gly Thr Ala Cys Gly Ala Cys Thr Thr
Thr Thr Ala Ala 115 120 125Cys Ala
Ala Cys Thr Ala Gly Cys Ala Cys Thr Ala Cys Ala Thr Cys 130
135 140Gly Thr Cys Gly Ala Cys Ala Ala Ala Ala Gly
Gly Thr Ala Thr Ala145 150 155
160Cys Ala Cys Cys Ala Ala Gly Gly Gly Ala Thr Thr Thr Cys Cys Cys
165 170 175Cys Thr Cys Thr
Ala Cys Gly Cys Cys Cys Cys Cys Thr Gly Thr Gly 180
185 190Ala Cys Cys Thr Thr Ala Thr Thr Cys Ala Thr
Cys Ala Cys Ala Ala 195 200 205Ala
Gly Gly Cys Gly Ala Ala Ala Ala Gly Gly Thr Ala Ala Gly Thr 210
215 220Gly Cys Thr Thr Ala Ala Ala Gly Thr Thr
Gly Ala Thr Cys Thr Thr225 230 235
240Thr Gly Ala Thr Gly Thr Ala Gly Thr Ala Cys Thr Gly Thr Cys
Cys 245 250 255Thr Ala Ala
Thr Thr Gly Thr Gly Ala Ala Gly Thr Thr Gly Thr Ala 260
265 270Thr Ala Thr Gly Thr Cys Cys Ala Thr Ala
Thr Thr Ala Cys Thr Thr 275 280
285Gly Thr Thr Cys Gly Ala Ala Cys Ala Cys Thr Ala Gly Thr Cys Ala 290
295 300Cys Ala Ala Cys Ala Thr Thr Gly
Gly Thr Cys Cys Thr Thr Ala Gly305 310
315 320Gly Ala Gly Thr Ala Thr Cys Thr Thr Cys Ala Thr
Gly Ala Ala Gly 325 330
335Gly Thr Gly Cys Cys Thr Cys Ala Ala Ala Ala Thr Ala Ala Thr Gly
340 345 350Cys Thr Cys Cys Cys Ala
Ala Ala Ala Thr Thr Ala Ala Ala Ala Thr 355 360
365Ala Cys Thr Ala Cys Ala Ala Cys Ala Thr Ala Ala Ala Gly
Thr Gly 370 375 380Thr Thr Thr Ala Gly
Gly Gly Thr Ala Cys Thr Ala Ala Ala Ala Ala385 390
395 400Ala Ala Cys Ala Ala Ala Thr Cys Gly Ala
Ala Cys Thr Cys Cys Ala 405 410
415Cys Cys Ala Gly Thr Thr Ala Ala Gly Cys Cys Cys Thr Ala Thr Ala
420 425 430Thr Cys Ala Thr Gly
Thr Ala Thr Thr Thr Thr Ala Gly Ala Ala Ala 435
440 445Ala Thr Ala Ala Ala Gly Thr Ala Thr Ala Thr Thr
Ala Thr Thr Ala 450 455 460Gly Ala Ala
Ala Ala Cys Ala Ala Ala Ala Gly Gly Thr Thr Cys Ala465
470 475 480Gly Thr Ala Thr Ala Thr Thr
Gly Thr Ala Gly Ala Ala Ala Ala Ala 485
490 495Ala Thr Ala Gly Gly Ala Cys Ala Cys Thr Ala Ala
Thr Cys Thr Ala 500 505 510Gly
Gly Gly Thr Gly Thr Ala Gly Thr Ala Thr Ala Thr Cys Thr Gly 515
520 525Gly Ala Thr Cys Ala Cys Thr Thr Thr
Ala Thr Thr Gly Thr Cys Thr 530 535
540Ala Ala Cys Cys Thr Ala Thr Gly Thr Thr Thr Thr Thr Thr Thr Gly545
550 555 560Thr Thr Ala Ala
Ala Ala Ala Ala Thr Thr Thr Ala Thr Ala Ala Ala 565
570 575Ala Cys Ala Gly Gly Ala Thr Thr Ala Cys
Cys Thr Gly Thr Thr Thr 580 585
590Thr Thr Ala Thr Gly Ala Gly Thr Thr Gly Ala Ala Cys Cys Cys Ala
595 600 605Ala Thr Ala Thr Cys Thr Thr
Ala Ala Ala Ala Cys Ala Ala Cys Ala 610 615
620Cys Ala Ala Thr Gly Ala Thr Thr Ala Ala Ala Gly Gly Cys Thr
Thr625 630 635 640Thr Ala
Thr Thr Gly Gly Ala Gly Ala Thr Ala Thr Thr Cys Thr Ala
645 650 655Ala Thr Gly Ala Gly Cys Cys
Cys Ala Ala Thr Gly Thr Ala Thr Thr 660 665
670Thr Cys Ala Thr Thr Ala Ala Ala Ala Thr Thr Thr Ala Thr
Gly Thr 675 680 685Thr Thr Gly Thr
Cys Cys Ala Ala Ala Thr Cys Cys Ala Thr Thr Thr 690
695 700Cys Ala Thr Ala Ala Cys Ala Thr Thr Thr Thr Thr
Thr Gly Gly Thr705 710 715
720Ala Cys Thr Thr Thr Ala Thr Cys Ala Thr Ala Ala Ala Cys Cys Thr
725 730 735Thr Ala Ala Thr Cys
Ala Ala Ala Thr Cys Ala Ala Thr Ala Ala Ala 740
745 750Ala Ala Cys Ala Thr Gly Thr Gly Thr Ala Gly Ala
Thr Cys Ala Thr 755 760 765Cys Cys
Thr Thr Thr Thr Gly Thr Thr Cys Thr Cys Thr Ala Thr Ala 770
775 780Cys Thr Gly Gly Thr Cys Thr Ala Thr Cys Ala
Cys Thr Thr Gly Thr785 790 795
800Cys Thr Thr Ala Thr Thr Ala Ala Ala Ala Ala Thr Gly Ala Cys Thr
805 810 815Thr Thr Cys Ala
Thr Gly Gly Thr Thr Gly Ala Thr Cys Thr Thr Thr 820
825 830Thr Ala Gly Gly Thr Ala Thr Gly Ala Ala Ala
Cys Cys Gly Ala Thr 835 840 845Thr
Thr Gly Gly Thr Thr Ala Gly Ala Ala Gly Ala Gly Ala Thr Cys 850
855 860Cys Thr Cys Gly Cys Thr Ala Thr Thr Cys
Cys Ala Thr Thr Gly Ala865 870 875
880Gly Gly Gly Ala Gly Gly Gly Ala Gly Cys Ala Thr Thr Cys Gly
Ala 885 890 895Cys Ala Gly
Gly Ala Gly Cys Thr Ala Thr Ala Thr Thr Ala Thr Ala 900
905 910Thr Gly Cys Thr Cys Ala Ala Ala Gly Ala
Ala Gly Ala Ala Ala Ala 915 920
925Gly Gly Ala Thr Gly Thr Thr Thr Ala Cys Thr Thr Thr Thr Cys Cys 930
935 940Thr Thr Thr Gly Gly Ala Ala Gly
Thr Gly Thr Cys Cys Ala Gly Gly945 950
955 960Ala Cys Cys Ala Thr Gly Gly Gly Gly Cys Cys Ala
Gly Cys Ala Ala 965 970
975Ala Gly Cys Thr Ala Gly Cys Cys Ala Ala Gly Gly Ala Ala Thr Gly
980 985 990Cys Ala Cys Thr Cys Ala
Thr Cys Ala Ala Ala Ala Thr Thr Thr Thr 995 1000
1005Gly Cys Cys Thr Ala Ala Ala Thr Ala Thr Thr Gly
Cys Thr Gly 1010 1015 1020Gly Gly Gly
Ala Cys Ala Cys Cys Cys Cys Thr Thr Cys Thr Thr 1025
1030 1035Cys Cys Ala Ala Thr Cys Gly Thr Cys Thr Gly
Thr Thr Thr Gly 1040 1045 1050Ala Thr
Gly Cys Gly Ala Thr Ala Gly Thr Thr Ala Cys Cys Cys 1055
1060 1065Ala Cys Thr Gly Thr Gly Thr Cys Cys Cys
Thr Gly Thr Thr Ala 1070 1075 1080Thr
Cys Ala Thr Cys Ala Gly Thr Gly Ala Cys Gly Ala Cys Ala 1085
1090 1095Thr Thr Gly Ala Gly Cys Thr Ala Cys
Cys Thr Thr Ala Thr Gly 1100 1105
1110Ala Gly Gly Ala Thr Gly Thr Gly Thr Thr Gly Gly Ala Thr Thr
1115 1120 1125Ala Thr Thr Cys Ala Ala
Ala Ala Thr Thr Cys Thr Cys Cys Ala 1130 1135
1140Thr Cys Thr Thr Thr Gly Thr Cys Cys Gly Thr Thr Cys Gly
Thr 1145 1150 1155Cys Thr Gly Ala Thr
Gly Cys Thr Gly Thr Thr Ala Ala Gly Ala 1160 1165
1170Ala Ala Gly Gly Thr Thr Ala Cys Cys Thr Gly Ala Thr
Gly Ala 1175 1180 1185Gly Ala Cys Thr
Gly Cys Thr Cys Ala Gly Thr Gly Gly Thr Gly 1190
1195 1200Thr Ala Ala Gly Cys Ala Ala Gly Cys Ala Ala
Cys Ala Ala Thr 1205 1210 1215Gly Gly
Ala Cys Ala Ala Ala Gly Ala Thr Gly Thr Gly Gly Gly 1220
1225 1230Ala Thr Ala Gly Gly Cys Thr Cys Ala Ala
Ala Gly Ala Gly Gly 1235 1240 1245Thr
Gly Gly Ala Thr Ala Ala Ala Cys Ala Thr Thr Thr Thr Gly 1250
1255 1260Ala Gly Thr Ala Thr Cys Ala Gly Thr
Ala Thr Cys Cys Ala Thr 1265 1270
1275Cys Ala Cys Ala Gly Ala Ala Gly Gly Ala Thr Gly Ala Thr Gly
1280 1285 1290Cys Ala Gly Thr Cys Cys
Ala Gly Ala Thr Gly Ala Thr Cys Thr 1295 1300
1305Gly Gly Cys Ala Ala Gly Cys Ala Thr Thr Gly Thr Cys Thr
Ala 1310 1315 1320Gly Ala Ala Ala Gly
Gly Thr Gly Cys Cys Ala Thr Cys Ala Ala 1325 1330
1335Thr Thr Ala Ala Gly Cys Thr Gly Ala Ala Gly Gly Thr
Thr Cys 1340 1345 1350Ala Cys Ala Gly
Ala Thr Cys Thr Ala Ala Thr Ala Gly Ala Thr 1355
1360 1365Thr Thr Thr Cys Ala Ala Gly Ala Thr Cys Thr
Ala Ala Cys Ala 1370 1375 1380Gly Ala
Gly Gly Ala Ala Ala Ala Thr Ala Ala Ala Cys Ala Gly 1385
1390 1395Ala Ala Ala Gly Gly Gly Gly Thr Gly Thr
Gly Thr Cys Thr Ala 1400 1405 1410Thr
Cys Thr Thr Gly Thr Cys Thr Cys Thr Ala Thr Thr Gly Gly 1415
1420 1425Cys Thr Ala Ala Thr Cys Thr Ala Ala
Thr Gly Thr Ala Ala Cys 1430 1435
1440Ala Cys Ala Thr Thr Thr Cys Ala Cys Thr Gly Ala Cys Ala Cys
1445 1450 1455Ala Gly Gly Cys Thr Cys
Thr Cys Ala Gly Cys Cys Thr Thr Thr 1460 1465
1470Cys Cys Cys Ala Gly Thr Thr Gly Cys Ala Cys Ala Ala Ala
Ala 1475 1480 1485Thr Ala Gly Ala Thr
Ala Gly Ala Thr Thr Gly Thr Ala Ala Thr 1490 1495
1500Ala Cys Thr Cys Ala Gly Gly Thr Thr Ala Thr Cys Thr
Thr Thr 1505 1510 1515Ala Gly Gly Ala
Ala Ala Gly Gly Thr Thr Thr Gly Thr Ala Cys 1520
1525 1530Cys Thr Thr Ala Ala Gly Ala Thr Thr Thr Gly
Thr Thr Gly Gly 1535 1540 1545Thr Thr
Cys Ala Gly Thr Thr Gly Ala Ala Gly Cys Ala Thr Ala 1550
1555 1560Thr Ala Cys Thr Gly Thr Cys Ala Ala Gly
Thr Cys Ala Ala Ala 1565 1570 1575Gly
Gly Thr Thr Gly Thr Thr Thr Thr Gly Thr Ala Ala Gly Gly 1580
1585 1590Thr Ala Thr Gly Thr Thr Ala Ala Ala
Thr Gly Thr Ala Ala Thr 1595 1600
1605Cys Ala Thr Gly Ala Ala Ala Gly Ala Ala Ala Gly Ala Cys Thr
1610 1615 1620Thr Gly Gly Thr Thr Thr
Thr Gly Cys Thr Gly Thr Thr Thr Cys 1625 1630
1635Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
Ala 1640 1645 1650Ala Ala Ala Ala Ala
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1655 1660
1665Ala Ala Ala Ala Ala Ala 1670281893DNARye
28caccagatcg acgattcgcc atgctccccc gtgcaccact ccggccatga gcctcgcgct
60ccatggcgcc gccccgtgct cgcgccctac tcctgccgct ggcggcggcc accgtcctag
120tcgcctccac catcttcctc ttcgccgccg ccggcgccgg ccgctggcgc cccgccgaca
180ccggcctccc cgtcccggca acccccgccg acttctcggc cgtccctatc ggtgtgagtg
240taacatccac cgccaagggt aaagagctct cctttcttga tgagaatggc cgccccgacg
300accccagctc cggctcggcg gcggctgctg aacctgggag atgcgacccc cgcgacgccg
360ccgtcagggt gttcgtttac gacatgccgc cggagttcca cttcgggctg ctcggctggg
420cgccacctcc cgggaacggc ggcggcgtct ggcctgacgt caggggcggc acagtcccgc
480gctaccccgg tgggcttaac cagcagcaca gcgtggagta ctggctcacg ctggacctcc
540tggcatcctc gtcggcagca ccgtgcggtc cggctgtgag ggttgccgac tctcgtgatg
600cggacctgat cttcgtcccc ttcttcgcgt ccctcagtta caaccgccac tccaaggccg
660tgccgccgga gaaggtaagc agagacatgt acctacagga gaagctcgtc aggtatctgg
720tggcgcaacc ggagtggaag aggtccgggg gtgccgacca tgtcgtcgtc gcgcaccacc
780ccaacagctt actccacgcc cggtcggcgc tgttcccagc agtgttcgtg ctgtctgact
840tcgggaggta ccaccccagg gttgccagct tggagaagga tctcattgcg ccataccgac
900atatggcaaa gacatttgtg aatgacacgg ccgggtttga tgatcggccg acattgttat
960acttccgggg agccatttac agaaaggagg gaggaaacat tcggcaggaa ctatataata
1020tgctcaaaga tgagagggat gttttctttt ccttcggaag cgtccaggac catggtgtca
1080gcaaagccag ccagggaatg cactcatcaa agttttgcct aaacattgct ggggataccc
1140catcttccaa tcgtctcttt gacgctatag taagccactg tgtccctgtt atcataagtg
1200acgacattga gctcccttac gaggatatcc tagattattc aaagttctcc atctttgttc
1260ggtcgtctga tgctattaaa aagggttact tgatgagact gattaaaggc ataaacaagc
1320atcgatggac aaggatgtgg aagaggctca aagaagtgga taaacatttt gagtaccagt
1380tcccatctca taaggatgat gcggcccaga tgatctggca agcattggct aggaaggtgc
1440cttcgatccg actgaaggca cataggttta gaagatcttc aagatctgaa agaggaaaca
1500aataaacagg aaggaggtgt atgtattgat gatgtgctcc agatgatagg gcaagcagtg
1560gctaggaaag gtgccttgat tcggatgaag gcacatatgt ttagtggatt ttcaagatct
1620gaaagaggaa ccaagtaaac aggaaaagtg tgtatgtatc gtgtttctag gctaatctaa
1680cacagcattc actgacactg gcacgccagt tgcactttcc cttcgaggaa cattgtgaag
1740tagaagattc aggttgagtg ttggttatac cttggaacaa acacaggttg agtgttgagt
1800gttgaatgta tgttagcaag gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1860aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa
189329480PRTRye 29Met Ala Pro Pro Arg Ala Arg Ala Leu Leu Leu Pro Leu Ala
Ala Ala1 5 10 15Thr Val
Leu Val Ala Ser Thr Ile Phe Leu Phe Ala Ala Ala Gly Ala 20
25 30Gly Arg Trp Arg Pro Ala Asp Thr Gly
Leu Pro Val Pro Ala Thr Pro 35 40
45Ala Asp Phe Ser Ala Val Pro Ile Gly Val Ser Val Thr Ser Thr Ala 50
55 60Lys Gly Lys Glu Leu Ser Phe Leu Asp
Glu Asn Gly Arg Pro Asp Asp65 70 75
80Pro Ser Ser Gly Ser Ala Ala Ala Ala Glu Pro Gly Arg Cys
Asp Pro 85 90 95Arg Asp
Ala Ala Val Arg Val Phe Val Tyr Asp Met Pro Pro Glu Phe 100
105 110His Phe Gly Leu Leu Gly Trp Ala Pro
Pro Pro Gly Asn Gly Gly Gly 115 120
125Val Trp Pro Asp Val Arg Gly Gly Thr Val Pro Arg Tyr Pro Gly Gly
130 135 140Leu Asn Gln Gln His Ser Val
Glu Tyr Trp Leu Thr Leu Asp Leu Leu145 150
155 160Ala Ser Ser Ser Ala Ala Pro Cys Gly Pro Ala Val
Arg Val Ala Asp 165 170
175Ser Arg Asp Ala Asp Leu Ile Phe Val Pro Phe Phe Ala Ser Leu Ser
180 185 190Tyr Asn Arg His Ser Lys
Ala Val Pro Pro Glu Lys Val Ser Arg Asp 195 200
205Met Tyr Leu Gln Glu Lys Leu Val Arg Tyr Leu Val Ala Gln
Pro Glu 210 215 220Trp Lys Arg Ser Gly
Gly Ala Asp His Val Val Val Ala His His Pro225 230
235 240Asn Ser Leu Leu His Ala Arg Ser Ala Leu
Phe Pro Ala Val Phe Val 245 250
255Leu Ser Asp Phe Gly Arg Tyr His Pro Arg Val Ala Ser Leu Glu Lys
260 265 270Asp Leu Ile Ala Pro
Tyr Arg His Met Ala Lys Thr Phe Val Asn Asp 275
280 285Thr Ala Gly Phe Asp Asp Arg Pro Thr Leu Leu Tyr
Phe Arg Gly Ala 290 295 300Ile Tyr Arg
Lys Glu Gly Gly Asn Ile Arg Gln Glu Leu Tyr Asn Met305
310 315 320Leu Lys Asp Glu Arg Asp Val
Phe Phe Ser Phe Gly Ser Val Gln Asp 325
330 335His Gly Val Ser Lys Ala Ser Gln Gly Met His Ser
Ser Lys Phe Cys 340 345 350Leu
Asn Ile Ala Gly Asp Thr Pro Ser Ser Asn Arg Leu Phe Asp Ala 355
360 365Ile Val Ser His Cys Val Pro Val Ile
Ile Ser Asp Asp Ile Glu Leu 370 375
380Pro Tyr Glu Asp Ile Leu Asp Tyr Ser Lys Phe Ser Ile Phe Val Arg385
390 395 400Ser Ser Asp Ala
Ile Lys Lys Gly Tyr Leu Met Arg Leu Ile Lys Gly 405
410 415Ile Asn Lys His Arg Trp Thr Arg Met Trp
Lys Arg Leu Lys Glu Val 420 425
430Asp Lys His Phe Glu Tyr Gln Phe Pro Ser His Lys Asp Asp Ala Ala
435 440 445Gln Met Ile Trp Gln Ala Leu
Ala Arg Lys Val Pro Ser Ile Arg Leu 450 455
460Lys Ala His Arg Phe Arg Arg Ser Ser Arg Ser Glu Arg Gly Asn
Lys465 470 475
480301869DNAZea mays 30agctaccgct ccgttcttgt acgatggttc ctgaggatgc
aaaatggtga gttgaatttg 60attcacctgc gtgctgtttt gctgcatggt ggtggtggag
aggaagatgc agccattgcc 120gccgcctgag cgccggaggg tcgttcgttt cgtggtcttc
atggccgtct ccctcctggc 180cctgttctgc tgggctctcg tcaattccag gatcaacgtc
gccatgcctt actctgcttt 240cgtgatgcgc gatgtcgaca agacgcccgc attcacaggc
ctagaagaca ggcagaggca 300ccccgccggc gacccagcgt ggacctcggc ggcgccgcag
gccgtgccgg tgaccagtaa 360cgtcacggcg ggctcggtga aattgggtga tccggtgctc
cgggagccgc tggcaggaga 420agcggaacgg gaacggagcg agaggtgcga cgcggacagc
gcggcgctca gggtgtatat 480gtacgacctg ccggcggagt tccatttcgg catgctcggg
tgggaacgga aggggaagct 540ggcgtggccc gacgtccgcg acgcccacgc cgcgccgcac
taccccggcg ggctcaacct 600gcagcacagc gtggcgtact ggctcacgct ggacatcctg
tcctccgccc tgccgcccgg 660cagcgacgtg gtcagagaca ggccctgtgt cgccgtcagg
gtgacgaacg cgagcctcgc 720caacgtcttc ttcgtgccgt tcttcgcgtc actgagctac
aaccgccact cgaagctccg 780ccgcggggag agggtgagca ggaacagggt cctgcaggcc
gagctggtca agtacctgat 840gcggaaggag gagtggagga ggtggggcgg caagaaccac
ctcatcgtgc cgcaccaccc 900caacagcttg atggaggcac ggaagaagct cagcgccgcc
atgttcgtgt tgtctgactt 960cgggaggtac tcgccggacg ttgccaacct caagaaggac
gtcatcgcac cgtacaagca 1020cgtcctccgc tccttaggcg atggcgactc gccatcgttc
gagcaacgtc ccatcctcgc 1080atacttccaa ggggccatcc atcggaaagc tggcgggaag
gttcgccaga agctgtacca 1140tctgctcaag gacgagcgcg acgtgcactt cacctacggc
agcgtccggc agaacggcat 1200ccggcgcgcc accgccggga tgtccacgtc gaagttctgc
ctcaacatcg ccggcgacac 1260gccgtcctcg aaccggctct tcgacgccat cgtcagccac
tgtgtcccgg tcatcatcag 1320cgacgacatc gagttgccat ttgaggacat gctcgactac
tcggagttct gcgtgttcgt 1380gcgctccgcc gacgccgcca agaagggatt cctactacgg
ctgctacggg gcatatcgcg 1440cgaggagtgg accaagatgt ggatgagatt gaagaaggtg
actcaccatt tcgagtacca 1500gtacccttca cggtcaggtg atgctgtcca gatgacatgg
agcgcagtgg cgcggaagat 1560gcattcggtg cagctgcagc ttcacaagcg cgctagattc
cacaggacgg tttctgtatg 1620aatatgatca aggccaccga gcgcggaagt ctggatgtgg
caagattcgt cagttgcact 1680gtgcagatga tttttttaga cttaactgac ataaaggtaa
taacgggtga ataggtgtca 1740tttttgtaat gtttacttcc ctaaaagaaa tagagatgtg
agacttgaaa gcataaaatg 1800cactatctta atctagtatt aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1860aaaaaaaaa
186931511PRTZea mays 31Met Val Val Val Glu Arg Lys
Met Gln Pro Leu Pro Pro Pro Glu Arg1 5 10
15Arg Arg Val Val Arg Phe Val Val Phe Met Ala Val Ser
Leu Leu Ala 20 25 30Leu Phe
Cys Trp Ala Leu Val Asn Ser Arg Ile Asn Val Ala Met Pro 35
40 45Tyr Ser Ala Phe Val Met Arg Asp Val Asp
Lys Thr Pro Ala Phe Thr 50 55 60Gly
Leu Glu Asp Arg Gln Arg His Pro Ala Gly Asp Pro Ala Trp Thr65
70 75 80Ser Ala Ala Pro Gln Ala
Val Pro Val Thr Ser Asn Val Thr Ala Gly 85
90 95Ser Val Lys Leu Gly Asp Pro Val Leu Arg Glu Pro
Leu Ala Gly Glu 100 105 110Ala
Glu Arg Glu Arg Ser Glu Arg Cys Asp Ala Asp Ser Ala Ala Leu 115
120 125Arg Val Tyr Met Tyr Asp Leu Pro Ala
Glu Phe His Phe Gly Met Leu 130 135
140Gly Trp Glu Arg Lys Gly Lys Leu Ala Trp Pro Asp Val Arg Asp Ala145
150 155 160His Ala Ala Pro
His Tyr Pro Gly Gly Leu Asn Leu Gln His Ser Val 165
170 175Ala Tyr Trp Leu Thr Leu Asp Ile Leu Ser
Ser Ala Leu Pro Pro Gly 180 185
190Ser Asp Val Val Arg Asp Arg Pro Cys Val Ala Val Arg Val Thr Asn
195 200 205Ala Ser Leu Ala Asn Val Phe
Phe Val Pro Phe Phe Ala Ser Leu Ser 210 215
220Tyr Asn Arg His Ser Lys Leu Arg Arg Gly Glu Arg Val Ser Arg
Asn225 230 235 240Arg Val
Leu Gln Ala Glu Leu Val Lys Tyr Leu Met Arg Lys Glu Glu
245 250 255Trp Arg Arg Trp Gly Gly Lys
Asn His Leu Ile Val Pro His His Pro 260 265
270Asn Ser Leu Met Glu Ala Arg Lys Lys Leu Ser Ala Ala Met
Phe Val 275 280 285Leu Ser Asp Phe
Gly Arg Tyr Ser Pro Asp Val Ala Asn Leu Lys Lys 290
295 300Asp Val Ile Ala Pro Tyr Lys His Val Leu Arg Ser
Leu Gly Asp Gly305 310 315
320Asp Ser Pro Ser Phe Glu Gln Arg Pro Ile Leu Ala Tyr Phe Gln Gly
325 330 335Ala Ile His Arg Lys
Ala Gly Gly Lys Val Arg Gln Lys Leu Tyr His 340
345 350Leu Leu Lys Asp Glu Arg Asp Val His Phe Thr Tyr
Gly Ser Val Arg 355 360 365Gln Asn
Gly Ile Arg Arg Ala Thr Ala Gly Met Ser Thr Ser Lys Phe 370
375 380Cys Leu Asn Ile Ala Gly Asp Thr Pro Ser Ser
Asn Arg Leu Phe Asp385 390 395
400Ala Ile Val Ser His Cys Val Pro Val Ile Ile Ser Asp Asp Ile Glu
405 410 415Leu Pro Phe Glu
Asp Met Leu Asp Tyr Ser Glu Phe Cys Val Phe Val 420
425 430Arg Ser Ala Asp Ala Ala Lys Lys Gly Phe Leu
Leu Arg Leu Leu Arg 435 440 445Gly
Ile Ser Arg Glu Glu Trp Thr Lys Met Trp Met Arg Leu Lys Lys 450
455 460Val Thr His His Phe Glu Tyr Gln Tyr Pro
Ser Arg Ser Gly Asp Ala465 470 475
480Val Gln Met Thr Trp Ser Ala Val Ala Arg Lys Met His Ser Val
Gln 485 490 495Leu Gln Leu
His Lys Arg Ala Arg Phe His Arg Thr Val Ser Val 500
505 510321744DNAArabidopsis thaliana 32cttcggcgtt
ttcatcgttt ctcatctacc tcgaaatttt ctcgtcactc taatcaaaaa 60tctcacttcg
ttattttctc gggaaaaatt tcccgatcac taaacctagt ttttagatat 120tttctctcct
ggattctgga tctacacaat ctttcttcct cttcttctat gaatctgttc 180atcgtaattt
cagattcttg atatatgatt ccttttctcc aatctcttct cttagctctc 240agagatgggt
gagaaaacaa actcacgtta tctcggtgta atcatcactc gaaaatctat 300aattttcttg
ttcatatcaa tcatcaccgt cctttcttgg ttttttatct tttcttccac 360aaaccctaac
cgagttctcg atcacatctc agtatcagaa tccacagatg tacctctcat 420catcatcaag
aactcaaaca gttctccaca aaacaacgca ccaaaacccc aaaacagaga 480aggagcagaa
acagaggaac ccattaaaga aaacagagga ggaacaaaaa cagagtcatc 540catgaatcaa
aacagaggcg aaaccctccg gtgtatccaa agggtttctc cttctccaag 600gccattgaaa
gtctacatgt atgatatgag tccagagttt cattttgggt tattgggttg 660gaaaccagag
agaaacggtg tcgtttggcc tgatatcaga gtcaatgttc ctcaccatcc 720aggtggtctt
aacttgcagc acagtgttga gtattggctc acattagatc ttttgttctc 780tgagcttcca
gaagattcta gaagctctcg ggccgcgata cgtgtaaaga actcgagcga 840agctgatgtc
gtgttcgtgc ccttcttctc ttcattgagc tataaccgat tctctaaggt 900taaccaaaag
cagaagaaga gccaggacaa agagttgcag gaaaatgtgg tgaaatacgt 960aacgtcccaa
aaagagtgga agacctcagg agggaaggat catgtgatca tggcgcatca 1020tccgaatagt
atgtcgacgg caaggcataa gctatttccg gcgatgtttg tggtcgctga 1080ctttggtaga
tactcgccac atgttgccaa tgttgacaaa gacattgtgg ctccatacaa 1140acaccttgtt
ccatcgtatg ttaatgacac atcgggcttt gatggccgtc cgatcttgct 1200ctacttccaa
ggagccatct accgcaaagc tggtggattt gtgagacaag agctatataa 1260tcttctcaaa
gaagaaaaag acgtccactt ctctttcgga agcgtaagga accacggcat 1320atctaaagcc
ggcgaaggaa tgagatcgtc caagttctgt ctcaacatag ccggggatac 1380accatcctcg
aatcgcctct tcgacgccat agctagtcac tgtatacccg tgatcattag 1440cgatgacatc
gagttaccat atgaggatgt cctcaactac aatgagttct gtctctttgt 1500cagatcatca
gacgctttaa agaaagggtt tctgatgggt cttgtcagga gtattggcag 1560agaagagtat
aataagatgt ggcttcggtt gaaggaagtg gagaggtatt tcgatttgcg 1620ttttccggtg
aaggatgacg agggagatta tgcagttcag atgatttgga aagctgttgc 1680caggaaagct
cctttggtga agatgaaggt tcacagattt cagaggttta caaggccttt 1740ttag
1744331500DNAArabidopsis thaliana 33atgggtgaga aaacaaactc acgttatctc
ggtgtaatca tcactcgaaa atctataatt 60ttcttgttca tatcaatcat caccgtcctt
tcttggtttt ttatcttttc ttccacaaac 120cctaaccgag ttctcgatca catctcagta
tcagaatcca cagatgtacc tctcatcatc 180atcaagaact caaacagttc tccacaaaac
aacgcaccaa aaccccaaaa cagagaagga 240gcagaaacag aggaacccat taaagaaaac
agaggaggaa caaaaacaga gtcatccatg 300aatcaaaaca gaggcgaaac cctccggtgt
atccaaaggg tttctccttc tccaaggcca 360ttgaaagtct acatgtatga tatgagtcca
gagtttcatt ttgggttatt gggttggaaa 420ccagagagaa acggtgtcgt ttggcctgat
atcagagtca atgttcctca ccatccaggt 480ggtcttaact tgcagcacag tgttgagtat
tggctcacat tagatctttt gttctctgag 540cttccagaag attctagaag ctctcgggcc
gcgatacgtg taaagaactc gagcgaagct 600gatgtcgtgt tcgtgccctt cttctcttca
ttgagctata accgattctc taaggttaac 660caaaagcaga agaagagcca ggacaaagag
ttgcaggaaa atgtggtgaa atacgtaacg 720tcccaaaaag agtggaagac ctcaggaggg
aaggatcatg tgatcatggc gcatcatccg 780aatagtatgt cgacggcaag gcataagcta
tttccggcga tgtttgtggt cgctgacttt 840ggtagatact cgccacatgt tgccaatgtt
gacaaagaca ttgtggctcc atacaaacac 900cttgttccat cgtatgttaa tgacacatcg
ggctttgatg gccgtccgat cttgctctac 960ttccaaggag ccatctaccg caaagctggt
ggatttgtga gacaagagct atataatctt 1020ctcaaagaag aaaaagacgt ccacttctct
ttcggaagcg taaggaacca cggcatatct 1080aaagccggcg aaggaatgag atcgtccaag
ttctgtctca acatagccgg ggatacacca 1140tcctcgaatc gcctcttcga cgccatagct
agtcactgta tacccgtgat cattagcgat 1200gacatcgagt taccatatga ggatgtcctc
aactacaatg agttctgtct ctttgtcaga 1260tcatcagacg ctttaaagaa agggtttctg
atgggtcttg tcaggagtat tggcagagaa 1320gagtataata agatgtggct tcggttgaag
gaagtggaga ggtatttcga tttgcgtttt 1380ccggtgaagg atgacgaggg agattatgca
gttcagatga tttggaaagc tgttgccagg 1440aaagctcctt tggtgaagat gaaggttcac
agatttcaga ggtttacaag gcctttttag 150034499PRTArabidopsis thaliana 34Met
Gly Glu Lys Thr Asn Ser Arg Tyr Leu Gly Val Ile Ile Thr Arg1
5 10 15Lys Ser Ile Ile Phe Leu Phe
Ile Ser Ile Ile Thr Val Leu Ser Trp 20 25
30Phe Phe Ile Phe Ser Ser Thr Asn Pro Asn Arg Val Leu Asp
His Ile 35 40 45Ser Val Ser Glu
Ser Thr Asp Val Pro Leu Ile Ile Ile Lys Asn Ser 50 55
60Asn Ser Ser Pro Gln Asn Asn Ala Pro Lys Pro Gln Asn
Arg Glu Gly65 70 75
80Ala Glu Thr Glu Glu Pro Ile Lys Glu Asn Arg Gly Gly Thr Lys Thr
85 90 95Glu Ser Ser Met Asn Gln
Asn Arg Gly Glu Thr Leu Arg Cys Ile Gln 100
105 110Arg Val Ser Pro Ser Pro Arg Pro Leu Lys Val Tyr
Met Tyr Asp Met 115 120 125Ser Pro
Glu Phe His Phe Gly Leu Leu Gly Trp Lys Pro Glu Arg Asn 130
135 140Gly Val Val Trp Pro Asp Ile Arg Val Asn Val
Pro His His Pro Gly145 150 155
160Gly Leu Asn Leu Gln His Ser Val Glu Tyr Trp Leu Thr Leu Asp Leu
165 170 175Leu Phe Ser Glu
Leu Pro Glu Asp Ser Arg Ser Ser Arg Ala Ala Ile 180
185 190Arg Val Lys Asn Ser Ser Glu Ala Asp Val Val
Phe Val Pro Phe Phe 195 200 205Ser
Ser Leu Ser Tyr Asn Arg Phe Ser Lys Val Asn Gln Lys Gln Lys 210
215 220Lys Ser Gln Asp Lys Glu Leu Gln Glu Asn
Val Val Lys Tyr Val Thr225 230 235
240Ser Gln Lys Glu Trp Lys Thr Ser Gly Gly Lys Asp His Val Ile
Met 245 250 255Ala His His
Pro Asn Ser Met Ser Thr Ala Arg His Lys Leu Phe Pro 260
265 270Ala Met Phe Val Val Ala Asp Phe Gly Arg
Tyr Ser Pro His Val Ala 275 280
285Asn Val Asp Lys Asp Ile Val Ala Pro Tyr Lys His Leu Val Pro Ser 290
295 300Tyr Val Asn Asp Thr Ser Gly Phe
Asp Gly Arg Pro Ile Leu Leu Tyr305 310
315 320Phe Gln Gly Ala Ile Tyr Arg Lys Ala Gly Gly Phe
Val Arg Gln Glu 325 330
335Leu Tyr Asn Leu Leu Lys Glu Glu Lys Asp Val His Phe Ser Phe Gly
340 345 350Ser Val Arg Asn His Gly
Ile Ser Lys Ala Gly Glu Gly Met Arg Ser 355 360
365Ser Lys Phe Cys Leu Asn Ile Ala Gly Asp Thr Pro Ser Ser
Asn Arg 370 375 380Leu Phe Asp Ala Ile
Ala Ser His Cys Ile Pro Val Ile Ile Ser Asp385 390
395 400Asp Ile Glu Leu Pro Tyr Glu Asp Val Leu
Asn Tyr Asn Glu Phe Cys 405 410
415Leu Phe Val Arg Ser Ser Asp Ala Leu Lys Lys Gly Phe Leu Met Gly
420 425 430Leu Val Arg Ser Ile
Gly Arg Glu Glu Tyr Asn Lys Met Trp Leu Arg 435
440 445Leu Lys Glu Val Glu Arg Tyr Phe Asp Leu Arg Phe
Pro Val Lys Asp 450 455 460Asp Glu Gly
Asp Tyr Ala Val Gln Met Ile Trp Lys Ala Val Ala Arg465
470 475 480Lys Ala Pro Leu Val Lys Met
Lys Val His Arg Phe Gln Arg Phe Thr 485
490 495Arg Pro Phe35566PRTOryza sativa 35Met Ala Leu Thr
Arg Arg Leu Leu Ile Asp Leu Ser Ser Arg Arg Arg1 5
10 15Leu Phe Asn Ala Gly Lys Phe Ser Thr Thr
His Lys Lys Lys Pro Val 20 25
30Leu His Glu Ala Val Ser Leu Ala Gly Phe Leu Arg Cys Ser Arg Ala
35 40 45Leu Val Ser Trp Met Val Ala Glu
Arg Lys Met Gln Pro Ser Pro Ala 50 55
60Ala Pro Pro Ala Ala Glu His Arg Arg Arg Ala Leu Leu Arg Tyr Val65
70 75 80Val Phe Leu Ala Val
Ser Leu Leu Ala Phe Ser Cys Trp Ala Leu Val 85
90 95Ser Ser Arg Ile Asp Gly Ala Val Leu Ala Ala
Thr Ala Gly Gly Glu 100 105
110His Asp Asp His Asp Gly Ile Ile Val Arg Ser Ser Thr Gln Ala Glu
115 120 125Met Pro Ala Arg Gly Gly Asn
Ala Thr Ser Arg Gly Ala Val Glu Val 130 135
140Gly Val Gly Thr Pro Ala Ala Met Ile Thr Arg Gln Pro Ser Ser
Gly145 150 155 160Glu Thr
Thr Thr Thr Ala Ala Leu Ala Ala Thr Cys Asp Ala Glu Ser
165 170 175Ala Leu Leu Arg Val Tyr Leu
Tyr Asp Leu Pro Pro Glu Phe His Phe 180 185
190Gly Met Leu Gly Trp Asp Gly Lys Ala Ala Gly Ala Ala Trp
Pro Asp 195 200 205Val Ala Gly Asp
Pro Arg Ala Val Pro Arg Tyr Pro Gly Gly Leu Asn 210
215 220Leu Gln His Ser Val Glu Tyr Trp Leu Thr Leu Asp
Ile Leu Ser Ser225 230 235
240Thr Thr Ser Gly Asp His Arg Arg Arg Arg Pro Cys Thr Ala Val Arg
245 250 255Val Thr Asn Ala Ser
Leu Ala Asp Val Phe Leu Val Pro Phe Phe Ala 260
265 270Ser Leu Ser Tyr Asn Arg Gln Ser Lys Ser Pro His
Gly Gly His Gly 275 280 285Ser Gly
Gly Arg Ser Asp Arg Gln Leu Gln Gly Glu Leu Val Arg Tyr 290
295 300Leu Ala Arg Arg Glu Glu Trp Arg Arg Trp Gly
Gly Ala Asp His Leu305 310 315
320Val Val Pro His His Pro Asn Ser Met Met Asp Ala Arg Arg Arg Leu
325 330 335Ser Ala Ala Met
Phe Val Leu Ser Asp Phe Gly Arg Tyr Pro Pro Asp 340
345 350Val Ala Asn Leu Arg Lys Asp Val Ile Ala Pro
Tyr Lys His Val Val 355 360 365Pro
Ser Leu Gly Asp Gly Asp Ser Pro Gly Phe Glu Gln Arg Pro Val 370
375 380Leu Ala Tyr Phe Gln Gly Ala Ile His Arg
Lys Asn Gly Gly Arg Val385 390 395
400Arg Gln Arg Leu Tyr Gln Leu Ile Lys Asp Glu Lys Asp Val His
Phe 405 410 415Thr Tyr Gly
Ser Val Arg Gln Asn Gly Ile Arg Arg Ala Thr Lys Gly 420
425 430Met Ala Ser Ser Lys Phe Cys Leu Asn Ile
Ala Gly Asp Thr Pro Ser 435 440
445Ser Asn Arg Leu Phe Asp Ala Ile Val Ser His Cys Val Pro Val Ile 450
455 460Ile Ser Asp Asp Ile Glu Leu Pro
Phe Glu Asp Val Leu Asp Tyr Ser465 470
475 480Ala Phe Cys Val Phe Val Arg Ala Ser Asp Ala Val
Lys Arg Gly Phe 485 490
495Leu Leu His Leu Leu Arg Gly Ile Ser Gln Glu Glu Trp Thr Ala Met
500 505 510Trp Arg Arg Leu Lys Glu
Val Ala His His Phe Glu Tyr Gln Tyr Pro 515 520
525Ser Gln Pro Gly Asp Ala Val Gln Met Ile Trp Gly Ala Val
Ala Arg 530 535 540Lys Met His Leu Val
Lys Leu Gln Leu His Lys Arg Gly Arg Tyr Gln545 550
555 560Arg Thr Phe Ser Glu Ser
56536475PRTOryza sativa 36Met Pro Pro Arg Ala Arg Thr Leu Leu Met Pro Leu
Ala Ala Ala Thr1 5 10
15Leu Leu Val Ala Ser Thr Ile Phe Leu Phe Ala Ala Thr Gly Ala Arg
20 25 30Trp Arg Pro Ala Asp Thr Gly
Leu Pro Val Pro Ala Ala Asp Phe Ser 35 40
45Ala Ala Val Leu Glu Ser Ala Val Thr Asp Thr Thr Ala Ala Ala
Lys 50 55 60Glu Leu Ser Phe Val Asp
Glu Asn Gly Arg Pro Asp Asp Pro Ala Ser65 70
75 80Ser Ser Ala Ala Ala Ala Arg Cys Asp Pro Thr
His Ala Ala Val Arg 85 90
95Val Phe Met Tyr Asp Leu Pro Pro Glu Phe His Phe Gly Ile Leu Gly
100 105 110Trp Ser Pro Pro Thr Asp
Gly Ala Ala Asp Ala Ala Met Trp Pro Asp 115 120
125Val Gly Ser Gly Ala Ala Ala Pro Arg Tyr Pro Gly Gly Leu
Asn Gln 130 135 140Gln His Ser Val Glu
Tyr Trp Leu Thr Leu Asp Leu Leu Ser Ser Ser145 150
155 160Ser Pro Pro Cys Gly Ala Ala Val Arg Val
Ala Asp Ser Arg Asp Ala 165 170
175Asp Val Val Phe Val Pro Phe Phe Ala Ser Leu Ser Tyr Asn Arg His
180 185 190Ser Arg Val Val Pro
Pro Glu Lys Val Ser Arg Asp Lys Glu Leu Gln 195
200 205Glu Lys Leu Val Arg Tyr Leu Met Ala Gln Pro Glu
Trp Lys Arg Ser 210 215 220Gly Gly Ala
Asp His Val Ile Val Ala His His Pro Asn Ser Leu Leu225
230 235 240His Ala Arg Ser Val Leu Phe
Pro Val Val Phe Val Leu Ser Asp Phe 245
250 255Gly Arg Tyr His Pro Arg Val Ala Ser Leu Glu Lys
Asp Val Ile Ala 260 265 270Pro
Tyr Lys His Met Ala Lys Thr Phe Val Asn Asp Ser Ala Gly Phe 275
280 285Asp Asp Arg Pro Thr Leu Leu Tyr Phe
Arg Gly Ala Ile Phe Arg Lys 290 295
300Glu Gly Gly Asn Ile Arg Gln Glu Leu Tyr Tyr Met Leu Lys Asp Glu305
310 315 320Lys Asp Val Tyr
Phe Ala Phe Gly Ser Val Gln Asp His Gly Ala Ser 325
330 335Lys Ala Ser Lys Gly Met His Ala Ser Lys
Phe Cys Leu Asn Ile Ala 340 345
350Gly Asp Thr Pro Ser Ser Asn Arg Leu Phe Asp Ala Ile Val Ser His
355 360 365Cys Val Pro Val Ile Ile Ser
Asp Asp Ile Glu Leu Pro Tyr Glu Asp 370 375
380Ala Leu Asp Tyr Ser Lys Phe Ser Ile Phe Val Arg Ser Ser Asp
Ala385 390 395 400Val Lys
Lys Gly Tyr Leu Met Arg Leu Ile Arg Gly Val Ser Lys His
405 410 415Gln Trp Thr Arg Met Trp Asn
Arg Leu Lys Glu Val Asp Lys His Phe 420 425
430Glu Tyr Gln Tyr Pro Ser Gln Lys Asp Asp Ala Val Gln Met
Ile Trp 435 440 445Gln Ala Leu Ala
Arg Lys Val Pro Ala Ile Arg Leu Lys Ser His Arg 450
455 460Ser Arg Arg Phe Ser Arg Tyr Asp Arg Gly Lys465
470 47537468PRTOryza sativa 37Met Ala Ala Ala
Ala Ala Ser Ala Ser Ala Ser Cys Arg Arg Arg Pro1 5
10 15Ile Ala Trp Phe Phe Ala Ile Ala Ala Leu
Leu Phe Phe Phe Ser Trp 20 25
30Tyr Leu Leu Leu Asp Ser Ala Ala Val Thr Pro Glu Pro Leu Leu Ala
35 40 45Ala Arg Gly Gln Gly Leu Arg Val
Gly Ser Ser Gly Arg Lys Cys Asp 50 55
60Pro Ala Thr Ala Ala Leu Arg Val Phe Met Tyr Asp Leu Pro Ala Glu65
70 75 80Phe His Phe Gly Leu
Leu Asp Trp Glu Pro Gln Gly Gly Gly Gly Gly 85
90 95Gly Gly Gly Gly Val Trp Pro Asp Val Arg Gly
Gly Gly Val Pro Glu 100 105
110Tyr Pro Gly Gly Leu Asn Leu Gln His Ser Ile Glu Tyr Trp Leu Thr
115 120 125Leu Asp Leu Leu Ala Ser Glu
Gln Gly Ala Pro Thr Pro Cys Gly Ala 130 135
140Val Arg Val Arg His Ala Ala Ala Ala Asp Val Val Phe Val Pro
Phe145 150 155 160Phe Ala
Ser Leu Ser Phe Asn Arg His Ser Lys Val Val Pro Pro Ala
165 170 175Arg Ala Ser Glu Asp Arg Ala
Leu Gln Arg Arg Leu Leu Asp Tyr Leu 180 185
190Ala Ala Arg Pro Glu Trp Arg Arg Ser Gly Gly Arg Asp His
Val Val 195 200 205Leu Ala His His
Pro Asn Gly Met Leu Asp Ala Arg Tyr Lys Leu Trp 210
215 220Pro Cys Val Phe Val Leu Cys Asp Phe Gly Arg Tyr
Pro Pro Ser Val225 230 235
240Ala Gly Leu Asp Lys Asp Val Ile Ala Pro Tyr Arg His Val Val Pro
245 250 255Asn Phe Ala Asn Asp
Ser Ala Gly Tyr Asp Asp Arg Pro Thr Leu Leu 260
265 270Tyr Phe Gln Gly Ala Ile Tyr Arg Lys Asp Gly Gly
Phe Ile Arg Gln 275 280 285Glu Leu
Tyr Tyr Leu Leu Lys Asp Glu Lys Asp Val His Phe Ser Phe 290
295 300Gly Ser Val Val Gly Asn Gly Ile Glu Gln Ala
Thr Gln Gly Met Arg305 310 315
320Ala Ser Lys Phe Cys Leu Asn Ile Ala Gly Asp Thr Pro Ser Ser Asn
325 330 335Arg Leu Phe Asp
Ser Ile Val Ser His Cys Val Pro Ile Ile Ile Ser 340
345 350Asp Glu Ile Glu Leu Pro Phe Glu Asp Val Leu
Asp Tyr Ser Lys Phe 355 360 365Cys
Ile Ile Val Arg Gly Ala Asp Ala Val Lys Lys Gly Phe Leu Met 370
375 380Asn Leu Ile Asn Gly Ile Ser Arg Glu Asp
Trp Thr Arg Met Trp Asn385 390 395
400Arg Leu Lys Glu Val Glu Arg His Phe Glu Tyr Gln Tyr Pro Ser
Gln 405 410 415Asn Asp Asp
Ala Val Gln Met Ile Trp Lys Ala Ile Ala Arg Lys Ala 420
425 430Pro Ser Ile Arg Leu Lys Val Asn Arg Leu
Arg Arg Phe Ser Arg Phe 435 440
445Glu Thr Asn Arg Thr Asp Glu Thr Pro Thr Arg Ser Ser Trp Leu Glu 450
455 460Asn Gln Pro Ser46538437PRTOryza
sativa 38Met Ala Ser Pro Ser Ser Arg Ala Val Ala Val Gly Gly Ala Leu Leu1
5 10 15Leu Leu Leu Val
Phe Ala Val Pro Thr Thr Phe Leu Tyr Leu Thr Ser 20
25 30Ala Pro Ala Ala Ser Ser Pro Ser Leu Leu Leu
Asn Leu Lys Pro Phe 35 40 45Gly
Ala Arg Cys Ala Pro Ala Ala Ala Ala Ala Pro Pro Leu Arg Val 50
55 60Phe Met Tyr Asp Leu Pro Arg Arg Phe His
Val Gly Met Met Asp Ala65 70 75
80Ser Ala Ser Gly Phe Pro Ala Trp Pro Pro Ser Ala Gly Gly Ile
Arg 85 90 95Arg Gln His
Ser Val Glu Tyr Trp Met Met Ala Ser Leu Gln Gly Gly 100
105 110Gly Gly Gly Gly Asn Gly Ser Ser Ser Glu
Glu Gly Arg Glu Ala Val 115 120
125Arg Val Thr Asp Pro Asp Ala Ala Glu Ala Phe Phe Val Pro Phe Phe 130
135 140Ser Ser Leu Ser Phe Asn Val His
Gly Arg Asn Met Thr Asp Pro Glu145 150
155 160Thr Glu Ala Asp Arg Leu Leu Gln Val Glu Leu Met
Glu Ile Leu Trp 165 170
175Lys Ser Lys Tyr Trp Gln Arg Ser Ala Gly Arg Asp His Val Ile Pro
180 185 190Met His His Pro Asn Ala
Phe Arg Phe Leu Arg Asp Met Val Asn Ala 195 200
205Ser Ile Leu Ile Val Ala Asp Phe Gly Arg Tyr Thr Lys Glu
Leu Ala 210 215 220Ser Leu Arg Lys Asp
Val Val Ala Pro Tyr Val His Val Val Asp Ser225 230
235 240Phe Leu Asn Asp Asp Pro Pro Asp Pro Phe
Asp Asp Arg Pro Thr Leu 245 250
255Leu Phe Phe Arg Gly Arg Thr Val Arg Lys Asp Glu Gly Lys Ile Arg
260 265 270Ala Lys Leu Ala Lys
Ile Leu Lys Gly Lys Asp Gly Val Arg Phe Glu 275
280 285Asp Ser Leu Ala Thr Gly Glu Gly Ile Lys Thr Ser
Thr Glu Gly Met 290 295 300Arg Ser Ser
Lys Phe Cys Leu His Pro Ala Gly Asp Thr Pro Ser Ser305
310 315 320Cys Arg Leu Phe Asp Ala Ile
Val Ser His Cys Val Pro Val Ile Val 325
330 335Ser Ser Arg Ile Glu Leu Pro Phe Glu Asp Glu Ile
Asp Tyr Ser Glu 340 345 350Phe
Ser Leu Phe Phe Ser Val Glu Glu Ala Leu Arg Pro Asp Tyr Leu 355
360 365Leu Asn Gln Leu Arg Gln Ile Gln Lys
Thr Lys Trp Val Glu Ile Trp 370 375
380Ser Lys Leu Lys Asn Val Ser His His Tyr Glu Phe Gln Asn Pro Pro385
390 395 400Arg Lys Gly Asp
Ala Val Asn Met Ile Trp Arg Gln Val Lys His Lys 405
410 415Val Pro Ala Val Asn Leu Ala Ile His Arg
Asn Arg Arg Leu Lys Ile 420 425
430Pro Asp Trp Trp Gly 4353929DNAartificial sequenceprimer
39ggggacaagt ttgtacaaaa aagcaggct
294029DNAartificial sequenceprimer 40ggggaccact ttgtacaaga aagctgggt
294154DNAartificial sequenceprimer
41ttaaacaagt ttgtacaaaa aagcaggctg caattaaccc tcactaaagg gaac
544253DNAartificial sequenceprimer 42ttaaaccact ttgtacaaga aagctgggtg
cgtaatacga ctcactatag ggc 534312856DNAartificial
sequencevector 43cgccttggcg cgccgatcat ccacaagttt gtacaaaaaa gctgaacgag
aaacgtaaaa 60tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta
cataatactg 120taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg
ctttacactt 180tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atttaaatac
gcgttgatcc 240ggcttactaa aagccagata acagtatgcg tatttgcgcg ctgatttttg
cggtataaga 300atatatactg atatgtatac ccgaagtatg tcaaaaagag gtatgctatg
aagcagcgta 360ttacagtgac agttgacagc gacagctatc agttgctcaa ggcatatatg
atgtcaatat 420ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg
ccgaacgctg 480gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg tttattgaaa
tgaacggctc 540ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc
tataaaagag 600agagccgtta tcgtctgttt gtggatgtac agagtgatat cattgacacg
cccggtcgac 660ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc
cgtgaacttt 720acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat
atggccagtg 780tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa
aatgacatca 840aaaacgccat taacctgatg ttctggggaa tataaatgtc aggctccctt
atacacagcc 900agtctgcagg tcgaccatag tgactggata tgttgtgttt tacagtatta
tgtagtctgt 960tttttatgca aaatctaatt taatatattg atatttatat cattttacgt
ttctcgttca 1020gctttcttgt acaaagtggt gttaacctag acttgtccat cttctggatt
ggccaactta 1080attaatgtat gaaataaaag gatgcacaca tagtgacatg ctaatcacta
taatgtgggc 1140atcaaagttg tgtgttatgt gtaattacta gttatctgaa taaaagagaa
agagatcatc 1200catatttctt atcctaaatg aatgtcacgt gtctttataa ttctttgatg
aaccagatgc 1260atttcattaa ccaaatccat atacatataa atattaatca tatataatta
atatcaattg 1320ggttagcaaa acaaatctag tctaggtgtg ttttgcgaat tgcggccgcc
accgcggtgg 1380agctcgaatt ccggtccggg tcacctttgt ccaccaagat ggaactgcgg
ccgctcatta 1440attaagtcag gcgcgcctct agttgaagac acgttcatgt cttcatcgta
agaagacact 1500cagtagtctt cggccagaat ggccatctgg attcagcagg cctagaaggc
catttaaatc 1560ctgaggatct ggtcttccta aggacccggg atatcggacc gattaaactt
taattcggtc 1620cgaagcttga agttcctatt ccgaagttcc tattctccag aaagtatagg
aacttcgcat 1680gcctgcagtg cagcgtgacc cggtcgtgcc cctctctaga gataatgagc
attgcatgtc 1740taagttataa aaaattacca catatttttt ttgtcacact tgtttgaagt
gcagtttatc 1800tatctttata catatattta aactttactc tacgaataat ataatctata
gtactacaat 1860aatatcagtg ttttagagaa tcatataaat gaacagttag acatggtcta
aaggacaatt 1920gagtattttg acaacaggac tctacagttt tatcttttta gtgtgcatgt
gttctccttt 1980ttttttgcaa atagcttcac ctatataata cttcatccat tttattagta
catccattta 2040gggtttaggg ttaatggttt ttatagacta atttttttag tacatctatt
ttattctatt 2100ttagcctcta aattaagaaa actaaaactc tattttagtt tttttattta
ataatttaga 2160tataaaatag aataaaataa agtgactaaa aattaaacaa atacccttta
agaaattaaa 2220aaaactaagg aaacattttt cttgtttcga gtagataatg ccagcctgtt
aaacgccgtc 2280gacgagtcta acggacacca accagcgaac cagcagcgtc gcgtcgggcc
aagcgaagca 2340gacggcacgg catctctgtc gctgcctctg gacccctctc gagagttccg
ctccaccgtt 2400ggacttgctc cgctgtcggc atccagaaat tgcgtggcgg agcggcagac
gtgagccggc 2460acggcaggcg gcctcctcct cctctcacgg caccggcagc tacgggggat
tcctttccca 2520ccgctccttc gctttccctt cctcgcccgc cgtaataaat agacaccccc
tccacaccct 2580ctttccccaa cctcgtgttg ttcggagcgc acacacacac aaccagatct
cccccaaatc 2640cacccgtcgg cacctccgct tcaaggtacg ccgctcgtcc tccccccccc
ccctctctac 2700cttctctaga tcggcgttcc ggtccatgca tggttagggc ccggtagttc
tacttctgtt 2760catgtttgtg ttagatccgt gtttgtgtta gatccgtgct gctagcgttc
gtacacggat 2820gcgacctgta cgtcagacac gttctgattg ctaacttgcc agtgtttctc
tttggggaat 2880cctgggatgg ctctagccgt tccgcagacg ggatcgattt catgattttt
tttgtttcgt 2940tgcatagggt ttggtttgcc cttttccttt atttcaatat atgccgtgca
cttgtttgtc 3000gggtcatctt ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg
gttgggcggt 3060cgttctagat cggagtagaa ttctgtttca aactacctgg tggatttatt
aattttggat 3120ctgtatgtgt gtgccataca tattcatagt tacgaattga agatgatgga
tggaaatatc 3180gatctaggat aggtatacat gttgatgcgg gttttactga tgcatataca
gagatgcttt 3240ttgttcgctt ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg
ttctagatcg 3300gagtagaata ctgtttcaaa ctacctggtg tatttattaa ttttggaact
gtatgtgtgt 3360gtcatacatc ttcatagtta cgagtttaag atggatggaa atatcgatct
aggataggta 3420tacatgttga tgtgggtttt actgatgcat atacatgatg gcatatgcag
catctattca 3480tatgctctaa ccttgagtac ctatctatta taataaacaa gtatgtttta
taattatttt 3540gatcttgata tacttggatg atggcatatg cagcagctat atgtggattt
ttttagccct 3600gccttcatac gctatttatt tgcttggtac tgtttctttt gtcgatgctc
accctgttgt 3660ttggtgttac ttctgcaggt cgactttaac ttagcctagg atccacacga
caccatgata 3720gaggtgaaac cgattaacgc agaggatacc tatgaactaa ggcatagaat
actcagacca 3780aaccagccga tagaagcgtg tatgtttgaa agcgatttac ttcgtggtgc
atttcactta 3840ggcggctatt acgggggcaa actgatttcc atagcttcat tccaccaggc
cgagcactca 3900gaactccaag gccagaaaca gtaccagctc cgaggtatgg ctaccttgga
aggttatcgt 3960gagcagaagg cgggatcgag tctaattaaa cacgctgaag aaattcttcg
taagaggggg 4020gcggacttgc tttggtgtaa tgcgcggaca tccgcctcag gctactacaa
aaagttaggc 4080ttcagcgagc agggagaggt attcgacacg ccgccagtag gacctcacat
cctgatgtat 4140aaaaggatca cataactagc tagtcagtta acctagactt gtccatcttc
tggattggcc 4200aacttaatta atgtatgaaa taaaaggatg cacacatagt gacatgctaa
tcactataat 4260gtgggcatca aagttgtgtg ttatgtgtaa ttactagtta tctgaataaa
agagaaagag 4320atcatccata tttcttatcc taaatgaatg tcacgtgtct ttataattct
ttgatgaacc 4380agatgcattt cattaaccaa atccatatac atataaatat taatcatata
taattaatat 4440caattgggtt agcaaaacaa atctagtcta ggtgtgtttt gcgaattcag
agctcgaatt 4500cattccgatt aatcgtggcc tcttgctctt caggatgaag agctatgttt
aaacgtgcaa 4560gcgctactag acaattcagt acattaaaaa cgtccgcaat gtgttattaa
gttgtctaag 4620cgtcaatttg tttacaccac aatatatcct gccaccagcc agccaacagc
tccccgaccg 4680gcagctcggc acaaaatcac cactcgatac aggcagccca tcagtccggg
acggcgtcag 4740cgggagagcc gttgtaaggc ggcagacttt gctcatgtta ccgatgctat
tcggaagaac 4800ggcaactaag ctgccgggtt tgaaacacgg atgatctcgc ggagggtagc
atgttgattg 4860taacgatgac agagcgttgc tgcctgtgat caaatatcat ctccctcgca
gagatccgaa 4920ttatcagcct tcttattcat ttctcgctta accgtgacag gctgtcgatc
ttgagaacta 4980tgccgacata ataggaaatc gctggataaa gccgctgagg aagctgagtg
gcgctatttc 5040tttagaagtg aacgttgacg atcgtcgacc gtaccccgat gaattaattc
ggacgtacgt 5100tctgaacaca gctggatact tacttgggcg attgtcatac atgacatcaa
caatgtaccc 5160gtttgtgtaa ccgtctcttg gaggttcgta tgacactagt ggttcccctc
agcttgcgac 5220tagatgttga ggcctaacat tttattagag agcaggctag ttgcttagat
acatgatctt 5280caggccgtta tctgtcaggg caagcgaaaa ttggccattt atgacgacca
atgccccgca 5340gaagctccca tctttgccgc catagacgcc gcgcccccct tttggggtgt
agaacatcct 5400tttgccagat gtggaaaaga agttcgttgt cccattgttg gcaatgacgt
agtagccggc 5460gaaagtgcga gacccatttg cgctatatat aagcctacga tttccgttgc
gactattgtc 5520gtaattggat gaactattat cgtagttgct ctcagagttg tcgtaatttg
atggactatt 5580gtcgtaattg cttatggagt tgtcgtagtt gcttggagaa atgtcgtagt
tggatgggga 5640gtagtcatag ggaagacgag cttcatccac taaaacaatt ggcaggtcag
caagtgcctg 5700ccccgatgcc atcgcaagta cgaggcttag aaccaccttc aacagatcgc
gcatagtctt 5760ccccagctct ctaacgcttg agttaagccg cgccgcgaag cggcgtcggc
ttgaacgaat 5820tgttagacat tatttgccga ctaccttggt gatctcgcct ttcacgtagt
gaacaaattc 5880ttccaactga tctgcgcgcg aggccaagcg atcttcttgt ccaagataag
cctgcctagc 5940ttcaagtatg acgggctgat actgggccgg caggcgctcc attgcccagt
cggcagcgac 6000atccttcggc gcgattttgc cggttactgc gctgtaccaa atgcgggaca
acgtaagcac 6060tacatttcgc tcatcgccag cccagtcggg cggcgagttc catagcgtta
aggtttcatt 6120tagcgcctca aatagatcct gttcaggaac cggatcaaag agttcctccg
ccgctggacc 6180taccaaggca acgctatgtt ctcttgcttt tgtcagcaag atagccagat
caatgtcgat 6240cgtggctggc tcgaagatac ctgcaagaat gtcattgcgc tgccattctc
caaattgcag 6300ttcgcgctta gctggataac gccacggaat gatgtcgtcg tgcacaacaa
tggtgacttc 6360tacagcgcgg agaatctcgc tctctccagg ggaagccgaa gtttccaaaa
ggtcgttgat 6420caaagctcgc cgcgttgttt catcaagcct tacagtcacc gtaaccagca
aatcaatatc 6480actgtgtggc ttcaggccgc catccactgc ggagccgtac aaatgtacgg
ccagcaacgt 6540cggttcgaga tggcgctcga tgacgccaac tacctctgat agttgagtcg
atacttcggc 6600gatcaccgct tccctcatga tgtttaactc ctgaattaag ccgcgccgcg
aagcggtgtc 6660ggcttgaatg aattgttagg cgtcatcctg tgctcccgag aaccagtacc
agtacatcgc 6720tgtttcgttc gagacttgag gtctagtttt atacgtgaac aggtcaatgc
cgccgagagt 6780aaagccacat tttgcgtaca aattgcaggc aggtacattg ttcgtttgtg
tctctaatcg 6840tatgccaagg agctgtctgc ttagtgccca ctttttcgca aattcgatga
gactgtgcgc 6900gactcctttg cctcggtgcg tgtgcgacac aacaatgtgt tcgatagagg
ctagatcgtt 6960ccatgttgag ttgagttcaa tcttcccgac aagctcttgg tcgatgaatg
cgccatagca 7020agcagagtct tcatcagagt catcatccga gatgtaatcc ttccggtagg
ggctcacact 7080tctggtagat agttcaaagc cttggtcgga taggtgcaca tcgaacactt
cacgaacaat 7140gaaatggttc tcagcatcca atgtttccgc cacctgctca gggatcaccg
aaatcttcat 7200atgacgccta acgcctggca cagcggatcg caaacctggc gcggcttttg
gcacaaaagg 7260cgtgacaggt ttgcgaatcc gttgctgcca cttgttaacc cttttgccag
atttggtaac 7320tataatttat gttagaggcg aagtcttggg taaaaactgg cctaaaattg
ctggggattt 7380caggaaagta aacatcacct tccggctcga tgtctattgt agatatatgt
agtgtatcta 7440cttgatcggg ggatctgctg cctcgcgcgt ttcggtgatg acggtgaaaa
cctctgacac 7500atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
cagacaagcc 7560cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
ccagtcacgt 7620agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
gtactgagag 7680tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
cgcatcaggc 7740gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
cggcgagcgg 7800tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
aacgcaggaa 7860agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
gcgttgctgg 7920cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
tcaagtcaga 7980ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
agctccctcg 8040tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
ctcccttcgg 8100gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
taggtcgttc 8160gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
gccttatccg 8220gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
gcagcagcca 8280ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
ttgaagtggt 8340ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
ctgaagccag 8400ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
gctggtagcg 8460gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
caagaagatc 8520ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
taagggattt 8580tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
aaatgaagtt 8640ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
tgcttaatca 8700gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
tgactccccg 8760tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
gcaatgatac 8820cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
gccggaaggg 8880ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
aattgttgcc 8940gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
gccattgctg 9000cagggggggg gggggggggg gacttccatt gttcattcca cggacaaaaa
cagagaaagg 9060aaacgacaga ggccaaaaag cctcgctttc agcacctgtc gtttcctttc
ttttcagagg 9120gtattttaaa taaaaacatt aagttatgac gaagaagaac ggaaacgcct
taaaccggaa 9180aattttcata aatagcgaaa acccgcgagg tcgccgcccc gtaacctgtc
ggatcaccgg 9240aaaggacccg taaagtgata atgattatca tctacatatc acaacgtgcg
tggaggccat 9300caaaccacgt caaataatca attatgacgc aggtatcgta ttaattgatc
tgcatcaact 9360taacgtaaaa acaacttcag acaatacaaa tcagcgacac tgaatacggg
gcaacctcat 9420gtcccccccc cccccccccc tgcaggcatc gtggtgtcac gctcgtcgtt
tggtatggct 9480tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
gttgtgcaaa 9540aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
cgcagtgtta 9600tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
cgtaagatgc 9660ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg 9720agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
aactttaaaa 9780gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
accgctgttg 9840agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
ttttactttc 9900accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
gggaataagg 9960gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
aagcatttat 10020cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
taaacaaata 10080ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
cattattatc 10140atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
agaattggtc 10200gacgatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc
ggattgaagg 10260cgagatccag caactcgcgc cagatcatcc tgtgacggaa ctttggcgcg
tgatgactgg 10320ccaggacgtc ggccgaaaga gcgacaagca gatcacgctt ttcgacagcg
tcggatttgc 10380gatcgaggat ttttcggcgc tgcgctacgt ccgcgaccgc gttgagggat
caagccacag 10440cagcccactc gaccttctag ccgacccaga cgagccaagg gatctttttg
gaatgctgct 10500ccgtcgtcag gctttccgac gtttgggtgg ttgaacagaa gtcattatcg
tacggaatgc 10560caagcactcc cgaggggaac cctgtggttg gcatgcacat acaaatggac
gaacggataa 10620accttttcac gcccttttaa atatccgtta ttctaataaa cgctcttttc
tcttaggttt 10680acccgccaat atatcctgtc aaacactgat agtttaaact gaaggcggga
aacgacaatc 10740tgatcatgag cggagaatta agggagtcac gttatgaccc ccgccgatga
cgcgggacaa 10800gccgttttac gtttggaact gacagaaccg caacgttgaa ggagccactc
agcaagctgg 10860tacgattgta atacgactca ctatagggcg aattgagcgc tgtttaaacg
ctcttcaact 10920ggaagagcgg ttacccggac cgaagcttga agttcctatt ccgaagttcc
tattctctag 10980aaagtatagg aacttcagat ctcgatgctc accctgttgt ttggtgttac
ttctgcaggt 11040cgactctaga ggatccacca tgagcccaga acgacgcccg gccgacatcc
gccgtgccac 11100cgaggcggac atgccggcgg tctgcaccat cgtcaaccac tacatcgaga
caagcacggt 11160caacttccgt accgagccgc aggaaccgca ggactggacg gacgacctcg
tccgtctgcg 11220ggagcgctat ccctggctcg tcgccgaggt ggacggcgag gtcgccggca
tcgcctacgc 11280gggcccctgg aaggcacgca acgcctacga ctggacggcc gagtcgaccg
tgtacgtctc 11340cccccgccac cagcggacgg gactgggctc cacgctctac acccacctgc
tgaagtccct 11400ggaggcacag ggcttcaaga gcgtggtcgc tgtcatcggg ctgcccaacg
acccgagcgt 11460gcgcatgcac gaggcgctcg gatatgcccc ccgcggcatg ctgcgggcgg
ccggcttcaa 11520gcacgggaac tggcatgacg tgggtttctg gcagctggac ttcagcctgc
cggtaccgcc 11580ccgtccggtc ctgcccgtca ccgagatctg atccgtcgac caacctagac
ttgtccatct 11640tctggattgg ccaacttaat taatgtatga aataaaagga tgcacacata
gtgacatgct 11700aatcactata atgtgggcat caaagttgtg tgttatgtgt aattactagt
tatctgaata 11760aaagagaaag agatcatcca tatttcttat cctaaatgaa tgtcacgtgt
ctttataatt 11820ctttgatgaa ccagatgcat ttcattaacc aaatccatat acatataaat
attaatcata 11880tataattaat atcaattggg ttagcaaaac aaatctagtc taggtgtgtt
ttgcgaattg 11940cggccgcgat ctggggaatt cccatggaca ccggtaattc ccatgatctt
ctctccttca 12000tcaatggatg ccatgtttca taacaataac accaaatgtt tgatgagcta
ccaacaattg 12060cgcaaagact atggctaagc tcgagctcgc tcgctacaag ttgttgactt
tcaaatacaa 12120gtttgttttt ggaacaccaa atattctaca tgatctttca ctaagttgcg
caccactatc 12180aaaagattat ctaggccatt attcaagtaa agagtgaaca cgtctaagac
ccacaaccac 12240accaaataga atacgcatac atgcaacata ttgtgcaaga agtatccaac
tggactccca 12300tgtattctaa aactattttc gtagagttaa agttatgaca aacttatcaa
ataaaaattt 12360gaacgctgga ccaaaacttt catctttcaa atccaccatc gtctatcctc
ataaattgtt 12420ttgattataa cacatctacg taaatcattt gttttgaaca atactaattt
aattttatta 12480agtcaaataa cctgcttaga aaataatccc tccacctcat ttaacaattt
cttgtcaaac 12540acacaccaag aaaaaaatta atgaaagaga aaagaaatga aaaggacatg
gagttgaata 12600ctagcaaaat tgattgaagg aagattcaca attgaaattg aaaccattta
atttattttc 12660gggtccataa taataaattg gtaagaataa aaacccgatc aagtccggta
cagtacaatt 12720ccactccacc aactccttac ttaaacccct atttataccc actctcatcc
tcactcttcc 12780ttcacctctc acactctctt ctctctctca aaaccctcac acaaacgctg
cgtttagtgt 12840aagaaattca atccgg
1285644825DNAZea mays 44aaatccttac agaattgctg tagtttcata
gtgctagatg tggacagcaa agcgccgctg 60tatgcttctg cttttctttt ttggtgtgtg
tagccacatc ctttgttcct gcccggcgcc 120atcccacttg gttgtttttt tttatgattg
aaagccttca tgcttcctcg gtcaatcacc 180ggtgcgcact gggagcatcg ccggaaaaaa
aattcttcgg ctaagagtaa cttctttctc 240cttttcttct ctgatctcgc gagcagtgct
gataacgtgt tgtaatctac ttagcggtaa 300cgagattgag agagacaaaa tgacagaact
attgtcttta ttgcagagtg tcatgtattt 360atacagggga tacaaagtct cccaaggggt
gtgtcccttg ggagtaactg ccagttgatc 420acaggacaat attttgtaac aaaacgtaca
catcgtcaaa atagcgaggc atgaaactgg 480ccttggccat ggacgcgtga agcgcgccat
gcgttggata tgtggtcaat aagtatatac 540aatacaatgt ttaacagagc tgatagtact
gctttggcac atttttgtcc acgcttcatg 600agagataaaa cacctgcacg taaattcaca
tgctgcactg aaggcccgat cactgaggag 660cgaactgccg taactccctt ctatatatac
ccccagtccc tgtttcagtt ttcgtcaagc 720tagcagcacc aagttgtcga tcacttgcct
gctcttgagc tcgattaagc tatcatcagc 780tacagcatcc gatcccaaac tgcaactgta
gcagcgacaa ctgcc 82545860DNAZea mays 45ctggtaatta
ttggctgtag gattctaaac agagcctaaa tagctggaat agctctagcc 60ctcaatccaa
actaatgata tctatactta tgcaactcta aatttttatt ctaaaagtaa 120tatttcattt
ttgtcaacga gattctctac tctattccac aatcttttga agcaatattt 180accttaaatc
tgtactctat accaataatc atatattcta ttatttattt ttatctctct 240cctaaggagc
atccccctat gtctgcatgg cccccgcctc gggtcccaat ctcttgctct 300gctagtagca
cagaagaaaa cactagaaat gacttgcttg acttagagta tcagataaac 360atcatgttta
cttaacttta atttgtatcg gtttctacta tttttataat atttttgtct 420ctatagatac
tacgtgcaac agtataatca acctagttta atccagagcg aaggattttt 480tactaagtac
gtgactccat atgcacagcg ttccttttat ggttcctcac tgggcacagc 540ataaacgaac
cctgtccaat gttttcagcg cgaacaaaca gaaattccat cagcgaacaa 600acaacataca
tgcgagatga aaataaataa taaaaaaagc tccgtctcga taggccggca 660cgaatcgaga
gcctccatag ccagtttttt ccatcggaac ggcggttcgc gcacctaatt 720atatgcacca
cacgcctata aagccaacca acccgtcgga ggggcgcaag ccagacagaa 780gacagcccgt
cagcccctct cgtttttcat ccgccttcgc ctccaaccgc gtgcgctcca 840cgcctcctcc
aggaaagcga 86046899DNAZea
mays 46gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta
60taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt
120atacatatat ttaaacttta ctctacgaat aatataatct atagtactac aataatatca
180gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt
240ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg
300caaatagctt cacctatata atacttcatc cattttatta gtacatccat ttagggttta
360gggttaatgg tttttataga ctaatttttt tagtacatct attttattct attttagcct
420ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa
480tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta
540aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt
600ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca
660cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg
720ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag
780gcggcctcct cctcctctca cggcacggca gctacggggg attcctttcc caccgctcct
840tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttccc
89947879DNAMedicago sativa 47aattcccatg atcttctctc cttcatcaat ggatgccatg
tttcataaca ataacaccaa 60atgtttgatg agctaccaac aattgcgcaa agactatggc
taagctcgag ctcgctcgct 120acaagttgtt gactttcaaa tacaagtttg tttttggaac
accaaatatt ctacatgatc 180tttcactaag ttgcgcacca ctatcaaaag attatctagg
ccattattca agtaaagagt 240gaacacgtct aagacccaca accacaccaa atagaatacg
catacatgca acatattgtg 300caagaagtat ccaactggac tcccatgtat tctaaaacta
ttttcgtaga gttaaagtta 360tgacaaactt atcaaataaa aatttgaacg ctggaccaaa
actttcatct ttcaaatcca 420ccatcgtcta tcctcataaa ttgttttgat tataacacat
ctacgtaaat catttgtttt 480gaacaatact aatttaattt tattaagtca aataacctgc
ttagaaaata atccctccac 540ctcatttaac aatttcttgt caaacacaca ccaagaaaaa
aattaatgaa agagaaaaga 600aatgaaaagg acatggagtt gaatactagc aaaattgatt
gaaggaagat tcacaattga 660aattgaaacc atttaattta ttttcgggtc cataataata
aattggtaag aataaaaacc 720cgatcaagtc cggtacagta caattccact ccaccaactc
cttacttaaa cccctattta 780tacccactct catcctcact cttccttcac ctctcacact
ctcttctctc tctcaaaacc 840ctcacacaaa cgctgcgttt agtgtaagaa attcaatcc
87948318DNASolanum tuberosum 48agacttgtcc
atcttctgga ttggccaact taattaatgt atgaaataaa aggatgcaca 60catagtgaca
tgctaatcac tataatgtgg gcatcaaagt tgtgtgttat gtgtaattac 120tagttatctg
aataaaagag aaagagatca tccatatttc ttatcctaaa tgaatgtcac 180gtgtctttat
aattctttga tgaaccagat gcatttcatt aaccaaatcc atatacatat 240aaatattaat
catatataat taatatcaat tgggttagca aaacaaatct agtctaggtg 300tgttttgcga
attgcggc 318
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