Corn Event Mzdt09y

Abstract

A novel transgenic corn event designated MZDT09Y is disclosed. The invention relates to nucleic acids that are unique to event MZDT09Y and to methods of detecting the presence of event MZDT09Y based on DNA sequences of the recombinant constructs inserted into the corn genome that resulted in the MZDT09Y event and of genomic sequences flanking the insertion site. The invention further relates to corn plants comprising the transgenic genotype of event MZDT09Y and to methods for producing a corn plant by crossing a corn plant comprising the MZDT09Y genotype with itself or another corn variety. Seeds of corn plants comprising the MZDT09Y genotype are also objects of the invention.

Description

BACKGROUND OF THE INVENTION

[0001] The increasing world population and the dwindling supply of arable land available for agriculture fuels the need for research in the area of increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilize selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are often labor intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant's genome. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.

SUMMARY OF THE INVENTION

[0002] The following Summary lists several embodiments of the invention subject matter, and in many cases lists variations and permutations of these embodiments. This Summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the invention, whether listed in this Summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0003] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0004] The invention provides nucleotide sequences that when transgenically expressed in a plant increases plant vigor, yield and/or biomass as well as increased stress tolerance. It was discovered that the T6PP proteins described herein comprise modifications which are significantly associated with increased yield and/or increased tolerance to stress when transgenically expressed in a plant.

[0005] In one embodiment, the present invention encompasses a nucleic acid molecule, preferably isolated, comprising a nucleotide sequence that is unique to corn event MZDT09Y. The nucleotide sequence may comprise any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any of the complements thereof. The nucleic acid molecule is comprised in a corn seed deposited at the American Type Culture Collection under the accession number PTA-13025.

[0006] In another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer which function together in the presence of an event MZDT09Y DNA template in a sample to produce an amplicon diagnostic for event MZDT09Y. In one aspect, the pair of the first polynucleotide primer or a sequence of the second polynucleotide primer is chosen from SEQ ID NO: 7, or the complement thereof; or a sequence of the first polynucleotide primer is or is complementary to a corn plant genome sequence flanking the point of insertion of a heterologous DNA sequence inserted into the corn plant genome of event MZDT09Y, and a sequence of the second polynucleotide primer is or is complementary to the heterologous DNA sequence inserted into the genome of event MZDT09Y. In another aspect, the first polynucleotide primer comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and the complements thereof; and the second polynucleotide primer comprises at least 10 contiguous nucleotides from SEQ ID NO: 7, or the complements thereof. In another aspect, the first polynucleotide primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 32, and the complements thereof; and the second polynucleotide primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 33, and the complements thereof. In another aspect, the first polynucleotide primer consists of SEQ ID NO: 12 and the second polynucleotide primer consists of SEQ ID NO: 13; or the first polynucleotide primer consists of SEQ ID NO: 14 and the second polynucleotide primer consists of SEQ ID NO: 15; or the first polynucleotide primer consists of SEQ ID NO: 32 and the second polynucleotide primer consists of SEQ ID NO: 33.

[0007] In another embodiment, the present invention encompasses a method of detecting the presence of a nucleic acid molecule that is unique to event MZDT09Y in a sample comprising corn nucleic acids. In one aspect, the method comprises contacting the sample with a pair of primers that, when used in a nucleic-acid amplification reaction with genomic DNA from event MZDT09Y produces an amplicon that is diagnostic for event MZDT09Y; performing a nucleic acid amplification reaction, thereby producing the amplicon; and detecting the amplicon. In another aspect, the method comprises contacting the sample with a probe that hybridizes under high stringency conditions with genomic DNA from event MZDT09Y and does not hybridize under high stringency conditions with DNA of a control corn plant; subjecting the sample and probe to high stringency hybridization conditions; and detecting hybridization of the probe to the nucleic acid molecule.

[0008] In another embodiment, the present invention encompasses a kit for detecting nucleic acids that are unique to event MZDT09Y comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event MZDT09Y in the sample. In one aspect, the nucleic acid molecule of sufficient length of continuous polynucleotides comprises a nucleotide sequence selected from the group consisting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8; and the complements thereof. In another aspect, the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 12-15, SEQ ID NOs: 32-34, and the complements thereof.

[0009] In another embodiment, the present invention encompasses a transgenic corn plant, or cells or tissues thereof, comprising a nucleic acid molecule that is unique to corn event MZDT09Y. The nucleotide sequence may comprise any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any of the complements thereof. In another embodiment, the present invention encompasses a corn seed comprising a nucleic acid molecule that is unique to corn event MZDT09Y. An example of the seed is deposited at the American Type Culture Collection under the accession number PTA-13025.

[0010] In another embodiment, the present invention encompasses a biological sample derived from an event MZDT09Y corn plant, tissue, or seed, wherein the sample comprises a nucleotide sequence which is or is complementary to a nucleotide sequence selected from the group consisting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof; and wherein the sequence is detectable in the sample using a nucleic acid amplification or nucleic acid hybridization method. In one aspect, the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products. In another embodiment, the present invention encompasses an extract derived from the biological sample. In one aspect, the extract is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products.

[0011] In another embodiment, the present invention encompasses a method for producing a corn plant with increased yield, the method comprising sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises event MZDT09Y DNA, thereby producing a plurality of first generation progeny plants; selecting a first generation progeny plant with increased yield; selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and selecting from the second generation progeny plants, a plant with increased yield; wherein the second generation progeny plants comprise a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof. In one aspect, the increased yield is indicated by an increase as compared to a control plant of any one of the following: increased grain yield, increased seed, increased seed weight, increased biomass, increased sugar, increased oil, increased plant vigor, increased yield under non-optimal conditions, increased yield under stress conditions and increased yield under water stress conditions.

[0012] In another embodiment, the present invention encompasses a method for producing a corn plant with increased tolerance to abiotic stress, the method comprising sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises event MZDT09Y DNA, thereby producing a plurality of first generation progeny plants; selecting a first generation progeny plant with increased tolerance to abiotic stress; selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and selecting from the second generation progeny plants, a plant with increase tolerance to abiotic stress; wherein the second generation progeny plants comprise a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof. In one aspect, the abiotic stress comprises stress selected from the group consisting of water stress, heat stress or cold stress. In another aspect, the water stress is caused by drought.

[0013] In another embodiment, the present invention encompasses a method of producing hybrid corn seeds comprising planting seeds of a first inbred corn line comprising event MZDT09Y and seeds of a second inbred line having a genotype different from the first inbred corn line; cultivating corn plants resulting from the planting until time of flowering; emasculating the flowers of plants of one of the corn inbred lines; sexually crossing the two different inbred lines with each other; harvesting the hybrid seed produced thereby. The hybrid seed produced by this method comprise a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof. In one aspect, the plants of the first inbred corn line are the female parents or male parents. In another embodiment, the present invention encompasses hybrid seed produced by the above method of producing hybrid seed.

[0014] These and other features, objects and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying sequences, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention. The description of preferred embodiments is not intended to limit the invention to cover all modifications, equivalents and alternatives. Reference should therefore be made to the embodiments recited herein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0015] SEQ ID NO: 1 is the 20 by 5'- junction sequence.

[0016] SEQ ID NO: 2 is the 20 by 3'- junction sequence.

[0017] SEQ ID NO: 3 is the 60 by 5'- genome plus insert sequence.

[0018] SEQ ID NO: 4 is the 60 by 3'- insert plus genome sequence.

[0019] SEQ ID NO: 5 is the 5'- flanking genomic sequence.

[0020] SEQ ID NO: 6 is the 3'- flanking genomic sequence.

[0021] SEQ ID NO: 7 is the heterologous insert sequence.

[0022] SEQ ID NO: 8 is the heterologous insert sequence plus genomic flanking sequences.

[0023] SEQ ID NO: 9 is the OsMADS6 promoter.

[0024] SEQ ID NO: 10 is the sequence of the modified T6PP gene.

[0025] SEQ ID NO: 11 is the sequence of the modified T6PP protein.

[0026] SEQ ID NOs: 12-22 are primers useful in the present invention.

[0027] SEQ ID NOs: 23-28 are amplicons useful in the present invention.

[0028] SEQ ID NO: 29 is the unmodified rice T6PP (OsT6PP) cDNA sequence.

[0029] SEQ ID NO: 30 is the binary construct 15777.

[0030] SEQ ID NO: 31 is the binary construct 15769.

[0031] SEQ ID NOs: 32-34 are TaqMan primers and probe

[0032] SEQ ID NO: 35 is the amplicon produced by the TaqMan PCR reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, plant quantitative genetics, statistics and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Langenheim and Thimann, (1982) Botany: Plant Biology and Its Relation to Human Affairs, John Wiley; Cell Culture and Somatic Cell Genetics of Plants, vol. 1, Vasil, ed. (1984); Stanier, et al., (1986) The Microbial World, 5th ed., Prentice-Hall; Dhringra and Sinclair, (1985) Basic Plant Pathology Methods, CRC Press; Maniatis, et al., (1982) Molecular Cloning: A Laboratory Manual; DNA Cloning, vols. I and II, Glover, ed. (1985); Oligonucleotide Synthesis, Gait, ed. (1984); Nucleic Acid Hybridization, Hames and Higgins, eds. (1984); and the series Methods in Enzymology, Colowick and Kaplan, eds, Academic Press, Inc., San Diego, Calif.

[0034] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.

[0035] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, plant species or genera, constructs, and reagents described as such. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

[0036] As used herein the singular forms "a", "and", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art.

[0037] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent.

[0038] As used herein, the word "or" means any one member of a particular list and also includes any combination of members on that list.

[0039] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of means "including and limited to".

[0040] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0041] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0042] By "microbe" is meant any microorganism (including both eukaryotic and prokaryotic microorganisms), such as fungi, yeast, bacteria, actinomycetes, algae and protozoa, as well as other unicellular structures.

[0043] By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), O-Beta Replicase systems, transcription-based amplification system (TAS) and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, Persing, et al., eds., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

[0044] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; one exception is Micrococcus rubens, for which GTG is the methionine codon (Ishizuka, et al., (1993) J. Gen. Microbiol. 139:425-32) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and incorporated herein by reference.

[0045] A "control plant" or "control" as used herein may be a non-transgenic plant of the parental line used to generate a transgenic plant herein. A control plant may in some cases be a transgenic plant line that includes an empty vector or marker gene, but does not contain the recombinant polynucleotide of the present invention that is expressed in the transgenic plant being evaluated. A control plant in other cases is a transgenic plant expressing the gene with a constitutive promoter. In general, a control plant is a plant of the same line or variety as the transgenic plant being tested, lacking the specific trait-conferring, recombinant DNA that characterizes the transgenic plant. Such a progenitor plant that lacks that specific trait-conferring recombinant DNA can be a natural, wild-type plant, an elite, non-transgenic plant, or a transgenic plant without the specific trait-conferring, recombinant DNA that characterizes the transgenic plant. The progenitor plant lacking the specific, trait-conferring recombinant DNA can be a sibling of a transgenic plant having the specific, trait-conferring recombinant DNA. Such a progenitor sibling plant may include other recombinant DNA

[0046] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" when the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably 60-90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0047] The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

[0048] 2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0049] See also, Creighton, Proteins, W. H. Freeman and Co. (1984).

[0050] As used herein the terms "modified" or "modification" interchangeably refer to deliberate or random substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence which alters, adds or deletes at least one amino acid residue within a given polypeptide. A "modified T6PP" as used herein refers to any nucleic acid encoding a T6PP or peptides, polypeptides or protein having T6PP activity either of which having been modified so that the resultant T6PP confers decreased T6PP activity and/or decreased binding to T6P.

[0051] By "encoding" or "encoded," with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as is present in some plant, animal and fungal mitochondria, the bacterium Mycoplasma capricolumn (Yamao, et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

[0052] When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledonous plants or dicotyledonous plants as these preferences have been shown to differ (Murray, et al., (1989) Nucleic Acids Res. 17:477-98 and herein incorporated by reference). Thus, the maize preferred codon for a particular amino acid might be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.

[0053] As used herein, a "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence. A heterologous nucleic acid refers to a nucleic acid that originates from a foreign species with respect to a host cell or from the same species as the host cell, provided the heterologous nucleic acid sequence is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. A heterologous protein may originate from a foreign species, or if from the same species, is substantially modified from its original form by deliberate human intervention.

[0054] By "host cell" is meant a cell, which comprises a heterologous nucleic acid sequence of the invention, which comprises an expression cassette and supports the replication and/or expression of the expression cassette. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, plant, amphibian or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells, including but not limited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola, barley, millet and tomato. A particularly preferred monocotyledonous host cell is a maize host cell.

[0055] As used herein, the term transgenic "event" refers to a recombinant plant produced by transformation and regeneration of a plant cell or tissue with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term "event" refers to the original transformant and/or progeny of the transformant that include the heterologous DNA. The term "event" also refers to progeny produced by a sexual outcross between the transformant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and the flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. The term "event" also refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome of a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected. Thus, "event MZDT09Y", "MZDT09Y", "09Y" or "09Y event" may be used interchangeably.

[0056] The term "hybridization complex" includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.

[0057] The term "introduced" in the context of inserting a nucleic acid into a cell, means "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid 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).

[0058] As used herein "gene stack" refers to the introduction of two or more genes into the genome of an organism. In certain aspects of the invention it may be desirable to stack any abiotic stress gene (e.g. cold shock proteins, genes associated with ABA response) with the T6PPs as described herein. Likewise, it may also be desirable to stack the T6PPs as described herein with genes conferring insect resistance, disease resistance, increased yield or any other beneficial trait (e.g. increased plant height, etc.) known in the art.

[0059] The terms "isolated" refers to material, such as a nucleic acid or a protein, which is substantially or essentially free from components which normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment. In contrast, a non-isolated nucleic acid, such as DNA or RNA, is found in the state in which it exists in nature. An isolated nucleic acid may be in a transgenic plant and still be considered "isolated." Nucleic acids, which are "isolated," as defined herein, are also referred to as "heterologous" nucleic acids.

[0060] As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

[0061] By "nucleic acid library" is meant a collection of isolated DNA or RNA molecules, which comprise in one case a substantial representation of the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, (1987) Guide To Molecular Cloning Techniques, from the series Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, Calif.; Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1-3; and Current Protocols in Molecular Biology, Ausubel, et al., eds, Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994 Supplement). In another instance "nucleic acid library" as defined herein may also be understood to represent libraries comprising a prescribed faction or rather not substantially representing an entire genome of a specified organism. For example, small RNAs, mRNAs and methylated DNA. A nucleic acid library as defined herein might also encompass variants of a particular molecule (e.g. a collection of variants for a particular protein).

[0062] As used herein "operably linked" includes reference to a functional linkage between a first sequence, such as a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0063] As used herein, the term "plant" includes reference to whole plants, plant organs, tissues (e.g., leaves, stems, roots, etc.), seeds and plant cells and progeny of same. Plant cell, as used herein includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. The class of plants, which can be used in the methods of the invention, is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants including species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium and Triticum. A particularly preferred plant is Zea mays.

[0064] As used herein, "yield" may include reference to bushels per acre of a grain crop at harvest, as adjusted for grain moisture (15% typically for maize, for example), and the volume of biomass generated (for forage crops such as alfalfa and plant root size for multiple crops). Grain moisture is measured in the grain at harvest. The adjusted test weight of grain is determined to be the weight in pounds per bushel, adjusted for grain moisture level at harvest. Biomass is measured as the weight of harvestable plant material generated. Yield can be affected by many properties including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, carbon assimilation, plant architecture, percent seed germination, seedling vigor, and juvenile traits. Yield can also be affected by efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Yield of a plant of the can be measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tons per acre, or kilo per hectare. For example, corn yield may be measured as production of shelled corn kernels per unit of production area, for example in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, for example at 15.5 percent moisture. Moreover a bushel of corn is defined by law in the State of Iowa as 56 pounds by weight, a useful conversion factor for corn yield is: 100 bushels per acre is equivalent to 6.272 metric tons per hectare. Other measurements for yield are common practice in the art In certain embodiments of the invention yield may be increased in stressed and/or non-stressed conditions.

[0065] As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.

[0066] The terms "polypeptide," "peptide" 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.

[0067] As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples are promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheids or sclerenchyma. Such promoters are referred to as "tissue preferred." A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "regulatable" promoter is a promoter, which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Another type of promoter is a developmentally regulated promoter, for example, a promoter that drives expression during pollen development. Tissue preferred, cell type specific, developmentally regulated and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter, which is active under most environmental conditions in most cells.

[0068] Any suitable promoter sequence can be used by the nucleic acid construct of the present invention. According to some embodiments of the invention, the promoter is a constitutive promoter, a tissue-specific, or an abiotic stress-inducible promoter.

[0069] Suitable constitutive promoters include, for example, CaMV 35S promoter (SEQ ID NO:1546; Odell et al., Nature 313:810-812, 1985); Arabidopsis At6669 promoter (SEQ ID NO:1652; see PCT Publication No. W004081173A2); maize Ubi 1 (Christensen et al., Plant Mol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); GOS2 (de Pater et al., Plant J November; 2(6):837-44, 1992); ubiquitin (Christensen et al., Plant Mol. Biol. 18: 675-689, 1992); Rice cyclophilin (Bucholz et al., Plant Mol Biol. 25(5):837-43, 1994); Maize H3 histone (Lepetit et al., Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al., Plant J. 10(1);107-121, 1996), constitutive root tip CT2 promoter (SEQ ID NO:1535; see also PCT application No. IL/2005/000627) and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Pat. Nos. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[0070] Suitable tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90:9586-9590, 1993], seed-preferred promoters [e.g., from seed specific genes (Simon, et al., Plant Mol. Biol. 5. 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235-245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al., Plant Mol Biol, 143).323-32 1990), napA (Stalberg, et al., Planta 199: 515-519, 1996), Wheat SPA (Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oleosin (Cummins, etal., Plant Mol. Biol. 19: 873-876, 1992)], endosperm specific promoters [e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMBO3:1409-15, 1984), Barley ltrl promoter, barley B1, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), Barley DOF (Mena et al., The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33, rice -globulin Glb-1 (Wu et al., Plant Cell Physiology 39(8) 885-889, 1998), rice alpha-globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice ADP-glucose PP (Trans Res 6:157-68, 1997), maize ESR gene family (Plant J 12:235-46, 1997), sorgum gamma-kafirin (Plant Mol. Biol 32:1029-35, 1996)], embryo specific promoters [e.g., rice OSH1 (Sato et al., Proc. Nati. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma of al, Plant Mol. Biol. 39:257-71, 1999), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], and flower-specific promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al., Mol. Gen Genet. 217:240-245; 1989), apetala-3].

[0071] Suitable abiotic stress-inducible promoters include, but not limited to, salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as maize rabl7 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266, 1993), maize rab28 gene promoter (Busk et. al., Plant J. 11:1285-1295, 1997) and maize Ivr2 gene promoter (Pelleschi et. al., Plant Mol. Biol. 39:373-380, 1999); heat-inducible promoters such as heat tomato hsp80-promoter from tomato (U.S. Pat. No. 5,187,267).

[0072] The term "Enzymatic activity" is meant to include demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups. The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, or sense or anti-sense, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes the complementary sequence thereof.

[0073] A "structural gene" is that portion of a gene comprising a DNA segment encoding a protein, polypeptide or a portion thereof, and excluding the 5' sequence which drives the initiation of transcription. The structural gene may alternatively encode a nontranslatable product. The structural gene may be one which is normally found in the cell or one which is not normally found in the cell or cellular location wherein it is introduced, in which case it is termed a "heterologous gene". A heterologous gene may be derived in whole or in part from any source known to the art, including a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. A structural gene may contain one or more modifications that could affect biological activity or its characteristics, the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions and substitutions of one or more nucleotides. The structural gene may constitute an uninterrupted coding sequence or it may include one or more introns, bounded by the appropriate splice junctions. The structural gene may be translatable or non-translatable, including in an anti-sense orientation. The structural gene may be a composite of segments derived from a plurality of sources and from a plurality of gene sequences (naturally occurring or synthetic, where synthetic refers to DNA that is chemically synthesized).

[0074] "Derived from" is used to mean taken, obtained, received, traced, replicated or descended from a source (chemical and/or biological). A derivative may be produced by chemical or biological manipulation (including, but not limited to, substitution, addition, insertion, deletion, extraction, isolation, mutation and replication) of the original source.

[0075] "Chemically synthesized", as related to a sequence of DNA, means that portions of the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983), Weissman (ed.), Praeger Publishers, New York, Chapter 1); automated chemical synthesis can be performed using one of a number of commercially available machines.

[0076] As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention or may have reduced or eliminated expression of a native gene. The term "recombinant" as used herein 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.

[0077] As used herein, an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements, which permit transcription of a particular nucleic acid in a target cell. The expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed and a promoter.

[0078] The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide (collectively "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0079] The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 40% sequence identity, preferably 60-90% sequence identity and most preferably 100% sequence identity (i.e., complementary) with each other.

[0080] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which can be up to 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Optimally, the probe is approximately 500 nucleotides in length, but can vary greatly in length from less than 500 nucleotides to equal to the entire length of the target sequence.

[0081] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide or Denhardt's. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37.degree. C. and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C. and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C. and a wash in 0.1.times.SSC at 60 to 65.degree. C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T.sub.m can be approximated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-84: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC).sup.-0.61 (% form)--500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point (T.sub.m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4.degree. C. lower than the thermal melting point (T.sub.m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than the thermal melting point (T.sub.m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20.degree. C. lower than the thermal melting point (T.sub.m). Using the equation, hybridization and wash compositions, and desired T.sub.m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993); and Current Protocols in Molecular Biology, chapter 2, Ausubel, et al., eds, Greene Publishing and Wiley-Interscience, New York (1995). Unless otherwise stated, in the present application high stringency is defined as hybridization in 4.times.SSC, 5.times.Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65.degree. C. and a wash in 0.1.times.SSC, 0.1% SDS at 65.degree. C.

[0082] As used herein, "transgenic plant" includes reference to a plant, which comprises within its genome a heterologous nucleic acid sequence. Generally, the heterologous nucleic acid sequence is stably integrated within the genome such that the nucleic acid sequence is passed on to successive generations. The heterologous nucleic acid sequence may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of a heterologous nucleic acid sequence, including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. 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.

[0083] As used herein, "vector" includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.

[0084] "Overexpression" refers to the level of expression in transgenic organisms that exceeds levels of expression in normal or untransformed organisms.

[0085] "Plant tissue" includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells and culture such as single cells, protoplast, embryos, and callus tissue. The plant tissue may be in plants or in organ, tissue or cell culture.

[0086] "Preferred expression", "Preferential transcription" or "preferred transcription" interchangeably refers to the expression of gene products that are preferably expressed at a higher level in one or a few plant tissues (spatial limitation) and/or to one or a few plant developmental stages (temporal limitation) while in other tissues/developmental stages there is a relatively low level of expression.

[0087] "Primary transformant" and "TO generation" refer to transgenic plants that are of the same genetic generation as the tissue that was initially transformed (i.e., not having gone through meiosis and fertilization since transformation). "Secondary transformants" and the "T1, T2, T3, etc. generations" refer to transgenic plants derived from primary transformants through one or more meiotic and fertilization cycles. They may be derived by self-fertilization of primary or secondary transformants or crosses of primary or secondary transformants with other transformed or untransformed plants.

[0088] A "selectable marker gene" refers to a gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the ability to grow of non-transformed cells. The selective advantage possessed by the transformed cells may also be due to their enhanced capacity, relative to non-transformed cells, to utilize an added compound as a nutrient, growth factor or energy source. A selective advantage possessed by a transformed cell may also be due to the loss of a previously possessed gene in what is called "negative selection". In this, a compound is added that is toxic only to cells that did not lose a specific gene (a negative selectable marker gene) present in the parent cell (typically a transgene).

[0089] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. "Transiently transformed" refers to cells in which transgenes and foreign DNA have been introduced (for example, by such methods as Agrobacterium-mediated transformation or biolistic bombardment), but not selected for stable maintenance. "Stably transformed" refers to cells that have been selected and regenerated on a selection media following transformation.

[0090] "Transformed," "transgenic," and "recombinant" are used interchangeably and each refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

[0091] The term "translational enhancer sequence" refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon. The translational enhancer sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. "Visible marker" refers to a gene whose expression does not confer an advantage to a transformed cell but can be made detectable or visible. Examples of visible markers include but are not limited to .beta.-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP).

[0092] "Wild-type" refers to the normal gene, virus, or organism found in nature without any mutation or modification.

[0093] As used herein, "plant material," "plant part" or "plant tissue" means plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, tubers, rhizomes and the like.

[0094] As used herein "Protein extract" refers to partial or total protein extracted from a plant part. Plant protein extraction methods are well known in the art.

[0095] As used herein "plant sample" or "biological sample" refers to either intact or non-intact (e g milled seed or plant tissue, chopped plant tissue, lyophilized tissue) plant tissue. It may also be an extract comprising intact or non-intact seed or plant tissue. The biological sample or extract may be selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products.

[0096] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides or polypeptides: (a) "reference sequence," (b) "comparison window," (c) "sequence identity," (d) "percentage of sequence identity" and (e) "substantial identity."

[0097] As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence or the complete cDNA or gene sequence.

[0098] As used herein, "comparison window" means includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, and 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

[0099] Methods of alignment of nucleotide and amino acid sequences for comparison are well known in the art. The local homology algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, may conduct optimal alignment of sequences for comparison; by the homology alignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-53; by the search for similarity method (Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG.RTM. programs (Accelrys, Inc., San Diego, Calif.).). The CLUSTAL program is well described by Higgins and Sharp, (1988) Gene 73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) Computer Applications in the Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol. 24:307-31. The preferred program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60 which is similar to the method described by Higgins and Sharp, (1989) CABIOS 5:151-53 and hereby incorporated by reference). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0100] GAP uses the algorithm of Needleman and Wunsch, supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the Wisconsin Genetics Software Package are 8 and 2, respectively. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 100. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40 and 50 or greater.

[0101] GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0102] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402).

[0103] As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination.

[0104] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

[0105] As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0106] The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 55-100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90% and most preferably at least 95%.

[0107] Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. The degeneracy of the genetic code allows for many amino acids substitutions that lead to variety in the nucleotide sequence that code for the same amino acid, hence it is possible that the DNA sequence could code for the same polypeptide but not hybridize to each other under stringent conditions. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide, which the first nucleic acid encodes, is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

[0108] The phrase "abiotic stress" as used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant by abiotic factors (i.e. water availability, heat, cold, and etc). Accordingly, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, water deprivation, water deficit, drought, flooding, freezing, low or high temperature (e g , chilling or excessive heat), toxic chemical pollution, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution or UV irradiation.

[0109] The phrase "abiotic stress tolerance" as used herein refers to the ability of a plant to endure an abiotic stress without suffering a substantial alteration in metabolism, growth, productivity and/or viability.

[0110] As used herein "water deficit" means a period when water available to a plant is not replenished at the rate at which it is consumed by the plant. A long period of water deficit is colloquially called drought. Lack of rain or irrigation may not produce immediate water stress if there is an available reservoir of ground water to support the growth rate of plants. Plants grown in soil with ample groundwater can survive days without rain or irrigation without adverse effects on yield. Plants grown in dry soil are likely to suffer adverse effects with minimal periods of water deficit. Severe water deficit stress can cause wilt and plant death; moderate drought can reduce yield, stunt growth or retard development. Plants can recover from some periods of water deficit stress without significantly affecting yield. However, water deficit at the time of pollination can lower or reduce yield. Thus, a useful period in the life cycle of corn, for example, for observing response or tolerance to water deficit is the late vegetative stage of growth before tassel emergence or the transition to reproductive development. Tolerance to water deficit is determined by comparison to control plants. For instance, plants of this invention can produce a higher yield than control plants when exposed to water deficit. In the laboratory and in field trials drought can be simulated by giving plants of this invention and control plants less water than is given to sufficiently-watered control plants and measuring differences in traits. One aspect of the invention provides plants overexpressing the genes as disclosed herein which confers a higher tolerance to a water deficit.

[0111] As used herein, the phrase "water optimization" refers to any measure of a plant, its parts, or its structure that can be measured and/or quantified in order to assess an extent of or a rate of plant growth and development under different conditions of water availability. As such, a "water optimization trait" is any trait that can be shown to influence yield in a plant under different sets of growth conditions related to water availability. Exemplary measures of water optimization are grain yield at standard moisture percentage (YGSMN), grain moisture at harvest (GMSTP), grain weight per plot (GWTPN), and percent yield recovery (PYREC).

[0112] As used herein, the phrases "drought tolerance" and "drought tolerant" refer to a plant's ability to endure and/or thrive under conditions where water availability is suboptimal. . In general, a plant is labeled as "drought tolerant" if it displays "enhanced drought tolerance." As used herein, the phrase "enhanced drought tolerance" refers to a measurable improvement, enhancement, or increase in one or more water optimization phenotypes as compared to one or more control plants.

[0113] Water Use Efficiency (WUE) is a parameter frequently used to estimate the tradeoff between water consumption and CO.sub.2 uptake/growth (Kramer, 1983, Water Relations of Plants, Academic Press p. 405). WUE has been defined and measured in multiple ways. One approach is to calculate the ratio of whole plant dry weight, to the weight of water consumed by the plant throughout its life (Chu et al., 1992, Oecologia 89:580). Another variation is to use a shorter time interval when biomass accumulation and water use are measured (Mian et al., 1998, Crop Sci. 38:390). Another approach is to utilize measurements from restricted parts of the plant, for example, measuring only aerial growth and water use (Nienhuis et al 1994 Amer J Bot 81:943). WUE also has been defined as the ratio of CO.sub.2 uptake to water vapor loss from a leaf or portion of a leaf, often measured over a very short time period (e.g. seconds/minutes) (Kramer, 1983, p. 406). The ratio of .sup.13C/ .sup.12C fixed in plant tissue, and measured with an isotope ratio mass-spectrometer, also has been used to estimate WUE in plants using C-3 photosynthesis (Martin et al., 1999, Crop Sci. 1775). As used herein, the term "water use efficiency" refers to the amount of organic matter produced by a plant divided by the amount of water used by the plant in producing it, i.e. the dry weight of a plant in relation to the plant's water use. As used herein, the term "dry weight" refers to everything in the plant other than water, and includes, for example, carbohydrates, proteins, oils, and mineral nutrients. It is contemplated that the transgenic plants produced by the methods described herein will confer an increase in water use efficiency.

[0114] The phrase "biotic stress" as used herein refers to any adverse effect on metabolism, growth, reproduction and/or viability of a plant by biotic factors (i.e. insect pressure, disease and etc).

[0115] The phrase "biotic stress tolerance" as used herein refers to the ability of a plant to endure an biotic stress without suffering a substantial alteration in metabolism, growth, reproduction and/or viability.

[0116] As used herein the phrase "plant biomass" refers to the amount (measured in grams of air-dry or dry tissue) of a tissue produced from the plant in a growing season, which could also determine or affect the plant yield or the yield per growing area.

[0117] As used herein the phrase "plant vigor" refers to the amount (measured by weight) of tissue produced by the plant in a given time. Hence increased vigor could determine or affect the plant yield or the yield per growing time or growing area.

[0118] The term "early vigor" refers to active healthy well-balanced growth especially during early stages of plant growth, and may result from increased plant fitness due to, for example, the plants being better adapted to their environment (optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigor also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (e.g. crops growing in a uniform fashion, such as the crops reaching various stages of development at substantially the same time), and often higher yields. Therefore, early vigor may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.

[0119] As used herein, "seedling vigor" refers to the plant characteristic whereby the plant emerges from soil faster, has an increased germination rate (i.e., germinates faster), has faster and larger seedling growth and/or germinates faster under cold conditions as compared to the wild type or control under similar conditions. Seedling vigor has often been defined to comprise the seed properties that determine "the potential for rapid, uniform emergence and development of normal seedlings under a wide range of field conditions".

[0120] The life cycle of flowering plants in general can be divided into three growth phases: vegetative, inflorescence, and floral (late inflorescence phase). In the vegetative phase, the shoot apical meristem (SAM) generates leaves that later will ensure the resources necessary to produce fertile offspring. Upon receiving the appropriate environmental and developmental signals the plant switches to floral, or reproductive, growth and the SAM enters the inflorescence phase (I) and gives rise to an inflorescence with flower primordia. During this phase the fate of the SAM and the secondary shoots that arise in the axils of the leaves is determined by a set of meristem identity genes, some of which prevent and some of which promote the development of floral meristems. Once established, the plant enters the late inflorescence phase where the floral organs are produced. If the appropriate environmental and developmental signals are present the plant switches to floral, or reproductive, growth. If such signals are disrupted, the plant will not be able to enter reproductive growth, therefore maintaining vegetative growth.

[0121] "Germplasm" refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leafs, stems, pollen, or cells, which can be cultured into a whole plant.

[0122] Plants engineered for improved yield under various biotic and abiotic stresses is of special interest in the field of agriculture. For example, abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al., Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought, floods, salinity, extremes of temperature, chemical toxicity and oxidative stress. The ability to improve plant tolerance to abiotic stress would be of great economic advantage to farmers worldwide and would allow for the cultivation of crops during adverse conditions and in territories where cultivation of crops may not otherwise be possible.

[0123] In some instances plant yield is relative to the amount of plant biomass a particular plant may produce. A larger plant with a greater leaf area can typically absorb more light, nutrients and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period (Fasoula & Tollenaar 2005 Maydica 50:39). Increased plant biomass may also be highly desirable in processes such as the conversion of biomass (e.g. corn, grasses, sorghum, cane) to fuels such as for example ethanol or butanol.

[0124] The ability to increase plant yield would have many applications in areas such as agriculture, the production of ornamental plants, arboriculture, horticulture, biofuel production, pharmaceuticals, enzyme industries which use plants as factories for these molecules and forestry. Increasing yield may also find use in the production of microbes or algae for use in bioreactors (for the biotechnological production of substances such as pharmaceuticals, antibodies, vaccines, fuel or for the bioconversion of organic waste) and other such areas.

[0125] Plant breeders are often interested in improving specific aspects of yield depending on the crop or plant in question, and the part of that plant or crop which is of relative economic value. For example, a plant breeder may look specifically for improvements in plant biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or harvestable parts below ground. This is particularly relevant where the aboveground parts or below ground parts of a plant are for consumption. For many crops, particularly cereals, an improvement in seed yield is highly desirable. Increased seed yield may manifest itself in many ways with each individual aspect of seed yield being of varying importance to a plant breeder depending on the crop or plant in question and its end use.

[0126] It would be of great advantage to a plant breeder to be able to pick and choose the aspects of yield to be altered. It may also be highly desirable to be able to pick a gene suitable for altering a particular aspect of yield (e.g. seed yield, biomass weight, water use efficiency, yield under stress conditions). For example an increase in the fill rate, combined with increased thousand kernel weight would be highly desirable for a crop such as corn. For rice and wheat a combination of increased fill rate, harvest index and increased thousand kernel weight would be highly desirable.

[0127] It has now been discovered that the expression of several forms of trehalose-6-phosphate phosphatase (T6PP) in plants confers a significant increase in yield as well as confer resistance to various types of stress (i.e. abiotic stress). During the course of analyzing various T6PP variants transgenically expressed in plants it has been found that the transgenic plants showing the highest yield in seed also comprised T6PPs with modifications to amino acid residues associated with substrate binding. Not to be limited by theory, these proteins may have decreased activity as compared to a T6PP not containing these modifications. Further, not to be limited by theory, it appears that expressing a T6PP in plant with decreased activity results in a beneficial phenotype having significant increased yield in both stress (e.g. drought) and non-stressed field conditions. T6PP is an enzyme involved in the trehalose biosynthesis pathway. Trehalose, a non-reducing disaccharide consisting of two glucose molecules linked via alpha-1,1 bonds. The sugar trehalose can be found in many various organisms across multiple kingdoms (e.g. plants, bacteria, insects, etc). Trehalose has been shown to be involved in carbohydrate storage function and has been further associated to play a role in stress tolerance in bacteria, fungi and insects. In plants, trehalose was initially thought to be confined to extremophiles such as the resurrection plant Selaginella lepidophylla, however it is now widely accepted that trehalose metabolism is ubiquitous in the plant kingdom.

[0128] Trehalose is synthesized from UDP-glucose and Glucose-6-phosphate in two enzymatic reactions. First UDP-glucose and Glucose-6-phosphate are converted to UDP (uridine diphosphate) and alpha, alpha-trehalose 6-phosphate (T6P) by the enzyme T6PS (trehalose phosphate synthase). In a second step, which is catalyzed by the enzyme T6PP (trehalose phosphate phosphatase), T6P is de-phosphorylated to produce trehalose and orthophosphate.

[0129] In yeast, the two enzymatic activities (T6PS and T6PP activity) reside in a large protein complex, containing the active subunits, T6PS 1 and T6PS2, and the regulatory subunits, with T6PS1 having T6PS activity and T6PS2 having T6PP activity. In E. coli, the two enzymatic activities are found in separate protein complexes. In plants, the protein complex has not been characterized to date.

[0130] In Arabidopsis thaliana, trehalose biosynthetic enzymes have been classified into three classes:

[0131] Class I: containing four genes, AtT6PS1 to AtT6PS4 having high similarity to ScT6PS1;

[0132] Class II: having seven members, AtT6PS5 to AtT6PS1 1, with high sequence similarity to ScT6PS2; and

[0133] Class III:, containing 10 members, AtT6PPA to AtT6PPJ, encoding proteins with similarity to E. coli T6PS2 and the C-terminus of ScT6PS2 proteins.

[0134] Genes encoding proteins within these classes are also present in other plant species.

[0135] Within Class I and Class II, enzymatic activity has only been unambiguously determined for AtT6PS1, which displays T6PS activity (Blazquez et al. Plant J. March 1998;13(5):685-9.). Surprisingly, no T6PP activity has been reported to date for any of the other Class II T6PS proteins. In contrast, T6PP activity was previously described for AtT6PPA and AtT6PPB, two of the members of Class III (Vogel et al. Plant J. March 1998;13(5):673-83). Plant Class III T6PPs contain two phosphatase consensus sequence motifs found in all T6PP enzymes described to date (Thaller et al. Protein Sci. July 1998;7(7):1647-52).

[0136] The genetic manipulation of trehalose biosynthesis genes has been reported to lead to improved stress tolerance in plants, as well as causing striking developmental alterations. Overexpression of E. coli OtsA and OtsB genes (equivalents to T6PP and T6PS) in transgenic tobacco and potato plants was reported to cause developmental aberrations in roots and leaves as well as stunted plant growth. Fewer seeds were produced in the OtsA transgenic tobacco plants and the OtsB transgenic potato plants did not produced tubers (Goddijn et al. Plant Physiol. January 1997;113(1):181-90). Similar results have been described by others (Holmstrom et al. Nature, 379, 683-684; Romero et al. Planta, 201, 293-297; Pilont-Smits et al. 1998; J Plant Physiol. 152:525-532; Schluepmann et al. Proc Natl. Acad. Sci. U S A. 2003;100(11):6849-54). Mutants defective in T6PS and T6PP genes have also reportedly shown developmental defects. T6PS 1 knock out mutants in Arabidopsis showed impaired embryo development (Eastmond et al. Plant J. January 2002; 29(2):225-35). McSteen et. al. (Plant Cell 2006; 18; 518-522) mentions the isolation and characterization of a maize geneRAMOSA3 (RA3) reported to be responsible for meristem development and inflorescence development including branching. It is suggested that the gene, gene product, and regulatory regions may be used to manipulate branching, meristem growth, inflorescence development and arrangement. Negative phenotypes associated with the expression of a transgene can have detrimental effects to a plant's relative yield. For example, without seed set, seed filling, fertility of a plant etc. there would be no increase in seed yield. Patent application U.S. 2007/0006344 is a first account to this application's knowledge of a method which describes the expression of a T6PP in a plant to confer an increase in plant yield without any negative phenotypes and/or detrimental effects to the plant biological function. U.S. Patent Application 2007/0006344 describes the use of a trehalose-6-phosphate phosphatase operably linked to a OsMADS promoter that targets the preferential expression of the T6PP to maternal reproductive tissue of a plant which resulted in a significant yield increase in maize under stress and non-stress conditions. The following invention generally involves the identification of T6PP having modifications that confer improved yield and field efficacy in crop plants and further describes methods one may use to increase yield in a plant by utilizing modified T6PPs in a plant.

[0137] As used herein the term "reduced activity" refers to any decrease in T6PP activity.

[0138] T6PP proteins (at least in their native form) typically have trehalose-6-phosphate phosphatase activity. Polypeptides with trehalose-6-phosphate phosphatase activity belong to the enzymatic class of EC:3.1.3.12, according to the classification of the Enzyme Commission of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). Enzymes in class EC:3.1.3.12 catalyze the reaction: trehalose-6-phosphate+H.sub.2O=trehalose+phosphate. It is contemplated that any T6PP or protein having T6PP activity can be modified for decreased activity via methods such as point mutation(s), irradiation, etc to confer the positive effects as described herein when expressed transgenically in a plant.

[0139] The activity of a trehalose-6-phosphate phosphatase protein may be measured by determining the levels of the substrate processed and the levels of product accumulated in an in vitro reaction, that is, by determining the level of trehalose-6-phosphate consumption and/or trehalose accumulation from the reaction. Enzymatic methods to measure trehalose can be based on hydrolyzing trehalose to glucose, such as those described by Van Dijck et al. Biochem J. August 2002 15;366(Pt 1):63-71 and Zentella et al. Plant Physiol. April 1999;119(4):1473-82.

[0140] Trehalose-6-phosphate levels may also be measured by HPLC (High Performance Liquid Chromatography) methods as described by Avonce et al. Plant Physiol. November 2004; 136(3):3649-59; Schluepmann et al. 2003. Alternative methods based on determining the release of inorganic phosphate from trehalose-6-phosphate have also been described Klutts et al. J Biol Chem. January 2003 24;278(4):2093-100. An alternative method to determine trehalose-6-phosphate levels using liquid chromatography coupled to MS-Q3 (triple quadrupole MS) has been described by Lunn et al. Biochem J. July 2006 1;397(1):139-48.

[0141] It is known that upon stable or transient integration of nucleic acids into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).

[0142] The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.

[0143] The transfer of foreign genes into the genome of a plant, other than by breeding, is called transformation. Any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium -mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium -mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 Al, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens , for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0144] The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

[0145] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.

[0146] Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organization. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

[0147] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.

[0148] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

[0149] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.

[0150] The invention also includes host cells containing an isolated nucleic acid encoding a T6PP protein as defined hereinabove. Preferred host cells according to the invention are plant cells.

[0151] Host plants for the nucleic acids or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageous in all plants, which are capable of synthesizing the polypeptides used in the inventive method.

[0152] A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.

[0153] The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, ears, flowers, stems, and other biological samples. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch, proteins, or an extract derived from corn event MZDT09Y. The biological sample or extract is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn-by-products.

[0154] Reference herein to enhanced yield-related traits is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground. In particular, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of suitable control plants.

[0155] The terms "increase", "improving" or "improve" are interchangeable and shall mean in the sense of the application at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to the wild type plant as defined herein.

[0156] Increased seed yield may manifest itself as one or more of the following: a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per hectare or acre; b) increased number of flowers per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds); e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; and f) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight, and may also result from an increase in embryo and/or endosperm size.

[0157] An increase in seed yield may also be manifested as an increase in seed size and/or seed volume. Furthermore, an increase in seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter.

[0158] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants established per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.

[0159] Since the corn event MZDT09Y plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of control plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigor, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigor. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period) Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.

[0160] Performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding a T6PP protein as defined herein.

[0161] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects. Another abiotic stress may result from a nutrient deficiency, such as a shortage of nitrogen, phosphorus and potassium.

[0162] The methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to confer plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinization are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.

[0163] Performance of the methods of the invention gives plants grown under non-stress conditions or under drought conditions increased yield relative to suitable control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for enhancing yield-related traits in plants grown under non-stress conditions or under drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a modified T6PP polypeptide having decreased substrate binding and/or activity.

[0164] In one embodiment of the invention, the enhanced yield-related trait is manifested as an increase in one or more of the following: total number of seeds per plant, number of filled seeds per plant and seed weight per plant. Preferably, these increases are found in plants grown under non-stress conditions.

[0165] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0166] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0167] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

[0168] The methods according to the present invention result in plants having enhanced yield-related traits, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield- enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.

EXAMPLES

Example 1

Identification and Cloning of the Rice T6PP cDNA Sequence into a Binary Vector

[0169] The first vascular plant trehalose-6-phosphate phosphatase genes were cloned from Arabidopsis thaliana by complementation of a yeast T6PS2 deletion mutant (Vogel et al. 1998). The genes designated AtT6PPA and AtT6PPB (GenBank accessions AF007778 and AF007779) were shown at that time to have trehalose-6-phosphate phosphatase activity. The AtT6PPA and AtTTPB protein sequences were used in TBLASTN queries of maize and rice sequence databases. Sequence alignments organized the hits into individual genes. Three maize and three rice T6PP homologs were identified. The rice T6PP (OsT6PP) cDNA sequence as indicated by SEQ ID NO: 29 was amplified using high-fidelity PCR. The 50 .mu.L reaction mixture consisted of 1 .mu.L rice cDNA library (prepared from callus mRNA in Stratagene's Lambda Unizap Vector, primary library size >1.times.10.sup.6 pfu, amplified library titer>1.times.10.sup.12 pfu/mL), 200 .mu.M dNTPs, 1 .mu.L 20 .mu.M of oligonucleotide primer T6PP-EC-5 (5'-catggaccatggatttgagcaatagctcac-3') and 1 .mu.L 20 .mu.M of oligonucleotide primer T6PP-EC-3 (5'-atcgcagagctcacactgagtgcttcttcc-3'), 5 .mu.L 10.times.Cloned PFU buffer and 2.5 Units of Pfuturbo DNA polymerase. The thermocycling program was 95.degree. C. for 2 minutes followed by 40 cycles of (94.degree. C. for 15 seconds, 50.degree. C. for 1 minute, 72.degree. C. for 1 minute) followed by 72.degree. C. for 10 minutes. The rice T6PP product was cloned with the Zero Blunt TOPO PCR cloning kit. The pCR-Blunt-II-TOPO-OsT6PP is identified by digesting 5 .mu.L pCR-Blunt-II-TOPO-OsT6PP miniprep DNA with EcoRI in a 20 .mu.L reaction containing 2 .mu.g BSA and 2 .mu.L 10.times.EcoRI restriction endonuclease buffer. The reaction is incubated at 37.degree. C. for 2 hours and the pCR-Blunt-II-TOPO-OsT6PP (EcoRI) products are resolved on 1% TAE agarose. The pCR-Blunt-II-TOPO-OsT6PP clone is then sequenced. The OsT6PP cDNA is flanked by NcoI/SacI restriction endonuclease sites. The OsT6PP was then further cloned into a binary vector as described in Example 8 of U.S. Patent Application Publication 2007/0006344 (therein referred to OsT6PP-3 and indicated by nucleotide SEQ ID NO: 531 and protein SEQ ID NO: 532)

Example 2

Initial Evaluation of Rice T6PP Maize Events in the Greenhouse

[0170] Rice T6PP maize events comprising SEQ ID NO: 1 operably linked to a promoter having preferential expression in maternal reproductive tissue (i.e. OsMADS promoter) were generated and further evaluated in both the greenhouse and field as described in Examples 8-13 in U.S. Patent Application Publication 2007/0006344. Initial greenhouse and field evaluation of the maize events indicated some events having a yield increase in both non-drought and drought conditions (See U.S. Patent Application Publication 2007/0006344 herein incorporated by reference).

Example 3

Evaluation and Identification High Yielding T6PP Maize Events

[0171] The maize events shown to confer a yield increase in the trials described in Example 2 and more specifically in U.S. Patent Application Publication 2007/0006344, were further characterized for yield and field efficacy. These events contained either binary construct 15777 (SEQ ID NO: 30) or 15769 (SEQ ID NO: 31) as is described in U.S. Patent Application Publication 2007/0006344. Essentially binary construct 15769 comprises an expression cassette having a OsT6PP (indicated in SEQ ID NO: 29 of the current application) operably linked to a OsMADS6 promoter. Binary construct 15777 contains the same expression cassette (OsMADS6 promoter and OsT6PP coding sequence) with the addition of transcriptional enhancers upstream of the OsMADS6 promoter. The details and specifics of both these constructs may again be found in the U.S. Patent Application Publication 2007/0006344. Overall there were 645 TO maize events generated from 15769 and 587 maize events were generated comprising the 15777 binary construct. Following the course of generation of transgenic events during plant transformation, selection of events having successfully integrated into the genomic DNA, as well as growth in greenhouse and field conditions relatively a small number of events were carried forward for field trials based on the selection criteria as well as plant event survival and phenotype criteria. The relatively high level of attrition led to only 17 events showing field efficacy. Events derived from maize plants comprising the 15777 binary construct proved to be most efficacious in the field testing. Two events, MZDT09Y and MZDT08H, were selected based upon viability and performance in managed stress environments (yield preservation under drought at flowering) and in agronomic trials which measured yield. Overall, best performing events comprising construct 15777 demonstrated a significant bushel per acre yield advantage over control check samples.

Example 4

Corn Events MZDT09Y and MZDT08H Sequence Analysis

[0172] Corn event MZDT09Y and corn event MZDT08H were further analyzed by sequencing of the T6PP CDS. PCR was used to amplify the integrated OsT6PP coding sequence using primers that anneal to the 5' and 3' region of the coding sequence. The respective PCR amplicons resulted in the approximate 1.1 Kb band size as would be expected from the coding sequence of the OsT6PP as depicted in SEQ ID NO: 29 which was the sequence that was comprised in the relative expression cassette. The amplicons were further sequenced as is well established in the art. Sequencing data indicated that both events contained modifications. MZDT09Y contained a single point mutation at nucleotide 730 respective to SEQ ID NO: 29 (T*CATTA where * indicates point of mutation). This single mutation led to an amino acid mutation changing a His residue to an Asp residue. MZDT08H was found to contain two modifications at nucleotides 305 (TG*CTTCC where * indicates point of mutation) and 388 (CGCC*ATT where * indicates point of mutation) respective to SEQ ID NO: 29. Interestingly, these mutations are located in highly conserved domains of the T6PP protein.

[0173] Primer pairs used to sequence the heterologous insert are as follows: 09Y-IS-1 (SEQ ID NO: 26) was amplified using forward primer 09Y-RBFS-F1 (SEQ ID NO: 16) and reverse primer 09Y-IS-R1 (SEQ ID NO: 18); 09Y-IS-2 (SEQ ID NO: 27) was amplified using forward primer 09Y-IS-F2 (SEQ ID NO: 19) and reverse primer 09Y-IS-R2 (SEQ ID NO: 20); 09Y-IS-3 (SEQ ID NO: 28) was amplified using forward primer 09Y-IS-F6 (SEQ ID NO: 21) and reverse primer 09Y-IS-R6 (SEQ ID NO: 22).

[0174] The modified T6PP enzyme (SEQ ID NO: 11) of corn event MZDT09Y has approximately 10% activity relative to the unmodified T6PP enzyme, and yet corn event MZDT09Y is not affected by yield drag. However, the modified T6PP enzyme of MZDT08H does impart a slight yield drag on the corn plant. For this surprising and unexpected result, corn event MZDT09Y was selected for progression.

Example 5

Methods of Detecting Corn Event MZDT09Y by PCR

[0175] Corn event MZDT09Y can be detected by assaying for a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Polymerase chain reaction (PCR) is a standard method of amplifying DNA practiced by those of skill in the art, provided that the sequence of the template DNA is known. SEQ ID NOs: 1-4 are completely unique to MZDT09Y and are disclosed herein for the first time. Primer pairs different from those primers explicitly disclosed herein may be developed by one of ordinary skill in the art and still achieve the same function, that is, to identify corn event MZDT09Y.

[0176] Primers SEQ ID NO: 12 (forward primer MZDT09Y-RB1) and SEQ ID NO: 13 (reverse primer ESPCR0001) function together in a PCR reaction to produce a right border amplicon comprising SEQ ID NO: 23 and its complementary sequence, which is indicative of the presence of MZDT09Y template DNA. This amplicon also comprises SEQ

[0177] ID NO: 1 and SEQ ID NO: 3.

[0178] Primers SEQ ID NO: 14 (forward primer 09Y-LBFS-F1) and SEQ ID NO: 15 (reverse primer 09Y-LBFS-R4) function together in a PCR reaction to produce a left border amplicon comprising SEQ ID NO: 24 and its complementary sequence, which is indicative of the presence of MZDT09Y template DNA. This amplicon also comprises SEQ ID NO: 2 and SEQ ID NO: 4.

[0179] In addition to conventional, gel-based PCR, TaqMan.RTM. PCR (Invitrogen.TM.) which uses fluorescence to enable detection of amplification in real-time, may also be used to detect the presence of corn event MZDT09Y. Forward primer P23198 (SEQ ID NO: 32), reverse primer P23352 (SEQ ID NO: 33), and probe P23200 (SEQ ID NO: 34, labeled with FAM on its 5'-terminus, and with BHQ1 on its 3'-terminus) function together in a TaqMan PCR reaction in the presence of MZT09Y template DNA to produce an amplicon (SEQ ID NO: 35), and thereby fluorescence, diagnostic of corn event MZDT09Y.

Example 6

Methods of Detecting Corn Event MZDT09Y by Hybridization

[0180] Corn event MZDT09Y can be detected by assaying using hybridization techniques for a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Methods of hybridization, for example a Southern blot, can detect DNA, provided that the sequence of the template DNA is known. SEQ ID NOs: 1-4 are completely unique to MZDT09Y and are disclosed herein for the first time.

[0181] By way of example and not limitation, DNA samples are cut with restriction enzymes and run overnight on an agarose gel in 1.times.TBE buffer at about 32 volts. Gels are photographed, washed, and blotted onto nylon membrane with 10.times.SSC as the transfer solution. They are linked to the membrane with UV light and pre-hybridized with calf thymus DNA at 65.degree. C. Probes comprising SEQ ID NO: 1 or SEQ ID NO: 2 are labeled with radioactive Phosphorus 32. Probes are added and hybridized at 65.degree. C., 3 hrs to overnight. Blots are washed several times and exposed in a phosphorimager cassette. Images are developed and scored.

Deposit

[0182] Applicants have made a deposit of corn seed of event MZDT09Y disclosed above on Jun. 28, 2012, in accordance with the Budapest Treaty at the American Type

[0183] Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., 20110, USA under ATCC Accession No. PTA-13025. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or the effective life of the patent, whichever is longer, and will be replaced as necessary during that period. Applicants impose no restrictions on the availability of the deposited material from the ATCC; however, Applicants have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicants do not waive any infringement of their rights granted under this patent or under the Plant Variety Protection Act (7 USC 2321 et seq.).

[0184] All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent document was specifically and individually indicated to be incorporated by reference.

[0185] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to those of skill in the art that certain changes and modifications may be practiced within the scope of the appended claims.

Sequence CWU 1

1

35120DNAZea maysmisc_feature(1)..(20)5' genome-insert junction 1gtcgtggagg aaacactgat 20220DNAZea maysmisc_feature(1)..(20)3' insert-genome junction 2gttattaagt cggggagggg 20360DNAZea maysmisc_feature(1)..(60)longer 5' genome-insert sequence 3ggaggggagc gtgagttgga gtcgtggagg aaacactgat agtttaaact gaaggcggga 60460DNAZea maysmisc_feature(1)..(60)longer 3' insert-genome junction 4catggccgta tccgcaatgt gttattaagt cggggagggg agcgtgagtt gtagtcgcgg 6051024DNAZea maysmisc_feature(1)..(1024)5' genomic flanking sequence 5cttgaaagga tgaaccatga aaatttagtt gcgatgtttc catgtagtga accgagatgt 60cttatcagta agtaaatagg ttaacatcaa gaaagcacat ttaataaaaa attggaaagg 120ggacatacat taattaacta tctaaaagta gtacacagaa ttagacattt agacataata 180tggtttgcta aatacgtagt aatctatata aacaaaatta aagttgtgta tttctaagac 240atgcatacgt acacaaacag aacatattga aactagcctt tgttgaacaa tttttaacag 300caaatgtggt tttggtttgt taatattgca agttattatg agtattcaac ctagtaaatt 360gaataatcct tgcctgatat attgaatcta tttgtatata tctagctaga tacaattatt 420agaacatgct tttattgtct acatatgtac ccatgtaaca tgaatagaaa ataaataggt 480atgcttatcg tagagtatct atgaccatgt agcaagaaaa ataagttaat atagagataa 540atcaatgggc aaaaaataaa atttggggga aagaatctca ctgctactac tcttccacta 600caaagaacaa caaggttggt tccaattcca cttccggaac aatcagcgca attgctccat 660ccatccacag ctgccaccga gactaggtga cacgatccaa gccgccatgg cgtcggctaa 720gcctctacgc tctagactgt gctggtggca ctagggagac tacgaaggtt cacctgcacc 780ttgaaggact agttgaggaa ggtgttggcg aggatgttga ggggtttgga cgatgaagca 840ccgtctaggg aacaattgtt ctggcccgtg acgaggtgct gcaacgccat ctccgccgcc 900ggagagacgt gcacgctctc tgacgtcaca tgtgagaggc tatggttgta gcttcaatcg 960ttggcggtgt gggctggtcc ggctcgatgt gaggggaggg gagcgtgagt tggagtcgtg 1020gagg 102461037DNAZea maysmisc_feature(1)..(1037)3' genomic flanking sequence 6cggggagggg agcgtgagtt gtagtcgcgg tgtgatcttg acccgacgca tgatttaaca 60ggaggtgact cgtgaacctt ggacgacgat cgtgtggctg ggaaaccaag ggatgatatg 120gacgcctcga gtgacgagat ttgctcgtag ctctattgat agcaggggcg aagctacgac 180ttgttgatgg tgtcaaatgg caccgataag ttttagaaaa tacaatgtaa aatattgttt 240acaatgttga tggcattcct ataaaactgt gctgacacta gcaaaactta aggctggctt 300cgccccttat tgatagtaag gctatggcct ggagatcatg tatataatcg tttaaaatat 360tattttatat tatagactaa gttatgtata tagtaaaata aaatttaaaa tagaagatgg 420aacgagcaag gagataacct aatgtgctga gacatgagag cgttatgccg gaccgggcca 480gaatcttcgc gcctccccgg tccacagaaa ccgcggtccg ccagagcgcg gcgcgcccag 540cgtccctgct cccaaagtcc catgcggcat gcgcccggcc caattggaaa ataaagcacc 600gccgcatatc ccgccctttc cttttgtcgg atggcgcgtc acggcgtttt tccccataaa 660aaattcagaa atatataaac accagacatg cgcccgcgcg ctcttccctc cctgccgccc 720gcgtcctcct ctccacacca atgcggccac caccgcacgt cgccgcctcc acctcgcctg 780ccctccacgc cgggtcgtcc cggcgccggc ctcaccaccg cggccggacg atgatgtccg 840tctcctgctg ccacttggcg gcgctcgtgt ccatcctcgc ggcgggcgcg gctacggcgt 900tgctcacctt gtccctaccc tcttctcctg gcgcgtccac gacgaggaga accgacttcg 960ttggagcatt gtcagtggca aacgagaccc cgccaccgcc gcacttgtca gcgcctccgg 1020cacccgccac gccgccc 1037710435DNAArtificial SequenceHeterologous inserted DNA from plasmid 15777 7aaacactgat agtttaaact gaaggcggga aacgacaatc tgatcatgag cggagaatta 60agggagtcac gttatgaccc ccgccgatga cgcgggacaa gccgttttac gtttggaact 120gacagaaccg caacgctgca ggaattggcc gcagcggcca tttaaatcaa ttgggcgcgc 180cagctgcttg tggggaccag acaaaaaagg aatggtgcag aattgttagg cgcacctacc 240aaaagcatct ttgcctttat tgcaaagata aagcagattc ctctagtaca agtggggaac 300aaaataacgt ggaaaagagc tgtcctgaca gcccactcac taatgcgtat gacgaacgca 360gtgacgacca caaaactcga gacttttcaa caaagggtaa tatccggaaa cctcctcgga 420ttccattgcc cagctatctg tcactttatt gtgaagatag tggaaaagga aggtggctcc 480tacaaatgcc atcattgcga taaaggaaag gctatcgttg aagatgcctc tgccgacagt 540ggtcccaaag atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc 600acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgaacaa 660tcccactatc cttcggtacc ggaccgctag gacgatggtg tgatgtggga acacgaagaa 720aacatgagga aaaaatatta aaatgaattt cccacttaaa atgcatcaaa taaaaaaaat 780aaagaaacga ccgggaatag acacagggtt tgtgaactag ctagggcaaa catcatatgg 840tcccttgctg atgcacaagt acattgagat gtcatttcaa ttctgtgcat catatgcatg 900tggtcccttg ctgaatatta ctcttgaaat atctaccagt gccaatctat tgcatgactt 960aattaattca caggttttgt tgattacatt attagtaagc ttgagagcac aagctcaatg 1020gatttttcta taaatgggga tcattttgca attttctttg tcgtgcaaag ttagccttct 1080ttattactac ttctgttttt aaatatacga tcctattgac ttttggtcat atatttaacc 1140atgtatctta tttagatagt ttgcgcaaat atatatacct tcaatgataa aattagttac 1200aatgaaacaa atgatattta cgcaattctt tttactaaac aagtcacaag aagtacctgc 1260agcaatatat gttggaaccg tgcagtagat cgagcctagc tacgcaaaaa aacaaaaaga 1320gaaaaaaagg gaaaggaaaa acattaatca tgcatgagca gtatgcccgg caactggaat 1380ttgtcaaaga tatggggaga ggagaataat acaagtacta ctactaccta gctctaccat 1440gcatatgcac ccaaaggcaa actggattat tggataaagc acagatgctg gcaaaacaat 1500ccttaagcct cccctccctg cttctttatt tttgggcagc ctctaccgga cggtgccgtg 1560gtccattgga ccagtaggtg gcgacataca tggtttgggt taagtctagg agagcagtgt 1620gtgtgcgcgc gcaagagaga gagactgtga gtctgggagt agccctctcc cctcctttgg 1680ccatcttcct cgtgtatatg catatatgca tcatcgcaac ggtgtatatt tgtggtgtgg 1740cgggtgtggc attggattgc ccccattttg gctcgtgctt cccagttagg gtaaaacctg 1800tggtaaactt gctagcccca cgccaaagtt acccttcttt attgttgaaa gggagaggag 1860gtgtgtgaat tgtgatggag ggagagagag agagatagaa agagagatgt gtgtcaaagc 1920aagcaagaaa ccagtttcac aaagagctac tactagtact agtgtactac tgtggtacag 1980tgcccaatgt cctttctccg gactcgactc cactaatatt ctcctcttct cgcgcggctc 2040gttatattct cgtcatcatt ggaggcttta gcaagcaaga agagaggcag tggtggtggt 2100ggtggaggag gagctagcta gcctgtgctt gctgatcggt gctgagctga ggaatcgttc 2160gatcgatcgg gcgagtcgac gaggggaaga gttgagctga ggcgcatcga gaacaagatc 2220aacaggcagg tcaccttctc caagcgccgc aacggcctcc tcaagaaggc ctacgagctg 2280tccgttctct gcgacgccga ggtcgcgctc atcatcttct ccagccgcgg caagctctac 2340gagttcggca gcgccgggta taattaatac agacacaaca acacacacaa ccaacaaacc 2400agcatcaatt tgaacctgca gatctgctgt tttctctgat caattgcttc tttttttttg 2460ttcttttttg tttcttttat ctgctgcaac ggcgtcctgc tcctctgggg tttctcgttt 2520tctttttcat ttatttttag caggtgccaa gtagccgagc tactatactt acctggccat 2580gttaattatt ttattccgtc tgtctgtgtg tgtctgtgca tactactata gggacatggc 2640gcggtgttct tataaaccgg gaggccggat ccctaactag catgggagga tatcttttca 2700gcggatctat acaaacccta ctcctgctga cctctttctt ccagtttctc cgggtcttcc 2760ttggattatt attgcccatc ttccgggttg tgcgtgtgtc agagacagct cgaacgataa 2820atttctcaaa accagtacta gagagggtgt gttgtgtgtg agaactgagt ggagagttag 2880catgaaggct gcaaactaga aaggaaggta tgttctttcc tttttgatcc atcaggggag 2940ccccttctgg tattaagatc tttccggcac attgattttc atactttgtg atgaccctgg 3000aagaatcggc gtagcagcgt agcaccgctc cattttggtc ttaccctcac ctccccatgc 3060tatgaactga tcaatttcat tgttcttcat cacccttctc ctagctttcc acttccttcg 3120gatctcatgc catgtttctc agcatgaatc aaatttaatt cgtgttttct acttccatat 3180atactggaag aaatttaatt agatctattt ttgctcggga ggtcttcata ctttgagttc 3240tgatgccatc accttatttc cccccccccc ttctcttgtt ctatcttctt cctcatcttg 3300gcttgatcat tttgatctgt cagttatagc atgatgcatt ctcaatttga ctgtatgtaa 3360gttcaaccgg aaatatgttg aatggatttt ctatatatca acacttgatg tcaggcctgc 3420atctgtttcg cttgtggtgg tgtggccaaa attgtctata tttgatcttt gctcttcttt 3480ctcctcattt catgacgatt cctactacgg cttaaaccat tctttattct ttactaatca 3540tggatgttgc ttgactccta gttgtttcgt actagctcaa cttggagatc ttttcattat 3600ttgcctagtt ggtgggtacg tttgtgacag atctaaaatg gtgcacgaaa agttttactt 3660attatgaaaa aagggagctt aacagggtaa tttctctatt tattcgtgat gacatttttt 3720ccttgataag ggggattttt tataatctgc actcacatgt ttatatgtaa aatctagctc 3780ttttgttttg tttttggcat atttcccgct aagtatagag tttatgtgga taacattata 3840acttttcaag atccaatcca catctttgat tgtgaaaatc atacaatagg gaaaatcaac 3900tgaagggtta attagatgct atatgcatat atatatatat gtgcgcgcgc gcgcgcctga 3960atttaactat gtatgcatcc aactgtttca ttgaaaaaga tttgatattt ttcagtctat 4020tctttttcga gtatatatct aatatgtttc aatctgtttt gaccattata agataaagcc 4080tatattcacc aggcatttga gatgatcttt tcatgcatga aaaagctgtt gttatcactt 4140caactaacca gacgatctaa catgtatttg tataagaaac agaccttgat ttccttctgt 4200aaaatcatgc atgtgttcgt tttgaattgg agtcggcgcg cctgtgtttt gaccgtcagg 4260aaagtctttt ttttccctga atagtcaagg gtctatactt cttgaagcaa ttgggacact 4320aatcaattat tgtttatacc tcggaccatc ttttccttct tcacaccact aatcagttta 4380tgccttggac cattaattgt gttgttcaca agcttcttgt ttatggttta caaagcattc 4440gcctagattt gtgtgtgtct ctacacatcg atcactttta aatacttgtc gctttcagtt 4500attcttttaa cgtttggtta tttatcttat ttaaaaaaat tatcgtatta ttatttattt 4560tgtttgtgat ttactttatt atcaaaagta tttcaaatat gacttatctt tttttataag 4620tgcactaatt tttcaaataa gatgaatggt caaatgttac aagaaaaagt taaagcaacc 4680actaatttag ggcggaggta gtaaaaccta gttattgtaa ccaataattt tatcaatcta 4740taaatgcaac acaaagtaac ttcgtgatat ctaacacaaa gccacttcaa cgatgaaagc 4800tgactgcatg ttttatcaaa acacatgtga tcagtttgtt ggatgaaaaa aattatctat 4860gtcataaatc aagagttata atataagctt ctggctctac aagtaacatt tctatgtttt 4920tttttacgtt cttacatact atgttttgcc aaaaaaaaca tgatcatttt gttggacgaa 4980aagaaatagt aaatatagag tgacctttga tatcattata atataagctt ctgcctctat 5040aaataacatc tatgcacttt ttacgtcgta gtaatttgat atatgagaaa tttacatata 5100acatttttgt gcagcataac caccatggat ttgagcaata gctcacctgt catcaccgat 5160ccggtggcga tcagccagca gttgttgggc ggcctgcctt caaatctgat gcagttttca 5220gtcatgcccg gtggctactc cagctctggc atgaacgttg gtgtcagtag gctcaaaatc 5280gaggaagtcc ttgtcaatgg actgcttgat gccatgaaat cctcgtcacc tcgcaggagg 5340ctgaatgtag catttggcga ggacaattca tctgaagaag aagaccctgc ttacagcgct 5400tggatggcaa aatgtccttc tgctttggct tccttcaagc aaattgtagc cagtgcacaa 5460gggaagaaga ttgctgtgtt tctagactat gacggcacac tgtcgcctat tgtggatgat 5520cctgacaaag cagtgatgtc tcccgtgatg agagctgctg tgagaaatgt tgcgaagtac 5580ttccccactg caattgtcag cggaaggtcc cgcaataagg tgtttgaatt tgtaaaactg 5640aaggagcttt attatgctgg aagtcatggt atggacataa tggcaccttc agcaaatcat 5700gagcacagtg ctgaaaagag caaacaggcc aatctcttcc aacctgcaca cgactttctg 5760ccaatgatcg atgaggttac caagtccctc ttgcaagttg tcagtggaat tgaaggtgca 5820actgttgaga acaacaaatt ctgcgtttct gtagattatc gcaacgttgc agagaaggat 5880tggaaactgg tcgcacggct cgtaaacgaa gtgctggagg cttttcctcg tctcaaagta 5940accaatggac gaatggtttt agaggttcgt ccggtgatcg actgggacaa gggaaaggct 6000gtggagtttc tgctccagtc actcgggcta aatgactctg aaaatgtgat ccccatctac 6060attggagacg acagaactga cgaagacgct ttcaaggtac ttcgacagcg aaattgcggt 6120tatggaatac tagtttcaca ggttcccaag gaaactgaag ccttctactc gctgagagat 6180ccatctgaag tgatggagtt cctcaatttc ttggtgagat ggaagaagca ctcagtgtga 6240gctcgctaag cagccatcga tcagctgtca gaagttggag ctaataataa aagggatgtg 6300gagtgggcta catgtatctc ggatctctct gcgagccacc taatggtctt gcgtggccct 6360ttaatctgta tgtttttgtg tgtaagctac tgctagctgt ttgcaccttc tgcgtccgtg 6420gttgtgtttc cgtgctacct ttttatgttt tgatttggat cttgtttgaa aataatctta 6480ccagctttgg gtaaactgtt tattacgtac tctatatagc atatgtgacc gacgacaacg 6540gtttcatttt agatgatgtg tatggatgat ttctttccaa aatcacatct ttagtataag 6600agcaatttta ccatccaata ccaaatttta tactagaaaa tattttggga tatcaaaatt 6660tatggtacct ccagtaccaa atgttgaatg gtaaactttc ataatataca agtcactcta 6720ggatatttaa gacaattttt agttttttct tattgttgcc cttgttaaat acatgagaaa 6780ttttacatca cttaaaatgt atcaagaggt atcaaatttt tttaatacaa aatttagtac 6840tttctccgtt tatatatgaa tgtggacaat gcttgaaagt cttataacct gaaactgagg 6900tagtgtatcg agaagtacaa aattttacac taaaatccca gtacttactc aataactgta 6960aaattactct aaatatgtac tccctctatt tcagattata agtcgtttta actttagtca 7020aagttaaact gtttcaagtt taaccaagtt tgtagataaa agtagtaaca tattcaacac 7080aagacaaata tattataaaa acatattgaa ttatagattt aattaaatta atttggtatt 7140gcaagtatta ctaaatttgt ttataaattt ggtcgaattt aaaatagttt gactttaacc 7200aaagtcaaaa caaattataa tctaaaacaa aggtaataca ttgtatcact ctcatgaatg 7260gattgtaaca tacattaatt taattactat tttagttctt gtgcaaaagt tgaaaacgat 7320ttatgtttgg aatctttttg tggtgtatat atatgaaacc attcctctac catccttccc 7380caaccataat cctcacaacc gttagcccca ttgtgatctc acccagttgc tagcctcttt 7440tgtcaccttg tcacagctct cctccattca ttacacaatg gcatcggacc gcgatcgctt 7500aattaagctt gcatgcctgc agtgcagcgt gacccggtcg tgcccctctc tagagataat 7560gagcattgca tgtctaagtt ataaaaaatt accacatatt ttttttgtca cacttgtttg 7620aagtgcagtt tatctatctt tatacatata tttaaacttt actctacgaa taatataatc 7680tatagtacta caataatatc agtgttttag agaatcatat aaatgaacag ttagacatgg 7740tctaaaggac aattgagtat tttgacaaca ggactctaca gttttatctt tttagtgtgc 7800atgtgttctc cttttttttt gcaaatagct tcacctatat aatacttcat ccattttatt 7860agtacatcca tttagggttt agggttaatg gtttttatag actaattttt ttagtacatc 7920tattttattc tattttagcc tctaaattaa gaaaactaaa actctatttt agttttttta 7980tttaataatt tagatataaa atagaataaa ataaagtgac taaaaattaa acaaataccc 8040tttaagaaat taaaaaaact aaggaaacat ttttcttgtt tcgagtagat aatgccagcc 8100tgttaaacgc cgccgacgag tctaacggac accaaccagc gaaccagcag cgtcgcgtcg 8160ggccaagcga agcagacggc acggcatctc tgtcgctgcc tctggacccc tctcgagagt 8220tccgctccac cgttggactt gctccgctgt cggcatccag aaattgcgtg gcggagcggc 8280agacgtgagc cggcacggca ggcggcctcc tcctcctctc acggcaccgg cagctacggg 8340ggattccttt cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat aaatagacac 8400cccctccaca ccctctttcc ccaacctcgt gttgttcgga gcgcacacac acacaaccag 8460atctccccca aatccacccg tcggcacctc cgcttcaagg tacgccgctc gtcctccccc 8520cccccccctc tctaccttct ctagatcggc gttccggtcc atagttaggg cccggtagtt 8580ctacttctgt tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc tgttagcgtt 8640cgtacacgga tgcgacctgt acgtcagaca cgttctgatt gctaacttgc cagtgtttct 8700ctttggggaa tcctgggatg gctctagccg ttccgcagac gggatcgatt tcatgatttt 8760ttttgtttcg ttgcataggg tttggtttgc ccttttcctt tatttcaata tatgccgtgc 8820acttgtttgt cgggtcatct tttcatgctt ttttttgtct tggttgtgat gatgtggtct 8880ggttgggcgg tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat 8940taattttgga tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg 9000atggaaatat cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac 9060agagatgctt tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc 9120gttctagatc ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac 9180tgtatgtgtg tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc 9240taggataggt atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca 9300gcatctattc atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt 9360ataattattt tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt 9420tttttagccc tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct 9480caccctgttg tttggtgtta cttctgcagg gatccactag tccaccatgt ctccggagag 9540gagaccagtt gagattaggc cagctacagc agctgatatg gccgcggttt gtgatatcgt 9600taaccattac attgagacgt ctacagtgaa ctttaggaca gagccacaaa caccacaaga 9660gtggattgat gatctagaga ggttgcaaga tagataccct tggttggttg ctgaggttga 9720gggtgttgtg gctggtattg cttacgctgg gccctggaag gctaggaacg cttacgattg 9780gacagttgag agtactgttt acgtgtcaca taggcatcaa aggttgggcc taggatccac 9840attgtacaca catttgctta agtctatgga ggcgcaaggt tttaagtctg tggttgctgt 9900tataggcctt ccaaacgatc catctgttag gttgcatgag gctttgggat acacagcccg 9960gggtacattg cgcgcagctg gatacaagca tggtggatgg catgatgttg gtttttggca 10020aagggatttt gagttgccag ctcctccaag gccagttagg ccagttaccc agatctgaac 10080tagtgatatc ggcgccatgg gtcgacctgc agatcgttca aacatttggc aataaagttt 10140cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 10200cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 10260gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 10320ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatctgctag ccctgcagga 10380aatttaccgg tgcccgggcg gccagcatgg ccgtatccgc aatgtgttat taagt 10435812740DNAArtificial SequenceHeterologous insertion sequence from 15777, plus the 5' and 3' flanking sequences. 8gcacaacata attaaagttg ataccttatg atattcgtat tagatgtatt tgaggttaaa 60ttattattac tggtagtatt aacagaaaat atattaattt aaaaggtgta aagctataaa 120agtataaagg aatatgttaa ttatagtttt tcttgaaagg atgaaccatg aaaatttagt 180tgcgatgttt ccatgtagtg aaccgagatg tcttatcagt aagtaaatag gttaacatca 240agaaagcaca tttaataaaa aattggaaag gggacataca ttaattaact atctaaaagt 300agtacacaga attagacatt tagacataat atggtttgct aaatacgtag taatctatat 360aaacaaaatt aaagttgtgt atttctaaga catgcatacg tacacaaaca gaacatattg 420aaactagcct ttgttgaaca atttttaaca gcaaatgtgg ttttggtttg ttaatattgc 480aagttattat gagtattcaa cctagtaaat tgaataatcc ttgcctgata tattgaatct 540atttgtatat atctagctag atacaattat tagaacatgc ttttattgtc tacatatgta 600cccatgtaac atgaatagaa aataaatagg tatgcttatc gtagagtatc tatgaccatg 660tagcaagaaa aataagttaa tatagagata aatcaatggg caaaaaataa aatttggggg 720aaagaatctc actgctacta ctcttccact acaaagaaca acaaggttgg ttccaattcc 780acttccggaa caatcagcgc aattgctcca tccatccaca gctgccaccg agactaggtg 840acacgatcca agccgccatg gcgtcggcta agcctctacg ctctagactg tgctggtggc 900actagggaga ctacgaaggt tcacctgcac cttgaaggac tagttgagga aggtgttggc 960gaggatgttg aggggtttgg acgatgaagc accgtctagg gaacaattgt tctggcccgt 1020gacgaggtgc tgcaacgcca tctccgccgc cggagagacg tgcacgctct ctgacgtcac 1080atgtgagagg ctatggttgt agcttcaatc gttggcggtg tgggctggtc cggctcgatg 1140tgaggggagg ggagcgtgag ttggagtcgt ggaggaaaca ctgatagttt aaactgaagg 1200cgggaaacga caatctgatc atgagcggag aattaaggga gtcacgttat gacccccgcc 1260gatgacgcgg gacaagccgt tttacgtttg gaactgacag aaccgcaacg ctgcaggaat 1320tggccgcagc ggccatttaa atcaattggg cgcgccagct gcttgtgggg accagacaaa 1380aaaggaatgg tgcagaattg ttaggcgcac ctaccaaaag catctttgcc tttattgcaa 1440agataaagca gattcctcta gtacaagtgg ggaacaaaat aacgtggaaa agagctgtcc 1500tgacagccca ctcactaatg cgtatgacga acgcagtgac gaccacaaaa ctcgagactt 1560ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 1620ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 1680gaaaggctat cgttgaagat

gcctctgccg acagtggtcc caaagatgga cccccaccca 1740cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 1800gtgatatctc cactgacgta agggatgacg aacaatccca ctatccttcg gtaccggacc 1860gctaggacga tggtgtgatg tgggaacacg aagaaaacat gaggaaaaaa tattaaaatg 1920aatttcccac ttaaaatgca tcaaataaaa aaaataaaga aacgaccggg aatagacaca 1980gggtttgtga actagctagg gcaaacatca tatggtccct tgctgatgca caagtacatt 2040gagatgtcat ttcaattctg tgcatcatat gcatgtggtc ccttgctgaa tattactctt 2100gaaatatcta ccagtgccaa tctattgcat gacttaatta attcacaggt tttgttgatt 2160acattattag taagcttgag agcacaagct caatggattt ttctataaat ggggatcatt 2220ttgcaatttt ctttgtcgtg caaagttagc cttctttatt actacttctg tttttaaata 2280tacgatccta ttgacttttg gtcatatatt taaccatgta tcttatttag atagtttgcg 2340caaatatata taccttcaat gataaaatta gttacaatga aacaaatgat atttacgcaa 2400ttctttttac taaacaagtc acaagaagta cctgcagcaa tatatgttgg aaccgtgcag 2460tagatcgagc ctagctacgc aaaaaaacaa aaagagaaaa aaagggaaag gaaaaacatt 2520aatcatgcat gagcagtatg cccggcaact ggaatttgtc aaagatatgg ggagaggaga 2580ataatacaag tactactact acctagctct accatgcata tgcacccaaa ggcaaactgg 2640attattggat aaagcacaga tgctggcaaa acaatcctta agcctcccct ccctgcttct 2700ttatttttgg gcagcctcta ccggacggtg ccgtggtcca ttggaccagt aggtggcgac 2760atacatggtt tgggttaagt ctaggagagc agtgtgtgtg cgcgcgcaag agagagagac 2820tgtgagtctg ggagtagccc tctcccctcc tttggccatc ttcctcgtgt atatgcatat 2880atgcatcatc gcaacggtgt atatttgtgg tgtggcgggt gtggcattgg attgccccca 2940ttttggctcg tgcttcccag ttagggtaaa acctgtggta aacttgctag ccccacgcca 3000aagttaccct tctttattgt tgaaagggag aggaggtgtg tgaattgtga tggagggaga 3060gagagagaga tagaaagaga gatgtgtgtc aaagcaagca agaaaccagt ttcacaaaga 3120gctactacta gtactagtgt actactgtgg tacagtgccc aatgtccttt ctccggactc 3180gactccacta atattctcct cttctcgcgc ggctcgttat attctcgtca tcattggagg 3240ctttagcaag caagaagaga ggcagtggtg gtggtggtgg aggaggagct agctagcctg 3300tgcttgctga tcggtgctga gctgaggaat cgttcgatcg atcgggcgag tcgacgaggg 3360gaagagttga gctgaggcgc atcgagaaca agatcaacag gcaggtcacc ttctccaagc 3420gccgcaacgg cctcctcaag aaggcctacg agctgtccgt tctctgcgac gccgaggtcg 3480cgctcatcat cttctccagc cgcggcaagc tctacgagtt cggcagcgcc gggtataatt 3540aatacagaca caacaacaca cacaaccaac aaaccagcat caatttgaac ctgcagatct 3600gctgttttct ctgatcaatt gcttcttttt ttttgttctt ttttgtttct tttatctgct 3660gcaacggcgt cctgctcctc tggggtttct cgttttcttt ttcatttatt tttagcaggt 3720gccaagtagc cgagctacta tacttacctg gccatgttaa ttattttatt ccgtctgtct 3780gtgtgtgtct gtgcatacta ctatagggac atggcgcggt gttcttataa accgggaggc 3840cggatcccta actagcatgg gaggatatct tttcagcgga tctatacaaa ccctactcct 3900gctgacctct ttcttccagt ttctccgggt cttccttgga ttattattgc ccatcttccg 3960ggttgtgcgt gtgtcagaga cagctcgaac gataaatttc tcaaaaccag tactagagag 4020ggtgtgttgt gtgtgagaac tgagtggaga gttagcatga aggctgcaaa ctagaaagga 4080aggtatgttc tttccttttt gatccatcag gggagcccct tctggtatta agatctttcc 4140ggcacattga ttttcatact ttgtgatgac cctggaagaa tcggcgtagc agcgtagcac 4200cgctccattt tggtcttacc ctcacctccc catgctatga actgatcaat ttcattgttc 4260ttcatcaccc ttctcctagc tttccacttc cttcggatct catgccatgt ttctcagcat 4320gaatcaaatt taattcgtgt tttctacttc catatatact ggaagaaatt taattagatc 4380tatttttgct cgggaggtct tcatactttg agttctgatg ccatcacctt atttcccccc 4440cccccttctc ttgttctatc ttcttcctca tcttggcttg atcattttga tctgtcagtt 4500atagcatgat gcattctcaa tttgactgta tgtaagttca accggaaata tgttgaatgg 4560attttctata tatcaacact tgatgtcagg cctgcatctg tttcgcttgt ggtggtgtgg 4620ccaaaattgt ctatatttga tctttgctct tctttctcct catttcatga cgattcctac 4680tacggcttaa accattcttt attctttact aatcatggat gttgcttgac tcctagttgt 4740ttcgtactag ctcaacttgg agatcttttc attatttgcc tagttggtgg gtacgtttgt 4800gacagatcta aaatggtgca cgaaaagttt tacttattat gaaaaaaggg agcttaacag 4860ggtaatttct ctatttattc gtgatgacat tttttccttg ataaggggga ttttttataa 4920tctgcactca catgtttata tgtaaaatct agctcttttg ttttgttttt ggcatatttc 4980ccgctaagta tagagtttat gtggataaca ttataacttt tcaagatcca atccacatct 5040ttgattgtga aaatcataca atagggaaaa tcaactgaag ggttaattag atgctatatg 5100catatatata tatatgtgcg cgcgcgcgcg cctgaattta actatgtatg catccaactg 5160tttcattgaa aaagatttga tatttttcag tctattcttt ttcgagtata tatctaatat 5220gtttcaatct gttttgacca ttataagata aagcctatat tcaccaggca tttgagatga 5280tcttttcatg catgaaaaag ctgttgttat cacttcaact aaccagacga tctaacatgt 5340atttgtataa gaaacagacc ttgatttcct tctgtaaaat catgcatgtg ttcgttttga 5400attggagtcg gcgcgcctgt gttttgaccg tcaggaaagt cttttttttc cctgaatagt 5460caagggtcta tacttcttga agcaattggg acactaatca attattgttt atacctcgga 5520ccatcttttc cttcttcaca ccactaatca gtttatgcct tggaccatta attgtgttgt 5580tcacaagctt cttgtttatg gtttacaaag cattcgccta gatttgtgtg tgtctctaca 5640catcgatcac ttttaaatac ttgtcgcttt cagttattct tttaacgttt ggttatttat 5700cttatttaaa aaaattatcg tattattatt tattttgttt gtgatttact ttattatcaa 5760aagtatttca aatatgactt atcttttttt ataagtgcac taatttttca aataagatga 5820atggtcaaat gttacaagaa aaagttaaag caaccactaa tttagggcgg aggtagtaaa 5880acctagttat tgtaaccaat aattttatca atctataaat gcaacacaaa gtaacttcgt 5940gatatctaac acaaagccac ttcaacgatg aaagctgact gcatgtttta tcaaaacaca 6000tgtgatcagt ttgttggatg aaaaaaatta tctatgtcat aaatcaagag ttataatata 6060agcttctggc tctacaagta acatttctat gttttttttt acgttcttac atactatgtt 6120ttgccaaaaa aaacatgatc attttgttgg acgaaaagaa atagtaaata tagagtgacc 6180tttgatatca ttataatata agcttctgcc tctataaata acatctatgc actttttacg 6240tcgtagtaat ttgatatatg agaaatttac atataacatt tttgtgcagc ataaccacca 6300tggatttgag caatagctca cctgtcatca ccgatccggt ggcgatcagc cagcagttgt 6360tgggcggcct gccttcaaat ctgatgcagt tttcagtcat gcccggtggc tactccagct 6420ctggcatgaa cgttggtgtc agtaggctca aaatcgagga agtccttgtc aatggactgc 6480ttgatgccat gaaatcctcg tcacctcgca ggaggctgaa tgtagcattt ggcgaggaca 6540attcatctga agaagaagac cctgcttaca gcgcttggat ggcaaaatgt ccttctgctt 6600tggcttcctt caagcaaatt gtagccagtg cacaagggaa gaagattgct gtgtttctag 6660actatgacgg cacactgtcg cctattgtgg atgatcctga caaagcagtg atgtctcccg 6720tgatgagagc tgctgtgaga aatgttgcga agtacttccc cactgcaatt gtcagcggaa 6780ggtcccgcaa taaggtgttt gaatttgtaa aactgaagga gctttattat gctggaagtc 6840atggtatgga cataatggca ccttcagcaa atcatgagca cagtgctgaa aagagcaaac 6900aggccaatct cttccaacct gcacacgact ttctgccaat gatcgatgag gttaccaagt 6960ccctcttgca agttgtcagt ggaattgaag gtgcaactgt tgagaacaac aaattctgcg 7020tttctgtaga ttatcgcaac gttgcagaga aggattggaa actggtcgca cggctcgtaa 7080acgaagtgct ggaggctttt cctcgtctca aagtaaccaa tggacgaatg gttttagagg 7140ttcgtccggt gatcgactgg gacaagggaa aggctgtgga gtttctgctc cagtcactcg 7200ggctaaatga ctctgaaaat gtgatcccca tctacattgg agacgacaga actgacgaag 7260acgctttcaa ggtacttcga cagcgaaatt gcggttatgg aatactagtt tcacaggttc 7320ccaaggaaac tgaagccttc tactcgctga gagatccatc tgaagtgatg gagttcctca 7380atttcttggt gagatggaag aagcactcag tgtgagctcg ctaagcagcc atcgatcagc 7440tgtcagaagt tggagctaat aataaaaggg atgtggagtg ggctacatgt atctcggatc 7500tctctgcgag ccacctaatg gtcttgcgtg gccctttaat ctgtatgttt ttgtgtgtaa 7560gctactgcta gctgtttgca ccttctgcgt ccgtggttgt gtttccgtgc taccttttta 7620tgttttgatt tggatcttgt ttgaaaataa tcttaccagc tttgggtaaa ctgtttatta 7680cgtactctat atagcatatg tgaccgacga caacggtttc attttagatg atgtgtatgg 7740atgatttctt tccaaaatca catctttagt ataagagcaa ttttaccatc caataccaaa 7800ttttatacta gaaaatattt tgggatatca aaatttatgg tacctccagt accaaatgtt 7860gaatggtaaa ctttcataat atacaagtca ctctaggata tttaagacaa tttttagttt 7920tttcttattg ttgcccttgt taaatacatg agaaatttta catcacttaa aatgtatcaa 7980gaggtatcaa atttttttaa tacaaaattt agtactttct ccgtttatat atgaatgtgg 8040acaatgcttg aaagtcttat aacctgaaac tgaggtagtg tatcgagaag tacaaaattt 8100tacactaaaa tcccagtact tactcaataa ctgtaaaatt actctaaata tgtactccct 8160ctatttcaga ttataagtcg ttttaacttt agtcaaagtt aaactgtttc aagtttaacc 8220aagtttgtag ataaaagtag taacatattc aacacaagac aaatatatta taaaaacata 8280ttgaattata gatttaatta aattaatttg gtattgcaag tattactaaa tttgtttata 8340aatttggtcg aatttaaaat agtttgactt taaccaaagt caaaacaaat tataatctaa 8400aacaaaggta atacattgta tcactctcat gaatggattg taacatacat taatttaatt 8460actattttag ttcttgtgca aaagttgaaa acgatttatg tttggaatct ttttgtggtg 8520tatatatatg aaaccattcc tctaccatcc ttccccaacc ataatcctca caaccgttag 8580ccccattgtg atctcaccca gttgctagcc tcttttgtca ccttgtcaca gctctcctcc 8640attcattaca caatggcatc ggaccgcgat cgcttaatta agcttgcatg cctgcagtgc 8700agcgtgaccc ggtcgtgccc ctctctagag ataatgagca ttgcatgtct aagttataaa 8760aaattaccac atattttttt tgtcacactt gtttgaagtg cagtttatct atctttatac 8820atatatttaa actttactct acgaataata taatctatag tactacaata atatcagtgt 8880tttagagaat catataaatg aacagttaga catggtctaa aggacaattg agtattttga 8940caacaggact ctacagtttt atctttttag tgtgcatgtg ttctcctttt tttttgcaaa 9000tagcttcacc tatataatac ttcatccatt ttattagtac atccatttag ggtttagggt 9060taatggtttt tatagactaa tttttttagt acatctattt tattctattt tagcctctaa 9120attaagaaaa ctaaaactct attttagttt ttttatttaa taatttagat ataaaataga 9180ataaaataaa gtgactaaaa attaaacaaa taccctttaa gaaattaaaa aaactaagga 9240aacatttttc ttgtttcgag tagataatgc cagcctgtta aacgccgccg acgagtctaa 9300cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca agcgaagcag acggcacggc 9360atctctgtcg ctgcctctgg acccctctcg agagttccgc tccaccgttg gacttgctcc 9420gctgtcggca tccagaaatt gcgtggcgga gcggcagacg tgagccggca cggcaggcgg 9480cctcctcctc ctctcacggc accggcagct acgggggatt cctttcccac cgctccttcg 9540ctttcccttc ctcgcccgcc gtaataaata gacaccccct ccacaccctc tttccccaac 9600ctcgtgttgt tcggagcgca cacacacaca accagatctc ccccaaatcc acccgtcggc 9660acctccgctt caaggtacgc cgctcgtcct cccccccccc ccctctctac cttctctaga 9720tcggcgttcc ggtccatagt tagggcccgg tagttctact tctgttcatg tttgtgttag 9780atccgtgttt gtgttagatc cgtgctgtta gcgttcgtac acggatgcga cctgtacgtc 9840agacacgttc tgattgctaa cttgccagtg tttctctttg gggaatcctg ggatggctct 9900agccgttccg cagacgggat cgatttcatg attttttttg tttcgttgca tagggtttgg 9960tttgcccttt tcctttattt caatatatgc cgtgcacttg tttgtcgggt catcttttca 10020tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga 10080gtagaattct gtttcaaact acctggtgga tttattaatt ttggatctgt atgtgtgtgc 10140catacatatt catagttacg aattgaagat gatggatgga aatatcgatc taggataggt 10200atacatgttg atgcgggttt tactgatgca tatacagaga tgctttttgt tcgcttggtt 10260gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct agatcggagt agaatactgt 10320ttcaaactac ctggtgtatt tattaatttt ggaactgtat gtgtgtgtca tacatcttca 10380tagttacgag tttaagatgg atggaaatat cgatctagga taggtataca tgttgatgtg 10440ggttttactg atgcatatac atgatggcat atgcagcatc tattcatatg ctctaacctt 10500gagtacctat ctattataat aaacaagtat gttttataat tattttgatc ttgatatact 10560tggatgatgg catatgcagc agctatatgt ggattttttt agccctgcct tcatacgcta 10620tttatttgct tggtactgtt tcttttgtcg atgctcaccc tgttgtttgg tgttacttct 10680gcagggatcc actagtccac catgtctccg gagaggagac cagttgagat taggccagct 10740acagcagctg atatggccgc ggtttgtgat atcgttaacc attacattga gacgtctaca 10800gtgaacttta ggacagagcc acaaacacca caagagtgga ttgatgatct agagaggttg 10860caagatagat acccttggtt ggttgctgag gttgagggtg ttgtggctgg tattgcttac 10920gctgggccct ggaaggctag gaacgcttac gattggacag ttgagagtac tgtttacgtg 10980tcacataggc atcaaaggtt gggcctagga tccacattgt acacacattt gcttaagtct 11040atggaggcgc aaggttttaa gtctgtggtt gctgttatag gccttccaaa cgatccatct 11100gttaggttgc atgaggcttt gggatacaca gcccggggta cattgcgcgc agctggatac 11160aagcatggtg gatggcatga tgttggtttt tggcaaaggg attttgagtt gccagctcct 11220ccaaggccag ttaggccagt tacccagatc tgaactagtg atatcggcgc catgggtcga 11280cctgcagatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt 11340cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg 11400taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt 11460taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg 11520tcatctatgt tactagatct gctagccctg caggaaattt accggtgccc gggcggccag 11580catggccgta tccgcaatgt gttattaagt cggggagggg agcgtgagtt gtagtcgcgg 11640tgtgatcttg acccgacgca tgatttaaca ggaggtgact cgtgaacctt ggacgacgat 11700cgtgtggctg ggaaaccaag ggatgatatg gacgcctcga gtgacgagat ttgctcgtag 11760ctctattgat agcaggggcg aagctacgac ttgttgatgg tgtcaaatgg caccgataag 11820ttttagaaaa tacaatgtaa aatattgttt acaatgttga tggcattcct ataaaactgt 11880gctgacacta gcaaaactta aggctggctt cgccccttat tgatagtaag gctatggcct 11940ggagatcatg tatataatcg tttaaaatat tattttatat tatagactaa gttatgtata 12000tagtaaaata aaatttaaaa tagaagatgg aacgagcaag gagataacct aatgtgctga 12060gacatgagag cgttatgccg gaccgggcca gaatcttcgc gcctccccgg tccacagaaa 12120ccgcggtccg ccagagcgcg gcgcgcccag cgtccctgct cccaaagtcc catgcggcat 12180gcgcccggcc caattggaaa ataaagcacc gccgcatatc ccgccctttc cttttgtcgg 12240atggcgcgtc acggcgtttt tccccataaa aaattcagaa atatataaac accagacatg 12300cgcccgcgcg ctcttccctc cctgccgccc gcgtcctcct ctccacacca atgcggccac 12360caccgcacgt cgccgcctcc acctcgcctg ccctccacgc cgggtcgtcc cggcgccggc 12420ctcaccaccg cggccggacg atgatgtccg tctcctgctg ccacttggcg gcgctcgtgt 12480ccatcctcgc ggcgggcgcg gctacggcgt tgctcacctt gtccctaccc tcttctcctg 12540gcgcgtccac gacgaggaga accgacttcg ttggagcatt gtcagtggca aacgagaccc 12600cgccaccgcc gcacttgtca gcgcctccgg cacccgccac gccgccccct gctccacctt 12660ccccgcccgc cgacaggcct cgaaagcgag aggtggggtg ggaccgagtt gggttgggtg 12720tcctgtgtcg tggtttcctc 1274094444DNAOryza sativapromoter(1)..(4444) 9cggaccgcta ggacgatggt gtgatgtggg aacacgaaga aaacatgagg aaaaaatatt 60aaaatgaatt tcccacttaa aatgcatcaa ataaaaaaaa taaagaaacg accgggaata 120gacacagggt ttgtgaacta gctagggcaa acatcatatg gtcccttgct gatgcacaag 180tacattgaga tgtcatttca attctgtgca tcatatgcat gtggtccctt gctgaatatt 240actcttgaaa tatctaccag tgccaatcta ttgcatgact taattaattc acaggttttg 300ttgattacat tattagtaag cttgagagca caagctcaat ggatttttct ataaatgggg 360atcattttgc aattttcttt gtcgtgcaaa gttagccttc tttattacta cttctgtttt 420taaatatacg atcctattga cttttggtca tatatttaac catgtatctt atttagatag 480tttgcgcaaa tatatatacc ttcaatgata aaattagtta caatgaaaca aatgatattt 540acgcaattct ttttactaaa caagtcacaa gaagtacctg cagcaatata tgttggaacc 600gtgcagtaga tcgagcctag ctacgcaaaa aaacaaaaag agaaaaaaag ggaaaggaaa 660aacattaatc atgcatgagc agtatgcccg gcaactggaa tttgtcaaag atatggggag 720aggagaataa tacaagtact actactacct agctctacca tgcatatgca cccaaaggca 780aactggatta ttggataaag cacagatgct ggcaaaacaa tccttaagcc tcccctccct 840gcttctttat ttttgggcag cctctaccgg acggtgccgt ggtccattgg accagtaggt 900ggcgacatac atggtttggg ttaagtctag gagagcagtg tgtgtgcgcg cgcaagagag 960agagactgtg agtctgggag tagccctctc ccctcctttg gccatcttcc tcgtgtatat 1020gcatatatgc atcatcgcaa cggtgtatat ttgtggtgtg gcgggtgtgg cattggattg 1080cccccatttt ggctcgtgct tcccagttag ggtaaaacct gtggtaaact tgctagcccc 1140acgccaaagt tacccttctt tattgttgaa agggagagga ggtgtgtgaa ttgtgatgga 1200gggagagaga gagagataga aagagagatg tgtgtcaaag caagcaagaa accagtttca 1260caaagagcta ctactagtac tagtgtacta ctgtggtaca gtgcccaatg tcctttctcc 1320ggactcgact ccactaatat tctcctcttc tcgcgcggct cgttatattc tcgtcatcat 1380tggaggcttt agcaagcaag aagagaggca gtggtggtgg tggtggagga ggagctagct 1440agcctgtgct tgctgatcgg tgctgagctg aggaatcgtt cgatcgatcg ggcgagtcga 1500cgaggggaag agttgagctg aggcgcatcg agaacaagat caacaggcag gtcaccttct 1560ccaagcgccg caacggcctc ctcaagaagg cctacgagct gtccgttctc tgcgacgccg 1620aggtcgcgct catcatcttc tccagccgcg gcaagctcta cgagttcggc agcgccgggt 1680ataattaata cagacacaac aacacacaca accaacaaac cagcatcaat ttgaacctgc 1740agatctgctg ttttctctga tcaattgctt cttttttttt gttctttttt gtttctttta 1800tctgctgcaa cggcgtcctg ctcctctggg gtttctcgtt ttctttttca tttattttta 1860gcaggtgcca agtagccgag ctactatact tacctggcca tgttaattat tttattccgt 1920ctgtctgtgt gtgtctgtgc atactactat agggacatgg cgcggtgttc ttataaaccg 1980ggaggccgga tccctaacta gcatgggagg atatcttttc agcggatcta tacaaaccct 2040actcctgctg acctctttct tccagtttct ccgggtcttc cttggattat tattgcccat 2100cttccgggtt gtgcgtgtgt cagagacagc tcgaacgata aatttctcaa aaccagtact 2160agagagggtg tgttgtgtgt gagaactgag tggagagtta gcatgaaggc tgcaaactag 2220aaaggaaggt atgttctttc ctttttgatc catcagggga gccccttctg gtattaagat 2280ctttccggca cattgatttt catactttgt gatgaccctg gaagaatcgg cgtagcagcg 2340tagcaccgct ccattttggt cttaccctca cctccccatg ctatgaactg atcaatttca 2400ttgttcttca tcacccttct cctagctttc cacttccttc ggatctcatg ccatgtttct 2460cagcatgaat caaatttaat tcgtgttttc tacttccata tatactggaa gaaatttaat 2520tagatctatt tttgctcggg aggtcttcat actttgagtt ctgatgccat caccttattt 2580cccccccccc cttctcttgt tctatcttct tcctcatctt ggcttgatca ttttgatctg 2640tcagttatag catgatgcat tctcaatttg actgtatgta agttcaaccg gaaatatgtt 2700gaatggattt tctatatatc aacacttgat gtcaggcctg catctgtttc gcttgtggtg 2760gtgtggccaa aattgtctat atttgatctt tgctcttctt tctcctcatt tcatgacgat 2820tcctactacg gcttaaacca ttctttattc tttactaatc atggatgttg cttgactcct 2880agttgtttcg tactagctca acttggagat cttttcatta tttgcctagt tggtgggtac 2940gtttgtgaca gatctaaaat ggtgcacgaa aagttttact tattatgaaa aaagggagct 3000taacagggta atttctctat ttattcgtga tgacattttt tccttgataa gggggatttt 3060ttataatctg cactcacatg tttatatgta aaatctagct cttttgtttt gtttttggca 3120tatttcccgc taagtataga gtttatgtgg ataacattat aacttttcaa gatccaatcc 3180acatctttga ttgtgaaaat catacaatag ggaaaatcaa ctgaagggtt aattagatgc 3240tatatgcata tatatatata tgtgcgcgcg cgcgcgcctg aatttaacta tgtatgcatc 3300caactgtttc attgaaaaag atttgatatt tttcagtcta ttctttttcg agtatatatc 3360taatatgttt caatctgttt tgaccattat aagataaagc ctatattcac caggcatttg 3420agatgatctt ttcatgcatg aaaaagctgt tgttatcact tcaactaacc agacgatcta 3480acatgtattt gtataagaaa cagaccttga tttccttctg taaaatcatg catgtgttcg 3540ttttgaattg gagtcggcgc gcctgtgttt tgaccgtcag gaaagtcttt tttttccctg 3600aatagtcaag ggtctatact tcttgaagca attgggacac taatcaatta ttgtttatac 3660ctcggaccat cttttccttc ttcacaccac taatcagttt atgccttgga ccattaattg 3720tgttgttcac aagcttcttg tttatggttt acaaagcatt cgcctagatt tgtgtgtgtc 3780tctacacatc gatcactttt aaatacttgt cgctttcagt tattctttta acgtttggtt 3840atttatctta tttaaaaaaa ttatcgtatt attatttatt ttgtttgtga tttactttat 3900tatcaaaagt atttcaaata tgacttatct ttttttataa

gtgcactaat ttttcaaata 3960agatgaatgg tcaaatgtta caagaaaaag ttaaagcaac cactaattta gggcggaggt 4020agtaaaacct agttattgta accaataatt ttatcaatct ataaatgcaa cacaaagtaa 4080cttcgtgata tctaacacaa agccacttca acgatgaaag ctgactgcat gttttatcaa 4140aacacatgtg atcagtttgt tggatgaaaa aaattatcta tgtcataaat caagagttat 4200aatataagct tctggctcta caagtaacat ttctatgttt ttttttacgt tcttacatac 4260tatgttttgc caaaaaaaac atgatcattt tgttggacga aaagaaatag taaatataga 4320gtgacctttg atatcattat aatataagct tctgcctcta taaataacat ctatgcactt 4380tttacgtcgt agtaatttga tatatgagaa atttacatat aacatttttg tgcagcataa 4440ccac 4444101116DNAOryza sativagene(1)..(1116) 10atggatttga gcaatagctc acctgtcatc accgatccgg tggcgatcag ccagcagttg 60ttgggcggcc tgccttcaaa tctgatgcag ttttcagtca tgcccggtgg ctactccagc 120tctggcatga acgttggtgt cagtaggctc aaaatcgagg aagtccttgt caatggactg 180cttgatgcca tgaaatcctc gtcacctcgc aggaggctga atgtagcatt tggcgaggac 240aattcatctg aagaagaaga ccctgcttac agcgcttgga tggcaaaatg tccttctgct 300ttggcttcct tcaagcaaat tgtagccagt gcacaaggga agaagattgc tgtgtttcta 360gactatgacg gcacactgtc gcctattgtg gatgatcctg acaaagcagt gatgtctccc 420gtgatgagag ctgctgtgag aaatgttgcg aagtacttcc ccactgcaat tgtcagcgga 480aggtcccgca ataaggtgtt tgaatttgta aaactgaagg agctttatta tgctggaagt 540catggtatgg acataatggc accttcagca aatcatgagc acagtgctga aaagagcaaa 600caggccaatc tcttccaacc tgcacacgac tttctgccaa tgatcgatga ggttaccaag 660tccctcttgc aagttgtcag tggaattgaa ggtgcaactg ttgagaacaa caaattctgc 720gtttctgtag attatcgcaa cgttgcagag aaggattgga aactggtcgc acggctcgta 780aacgaagtgc tggaggcttt tcctcgtctc aaagtaacca atggacgaat ggttttagag 840gttcgtccgg tgatcgactg ggacaaggga aaggctgtgg agtttctgct ccagtcactc 900gggctaaatg actctgaaaa tgtgatcccc atctacattg gagacgacag aactgacgaa 960gacgctttca aggtacttcg acagcgaaat tgcggttatg gaatactagt ttcacaggtt 1020cccaaggaaa ctgaagcctt ctactcgctg agagatccat ctgaagtgat ggagttcctc 1080aatttcttgg tgagatggaa gaagcactca gtgtga 111611371PRTOryza sativa 11Met Asp Leu Ser Asn Ser Ser Pro Val Ile Thr Asp Pro Val Ala Ile1 5 10 15Ser Gln Gln Leu Leu Gly Gly Leu Pro Ser Asn Leu Met Gln Phe Ser 20 25 30Val Met Pro Gly Gly Tyr Ser Ser Ser Gly Met Asn Val Gly Val Ser 35 40 45Arg Leu Lys Ile Glu Glu Val Leu Val Asn Gly Leu Leu Asp Ala Met 50 55 60Lys Ser Ser Ser Pro Arg Arg Arg Leu Asn Val Ala Phe Gly Glu Asp65 70 75 80Asn Ser Ser Glu Glu Glu Asp Pro Ala Tyr Ser Ala Trp Met Ala Lys 85 90 95Cys Pro Ser Ala Leu Ala Ser Phe Lys Gln Ile Val Ala Ser Ala Gln 100 105 110Gly Lys Lys Ile Ala Val Phe Leu Asp Tyr Asp Gly Thr Leu Ser Pro 115 120 125Ile Val Asp Asp Pro Asp Lys Ala Val Met Ser Pro Val Met Arg Ala 130 135 140Ala Val Arg Asn Val Ala Lys Tyr Phe Pro Thr Ala Ile Val Ser Gly145 150 155 160Arg Ser Arg Asn Lys Val Phe Glu Phe Val Lys Leu Lys Glu Leu Tyr 165 170 175Tyr Ala Gly Ser His Gly Met Asp Ile Met Ala Pro Ser Ala Asn His 180 185 190Glu His Ser Ala Glu Lys Ser Lys Gln Ala Asn Leu Phe Gln Pro Ala 195 200 205His Asp Phe Leu Pro Met Ile Asp Glu Val Thr Lys Ser Leu Leu Gln 210 215 220Val Val Ser Gly Ile Glu Gly Ala Thr Val Glu Asn Asn Lys Phe Cys225 230 235 240Val Ser Val Asp Tyr Arg Asn Val Ala Glu Lys Asp Trp Lys Leu Val 245 250 255Ala Arg Leu Val Asn Glu Val Leu Glu Ala Phe Pro Arg Leu Lys Val 260 265 270Thr Asn Gly Arg Met Val Leu Glu Val Arg Pro Val Ile Asp Trp Asp 275 280 285Lys Gly Lys Ala Val Glu Phe Leu Leu Gln Ser Leu Gly Leu Asn Asp 290 295 300Ser Glu Asn Val Ile Pro Ile Tyr Ile Gly Asp Asp Arg Thr Asp Glu305 310 315 320Asp Ala Phe Lys Val Leu Arg Gln Arg Asn Cys Gly Tyr Gly Ile Leu 325 330 335Val Ser Gln Val Pro Lys Glu Thr Glu Ala Phe Tyr Ser Leu Arg Asp 340 345 350Pro Ser Glu Val Met Glu Phe Leu Asn Phe Leu Val Arg Trp Lys Lys 355 360 365His Ser Val 3701226DNAZea mays 12cttgaaagga tgaaccatga aaattt 261326DNAZea mays 13cgtgactccc ttaattctcc gctcat 261421DNAZea mays 14cctgttgccg gtcttgcgat g 211521DNAZea mays 15ccaccccacc tctcgctttc g 211627DNAZea mays 16agttgcgatg tttccatgta gtgaacc 271720DNAZea mays 17ccggaggcgc tgacaagtgc 201830DNAZea mays 18tgatcaagcc aagatgagga agaagataga 301930DNAZea mays 19agttagcatg aaggctgcaa actagaaagg 302030DNAZea mays 20aaatcgtttt caacttttgc acaagaacta 302126DNAZea mays 21agacgacaga actgacgaag acgctt 262225DNAZea mays 22gatcacaccg cgactacaac tcacg 25231095DNAZea mays 23cttgaaagga tgaaccatga aaatttagtt gcgatgtttc catgtagtga accgagatgt 60cttatcagta agtaaatagg ttaacatcaa gaaagcacat ttaataaaaa attggaaagg 120ggacatacat taattaacta tctaaaagta gtacacagaa ttagacattt agacataata 180tggtttgcta aatacgtagt aatctatata aacaaaatta aagttgtgta tttctaagac 240atgcatacgt acacaaacag aacatattga aactagcctt tgttgaacaa tttttaacag 300caaatgtggt tttggtttgt taatattgca agttattatg agtattcaac ctagtaaatt 360gaataatcct tgcctgatat attgaatcta tttgtatata tctagctaga tacaattatt 420agaacatgct tttattgtct acatatgtac ccatgtaaca tgaatagaaa ataaataggt 480atgcttatcg tagagtatct atgaccatgt agcaagaaaa ataagttaat atagagataa 540atcaatgggc aaaaaataaa atttggggga aagaatctca ctgctactac tcttccacta 600caaagaacaa caaggttggt tccaattcca cttccggaac aatcagcgca attgctccat 660ccatccacag ctgccaccga gactaggtga cacgatccaa gccgccatgg cgtcggctaa 720gcctctacgc tctagactgt gctggtggca ctagggagac tacgaaggtt cacctgcacc 780ttgaaggact agttgaggaa ggtgttggcg aggatgttga ggggtttgga cgatgaagca 840ccgtctaggg aacaattgtt ctggcccgtg acgaggtgct gcaacgccat ctccgccgcc 900ggagagacgt gcacgctctc tgacgtcaca tgtgagaggc tatggttgta gcttcaatcg 960ttggcggtgt gggctggtcc ggctcgatgt gaggggaggg gagcgtgagt tggagtcgtg 1020gaggaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 1080attaagggag tcacg 1095241373DNAZea mays 24cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta 60ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg 120caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 180tcgcgcgcgg tgtcatctat gttactagat ctgctagccc tgcaggaaat ttaccggtgc 240ccgggcggcc agcatggccg tatccgcaat gtgttattaa gtcggggagg ggagcgtgag 300ttgtagtcgc ggtgtgatct tgacccgacg catgatttaa caggaggtga ctcgtgaacc 360ttggacgacg atcgtgtggc tgggaaacca agggatgata tggacgcctc gagtgacgag 420atttgctcgt agctctattg atagcagggg cgaagctacg acttgttgat ggtgtcaaat 480ggcaccgata agttttagaa aatacaatgt aaaatattgt ttacaatgtt gatggcattc 540ctataaaact gtgctgacac tagcaaaact taaggctggc ttcgcccctt attgatagta 600aggctatggc ctggagatca tgtatataat cgtttaaaat attattttat attatagact 660aagttatgta tatagtaaaa taaaatttaa aatagaagat ggaacgagca aggagataac 720ctaatgtgct gagacatgag agcgttatgc cggaccgggc cagaatcttc gcgcctcccc 780ggtccacaga aaccgcggtc cgccagagcg cggcgcgccc agcgtccctg ctcccaaagt 840cccatgcggc atgcgcccgg cccaattgga aaataaagca ccgccgcata tcccgccctt 900tccttttgtc ggatggcgcg tcacggcgtt tttccccata aaaaattcag aaatatataa 960acaccagaca tgcgcccgcg cgctcttccc tccctgccgc ccgcgtcctc ctctccacac 1020caatgcggcc accaccgcac gtcgccgcct ccacctcgcc tgccctccac gccgggtcgt 1080cccggcgccg gcctcaccac cgcggccgga cgatgatgtc cgtctcctgc tgccacttgg 1140cggcgctcgt gtccatcctc gcggcgggcg cggctacggc gttgctcacc ttgtccctac 1200cctcttctcc tggcgcgtcc acgacgagga gaaccgactt cgttggagca ttgtcagtgg 1260caaacgagac cccgccaccg ccgcacttgt cagcgcctcc ggcacccgcc acgccgcccc 1320ctgctccacc ttccccgccc gccgacaggc ctcgaaagcg agaggtgggg tgg 13732512453DNAZea mays 25agttgcgatg tttccatgta gtgaaccgag atgtcttatc agtaagtaaa taggttaaca 60tcaagaaagc acatttaata aaaaattgga aaggggacat acattaatta actatctaaa 120agtagtacac agaattagac atttagacat aatatggttt gctaaatacg tagtaatcta 180tataaacaaa attaaagttg tgtatttcta agacatgcat acgtacacaa acagaacata 240ttgaaactag cctttgttga acaattttta acagcaaatg tggttttggt ttgttaatat 300tgcaagttat tatgagtatt caacctagta aattgaataa tccttgcctg atatattgaa 360tctatttgta tatatctagc tagatacaat tattagaaca tgcttttatt gtctacatat 420gtacccatgt aacatgaata gaaaataaat aggtatgctt atcgtagagt atctatgacc 480atgtagcaag aaaaataagt taatatagag ataaatcaat gggcaaaaaa taaaatttgg 540gggaaagaat ctcactgcta ctactcttcc actacaaaga acaacaaggt tggttccaat 600tccacttccg gaacaatcag cgcaattgct ccatccatcc acagctgcca ccgagactag 660gtgacacgat ccaagccgcc atggcgtcgg ctaagcctct acgctctaga ctgtgctggt 720ggcactaggg agactacgaa ggttcacctg caccttgaag gactagttga ggaaggtgtt 780ggcgaggatg ttgaggggtt tggacgatga agcaccgtct agggaacaat tgttctggcc 840cgtgacgagg tgctgcaacg ccatctccgc cgccggagag acgtgcacgc tctctgacgt 900cacatgtgag aggctatggt tgtagcttca atcgttggcg gtgtgggctg gtccggctcg 960atgtgagggg aggggagcgt gagttggagt cgtggaggaa acactgatag tttaaactga 1020aggcgggaaa cgacaatctg atcatgagcg gagaattaag ggagtcacgt tatgaccccc 1080gccgatgacg cgggacaagc cgttttacgt ttggaactga cagaaccgca acgctgcagg 1140aattggccgc agcggccatt taaatcaatt gggcgcgcca gctgcttgtg gggaccagac 1200aaaaaaggaa tggtgcagaa ttgttaggcg cacctaccaa aagcatcttt gcctttattg 1260caaagataaa gcagattcct ctagtacaag tggggaacaa aataacgtgg aaaagagctg 1320tcctgacagc ccactcacta atgcgtatga cgaacgcagt gacgaccaca aaactcgaga 1380cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc 1440actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata 1500aaggaaaggc tatcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 1560ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 1620gatgtgatat ctccactgac gtaagggatg acgaacaatc ccactatcct tcggtaccgg 1680accgctagga cgatggtgtg atgtgggaac acgaagaaaa catgaggaaa aaatattaaa 1740atgaatttcc cacttaaaat gcatcaaata aaaaaaataa agaaacgacc gggaatagac 1800acagggtttg tgaactagct agggcaaaca tcatatggtc ccttgctgat gcacaagtac 1860attgagatgt catttcaatt ctgtgcatca tatgcatgtg gtcccttgct gaatattact 1920cttgaaatat ctaccagtgc caatctattg catgacttaa ttaattcaca ggttttgttg 1980attacattat tagtaagctt gagagcacaa gctcaatgga tttttctata aatggggatc 2040attttgcaat tttctttgtc gtgcaaagtt agccttcttt attactactt ctgtttttaa 2100atatacgatc ctattgactt ttggtcatat atttaaccat gtatcttatt tagatagttt 2160gcgcaaatat atataccttc aatgataaaa ttagttacaa tgaaacaaat gatatttacg 2220caattctttt tactaaacaa gtcacaagaa gtacctgcag caatatatgt tggaaccgtg 2280cagtagatcg agcctagcta cgcaaaaaaa caaaaagaga aaaaaaggga aaggaaaaac 2340attaatcatg catgagcagt atgcccggca actggaattt gtcaaagata tggggagagg 2400agaataatac aagtactact actacctagc tctaccatgc atatgcaccc aaaggcaaac 2460tggattattg gataaagcac agatgctggc aaaacaatcc ttaagcctcc cctccctgct 2520tctttatttt tgggcagcct ctaccggacg gtgccgtggt ccattggacc agtaggtggc 2580gacatacatg gtttgggtta agtctaggag agcagtgtgt gtgcgcgcgc aagagagaga 2640gactgtgagt ctgggagtag ccctctcccc tcctttggcc atcttcctcg tgtatatgca 2700tatatgcatc atcgcaacgg tgtatatttg tggtgtggcg ggtgtggcat tggattgccc 2760ccattttggc tcgtgcttcc cagttagggt aaaacctgtg gtaaacttgc tagccccacg 2820ccaaagttac ccttctttat tgttgaaagg gagaggaggt gtgtgaattg tgatggaggg 2880agagagagag agatagaaag agagatgtgt gtcaaagcaa gcaagaaacc agtttcacaa 2940agagctacta ctagtactag tgtactactg tggtacagtg cccaatgtcc tttctccgga 3000ctcgactcca ctaatattct cctcttctcg cgcggctcgt tatattctcg tcatcattgg 3060aggctttagc aagcaagaag agaggcagtg gtggtggtgg tggaggagga gctagctagc 3120ctgtgcttgc tgatcggtgc tgagctgagg aatcgttcga tcgatcgggc gagtcgacga 3180ggggaagagt tgagctgagg cgcatcgaga acaagatcaa caggcaggtc accttctcca 3240agcgccgcaa cggcctcctc aagaaggcct acgagctgtc cgttctctgc gacgccgagg 3300tcgcgctcat catcttctcc agccgcggca agctctacga gttcggcagc gccgggtata 3360attaatacag acacaacaac acacacaacc aacaaaccag catcaatttg aacctgcaga 3420tctgctgttt tctctgatca attgcttctt tttttttgtt cttttttgtt tcttttatct 3480gctgcaacgg cgtcctgctc ctctggggtt tctcgttttc tttttcattt atttttagca 3540ggtgccaagt agccgagcta ctatacttac ctggccatgt taattatttt attccgtctg 3600tctgtgtgtg tctgtgcata ctactatagg gacatggcgc ggtgttctta taaaccggga 3660ggccggatcc ctaactagca tgggaggata tcttttcagc ggatctatac aaaccctact 3720cctgctgacc tctttcttcc agtttctccg ggtcttcctt ggattattat tgcccatctt 3780ccgggttgtg cgtgtgtcag agacagctcg aacgataaat ttctcaaaac cagtactaga 3840gagggtgtgt tgtgtgtgag aactgagtgg agagttagca tgaaggctgc aaactagaaa 3900ggaaggtatg ttctttcctt tttgatccat caggggagcc ccttctggta ttaagatctt 3960tccggcacat tgattttcat actttgtgat gaccctggaa gaatcggcgt agcagcgtag 4020caccgctcca ttttggtctt accctcacct ccccatgcta tgaactgatc aatttcattg 4080ttcttcatca cccttctcct agctttccac ttccttcgga tctcatgcca tgtttctcag 4140catgaatcaa atttaattcg tgttttctac ttccatatat actggaagaa atttaattag 4200atctattttt gctcgggagg tcttcatact ttgagttctg atgccatcac cttatttccc 4260cccccccctt ctcttgttct atcttcttcc tcatcttggc ttgatcattt tgatctgtca 4320gttatagcat gatgcattct caatttgact gtatgtaagt tcaaccggaa atatgttgaa 4380tggattttct atatatcaac acttgatgtc aggcctgcat ctgtttcgct tgtggtggtg 4440tggccaaaat tgtctatatt tgatctttgc tcttctttct cctcatttca tgacgattcc 4500tactacggct taaaccattc tttattcttt actaatcatg gatgttgctt gactcctagt 4560tgtttcgtac tagctcaact tggagatctt ttcattattt gcctagttgg tgggtacgtt 4620tgtgacagat ctaaaatggt gcacgaaaag ttttacttat tatgaaaaaa gggagcttaa 4680cagggtaatt tctctattta ttcgtgatga cattttttcc ttgataaggg ggatttttta 4740taatctgcac tcacatgttt atatgtaaaa tctagctctt ttgttttgtt tttggcatat 4800ttcccgctaa gtatagagtt tatgtggata acattataac ttttcaagat ccaatccaca 4860tctttgattg tgaaaatcat acaataggga aaatcaactg aagggttaat tagatgctat 4920atgcatatat atatatatgt gcgcgcgcgc gcgcctgaat ttaactatgt atgcatccaa 4980ctgtttcatt gaaaaagatt tgatattttt cagtctattc tttttcgagt atatatctaa 5040tatgtttcaa tctgttttga ccattataag ataaagccta tattcaccag gcatttgaga 5100tgatcttttc atgcatgaaa aagctgttgt tatcacttca actaaccaga cgatctaaca 5160tgtatttgta taagaaacag accttgattt ccttctgtaa aatcatgcat gtgttcgttt 5220tgaattggag tcggcgcgcc tgtgttttga ccgtcaggaa agtctttttt ttccctgaat 5280agtcaagggt ctatacttct tgaagcaatt gggacactaa tcaattattg tttatacctc 5340ggaccatctt ttccttcttc acaccactaa tcagtttatg ccttggacca ttaattgtgt 5400tgttcacaag cttcttgttt atggtttaca aagcattcgc ctagatttgt gtgtgtctct 5460acacatcgat cacttttaaa tacttgtcgc tttcagttat tcttttaacg tttggttatt 5520tatcttattt aaaaaaatta tcgtattatt atttattttg tttgtgattt actttattat 5580caaaagtatt tcaaatatga cttatctttt tttataagtg cactaatttt tcaaataaga 5640tgaatggtca aatgttacaa gaaaaagtta aagcaaccac taatttaggg cggaggtagt 5700aaaacctagt tattgtaacc aataatttta tcaatctata aatgcaacac aaagtaactt 5760cgtgatatct aacacaaagc cacttcaacg atgaaagctg actgcatgtt ttatcaaaac 5820acatgtgatc agtttgttgg atgaaaaaaa ttatctatgt cataaatcaa gagttataat 5880ataagcttct ggctctacaa gtaacatttc tatgtttttt tttacgttct tacatactat 5940gttttgccaa aaaaaacatg atcattttgt tggacgaaaa gaaatagtaa atatagagtg 6000acctttgata tcattataat ataagcttct gcctctataa ataacatcta tgcacttttt 6060acgtcgtagt aatttgatat atgagaaatt tacatataac atttttgtgc agcataacca 6120ccatggattt gagcaatagc tcacctgtca tcaccgatcc ggtggcgatc agccagcagt 6180tgttgggcgg cctgccttca aatctgatgc agttttcagt catgcccggt ggctactcca 6240gctctggcat gaacgttggt gtcagtaggc tcaaaatcga ggaagtcctt gtcaatggac 6300tgcttgatgc catgaaatcc tcgtcacctc gcaggaggct gaatgtagca tttggcgagg 6360acaattcatc tgaagaagaa gaccctgctt acagcgcttg gatggcaaaa tgtccttctg 6420ctttggcttc cttcaagcaa attgtagcca gtgcacaagg gaagaagatt gctgtgtttc 6480tagactatga cggcacactg tcgcctattg tggatgatcc tgacaaagca gtgatgtctc 6540ccgtgatgag agctgctgtg agaaatgttg cgaagtactt ccccactgca attgtcagcg 6600gaaggtcccg caataaggtg tttgaatttg taaaactgaa ggagctttat tatgctggaa 6660gtcatggtat ggacataatg gcaccttcag caaatcatga gcacagtgct gaaaagagca 6720aacaggccaa tctcttccaa cctgcacacg actttctgcc aatgatcgat gaggttacca 6780agtccctctt gcaagttgtc agtggaattg aaggtgcaac tgttgagaac aacaaattct 6840gcgtttctgt agattatcgc aacgttgcag agaaggattg gaaactggtc gcacggctcg 6900taaacgaagt gctggaggct tttcctcgtc tcaaagtaac caatggacga atggttttag 6960aggttcgtcc ggtgatcgac tgggacaagg gaaaggctgt ggagtttctg ctccagtcac 7020tcgggctaaa tgactctgaa aatgtgatcc ccatctacat tggagacgac agaactgacg 7080aagacgcttt caaggtactt cgacagcgaa attgcggtta tggaatacta gtttcacagg 7140ttcccaagga aactgaagcc ttctactcgc tgagagatcc atctgaagtg atggagttcc 7200tcaatttctt ggtgagatgg aagaagcact cagtgtgagc tcgctaagca gccatcgatc 7260agctgtcaga agttggagct aataataaaa gggatgtgga gtgggctaca tgtatctcgg 7320atctctctgc gagccaccta atggtcttgc gtggcccttt aatctgtatg tttttgtgtg 7380taagctactg ctagctgttt gcaccttctg cgtccgtggt tgtgtttccg tgctaccttt 7440ttatgttttg atttggatct tgtttgaaaa taatcttacc agctttgggt aaactgttta 7500ttacgtactc tatatagcat atgtgaccga cgacaacggt ttcattttag atgatgtgta 7560tggatgattt ctttccaaaa tcacatcttt agtataagag caattttacc atccaatacc 7620aaattttata ctagaaaata ttttgggata tcaaaattta

tggtacctcc agtaccaaat 7680gttgaatggt aaactttcat aatatacaag tcactctagg atatttaaga caatttttag 7740ttttttctta ttgttgccct tgttaaatac atgagaaatt ttacatcact taaaatgtat 7800caagaggtat caaatttttt taatacaaaa tttagtactt tctccgttta tatatgaatg 7860tggacaatgc ttgaaagtct tataacctga aactgaggta gtgtatcgag aagtacaaaa 7920ttttacacta aaatcccagt acttactcaa taactgtaaa attactctaa atatgtactc 7980cctctatttc agattataag tcgttttaac tttagtcaaa gttaaactgt ttcaagttta 8040accaagtttg tagataaaag tagtaacata ttcaacacaa gacaaatata ttataaaaac 8100atattgaatt atagatttaa ttaaattaat ttggtattgc aagtattact aaatttgttt 8160ataaatttgg tcgaatttaa aatagtttga ctttaaccaa agtcaaaaca aattataatc 8220taaaacaaag gtaatacatt gtatcactct catgaatgga ttgtaacata cattaattta 8280attactattt tagttcttgt gcaaaagttg aaaacgattt atgtttggaa tctttttgtg 8340gtgtatatat atgaaaccat tcctctacca tccttcccca accataatcc tcacaaccgt 8400tagccccatt gtgatctcac ccagttgcta gcctcttttg tcaccttgtc acagctctcc 8460tccattcatt acacaatggc atcggaccgc gatcgcttaa ttaagcttgc atgcctgcag 8520tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg tctaagttat 8580aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta tctatcttta 8640tacatatatt taaactttac tctacgaata atataatcta tagtactaca ataatatcag 8700tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa ttgagtattt 8760tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct ttttttttgc 8820aaatagcttc acctatataa tacttcatcc attttattag tacatccatt tagggtttag 8880ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta ttttagcctc 8940taaattaaga aaactaaaac tctattttag tttttttatt taataattta gatataaaat 9000agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta aaaaaactaa 9060ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg ccgacgagtc 9120taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag cagacggcac 9180ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg ttggacttgc 9240tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg gcacggcagg 9300cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc caccgctcct 9360tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc ctctttcccc 9420aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa tccacccgtc 9480ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc taccttctct 9540agatcggcgt tccggtccat agttagggcc cggtagttct acttctgttc atgtttgtgt 9600tagatccgtg tttgtgttag atccgtgctg ttagcgttcg tacacggatg cgacctgtac 9660gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc ctgggatggc 9720tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt gcatagggtt 9780tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg ggtcatcttt 9840tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc gttctagatc 9900ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc tgtatgtgtg 9960tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg atctaggata 10020ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt tgttcgcttg 10080gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg agtagaatac 10140tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg tcatacatct 10200tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat acatgttgat 10260gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat atgctctaac 10320cttgagtacc tatctattat aataaacaag tatgttttat aattattttg atcttgatat 10380acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg ccttcatacg 10440ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt tggtgttact 10500tctgcaggga tccactagtc caccatgtct ccggagagga gaccagttga gattaggcca 10560gctacagcag ctgatatggc cgcggtttgt gatatcgtta accattacat tgagacgtct 10620acagtgaact ttaggacaga gccacaaaca ccacaagagt ggattgatga tctagagagg 10680ttgcaagata gatacccttg gttggttgct gaggttgagg gtgttgtggc tggtattgct 10740tacgctgggc cctggaaggc taggaacgct tacgattgga cagttgagag tactgtttac 10800gtgtcacata ggcatcaaag gttgggccta ggatccacat tgtacacaca tttgcttaag 10860tctatggagg cgcaaggttt taagtctgtg gttgctgtta taggccttcc aaacgatcca 10920tctgttaggt tgcatgaggc tttgggatac acagcccggg gtacattgcg cgcagctgga 10980tacaagcatg gtggatggca tgatgttggt ttttggcaaa gggattttga gttgccagct 11040cctccaaggc cagttaggcc agttacccag atctgaacta gtgatatcgg cgccatgggt 11100cgacctgcag atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc 11160ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac 11220atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac 11280atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg 11340gtgtcatcta tgttactaga tctgctagcc ctgcaggaaa tttaccggtg cccgggcggc 11400cagcatggcc gtatccgcaa tgtgttatta agtcggggag gggagcgtga gttgtagtcg 11460cggtgtgatc ttgacccgac gcatgattta acaggaggtg actcgtgaac cttggacgac 11520gatcgtgtgg ctgggaaacc aagggatgat atggacgcct cgagtgacga gatttgctcg 11580tagctctatt gatagcaggg gcgaagctac gacttgttga tggtgtcaaa tggcaccgat 11640aagttttaga aaatacaatg taaaatattg tttacaatgt tgatggcatt cctataaaac 11700tgtgctgaca ctagcaaaac ttaaggctgg cttcgcccct tattgatagt aaggctatgg 11760cctggagatc atgtatataa tcgtttaaaa tattatttta tattatagac taagttatgt 11820atatagtaaa ataaaattta aaatagaaga tggaacgagc aaggagataa cctaatgtgc 11880tgagacatga gagcgttatg ccggaccggg ccagaatctt cgcgcctccc cggtccacag 11940aaaccgcggt ccgccagagc gcggcgcgcc cagcgtccct gctcccaaag tcccatgcgg 12000catgcgcccg gcccaattgg aaaataaagc accgccgcat atcccgccct ttccttttgt 12060cggatggcgc gtcacggcgt ttttccccat aaaaaattca gaaatatata aacaccagac 12120atgcgcccgc gcgctcttcc ctccctgccg cccgcgtcct cctctccaca ccaatgcggc 12180caccaccgca cgtcgccgcc tccacctcgc ctgccctcca cgccgggtcg tcccggcgcc 12240ggcctcacca ccgcggccgg acgatgatgt ccgtctcctg ctgccacttg gcggcgctcg 12300tgtccatcct cgcggcgggc gcggctacgg cgttgctcac cttgtcccta ccctcttctc 12360ctggcgcgtc cacgacgagg agaaccgact tcgttggagc attgtcagtg gcaaacgaga 12420ccccgccacc gccgcacttg tcagcgcctc cgg 12453264307DNAZea mays 26agttgcgatg tttccatgta gtgaaccgag atgtcttatc agtaagtaaa taggttaaca 60tcaagaaagc acatttaata aaaaattgga aaggggacat acattaatta actatctaaa 120agtagtacac agaattagac atttagacat aatatggttt gctaaatacg tagtaatcta 180tataaacaaa attaaagttg tgtatttcta agacatgcat acgtacacaa acagaacata 240ttgaaactag cctttgttga acaattttta acagcaaatg tggttttggt ttgttaatat 300tgcaagttat tatgagtatt caacctagta aattgaataa tccttgcctg atatattgaa 360tctatttgta tatatctagc tagatacaat tattagaaca tgcttttatt gtctacatat 420gtacccatgt aacatgaata gaaaataaat aggtatgctt atcgtagagt atctatgacc 480atgtagcaag aaaaataagt taatatagag ataaatcaat gggcaaaaaa taaaatttgg 540gggaaagaat ctcactgcta ctactcttcc actacaaaga acaacaaggt tggttccaat 600tccacttccg gaacaatcag cgcaattgct ccatccatcc acagctgcca ccgagactag 660gtgacacgat ccaagccgcc atggcgtcgg ctaagcctct acgctctaga ctgtgctggt 720ggcactaggg agactacgaa ggttcacctg caccttgaag gactagttga ggaaggtgtt 780ggcgaggatg ttgaggggtt tggacgatga agcaccgtct agggaacaat tgttctggcc 840cgtgacgagg tgctgcaacg ccatctccgc cgccggagag acgtgcacgc tctctgacgt 900cacatgtgag aggctatggt tgtagcttca atcgttggcg gtgtgggctg gtccggctcg 960atgtgagggg aggggagcgt gagttggagt cgtggaggaa acactgatag tttaaactga 1020aggcgggaaa cgacaatctg atcatgagcg gagaattaag ggagtcacgt tatgaccccc 1080gccgatgacg cgggacaagc cgttttacgt ttggaactga cagaaccgca acgctgcagg 1140aattggccgc agcggccatt taaatcaatt gggcgcgcca gctgcttgtg gggaccagac 1200aaaaaaggaa tggtgcagaa ttgttaggcg cacctaccaa aagcatcttt gcctttattg 1260caaagataaa gcagattcct ctagtacaag tggggaacaa aataacgtgg aaaagagctg 1320tcctgacagc ccactcacta atgcgtatga cgaacgcagt gacgaccaca aaactcgaga 1380cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca gctatctgtc 1440actttattgt gaagatagtg gaaaaggaag gtggctccta caaatgccat cattgcgata 1500aaggaaaggc tatcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 1560ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 1620gatgtgatat ctccactgac gtaagggatg acgaacaatc ccactatcct tcggtaccgg 1680accgctagga cgatggtgtg atgtgggaac acgaagaaaa catgaggaaa aaatattaaa 1740atgaatttcc cacttaaaat gcatcaaata aaaaaaataa agaaacgacc gggaatagac 1800acagggtttg tgaactagct agggcaaaca tcatatggtc ccttgctgat gcacaagtac 1860attgagatgt catttcaatt ctgtgcatca tatgcatgtg gtcccttgct gaatattact 1920cttgaaatat ctaccagtgc caatctattg catgacttaa ttaattcaca ggttttgttg 1980attacattat tagtaagctt gagagcacaa gctcaatgga tttttctata aatggggatc 2040attttgcaat tttctttgtc gtgcaaagtt agccttcttt attactactt ctgtttttaa 2100atatacgatc ctattgactt ttggtcatat atttaaccat gtatcttatt tagatagttt 2160gcgcaaatat atataccttc aatgataaaa ttagttacaa tgaaacaaat gatatttacg 2220caattctttt tactaaacaa gtcacaagaa gtacctgcag caatatatgt tggaaccgtg 2280cagtagatcg agcctagcta cgcaaaaaaa caaaaagaga aaaaaaggga aaggaaaaac 2340attaatcatg catgagcagt atgcccggca actggaattt gtcaaagata tggggagagg 2400agaataatac aagtactact actacctagc tctaccatgc atatgcaccc aaaggcaaac 2460tggattattg gataaagcac agatgctggc aaaacaatcc ttaagcctcc cctccctgct 2520tctttatttt tgggcagcct ctaccggacg gtgccgtggt ccattggacc agtaggtggc 2580gacatacatg gtttgggtta agtctaggag agcagtgtgt gtgcgcgcgc aagagagaga 2640gactgtgagt ctgggagtag ccctctcccc tcctttggcc atcttcctcg tgtatatgca 2700tatatgcatc atcgcaacgg tgtatatttg tggtgtggcg ggtgtggcat tggattgccc 2760ccattttggc tcgtgcttcc cagttagggt aaaacctgtg gtaaacttgc tagccccacg 2820ccaaagttac ccttctttat tgttgaaagg gagaggaggt gtgtgaattg tgatggaggg 2880agagagagag agatagaaag agagatgtgt gtcaaagcaa gcaagaaacc agtttcacaa 2940agagctacta ctagtactag tgtactactg tggtacagtg cccaatgtcc tttctccgga 3000ctcgactcca ctaatattct cctcttctcg cgcggctcgt tatattctcg tcatcattgg 3060aggctttagc aagcaagaag agaggcagtg gtggtggtgg tggaggagga gctagctagc 3120ctgtgcttgc tgatcggtgc tgagctgagg aatcgttcga tcgatcgggc gagtcgacga 3180ggggaagagt tgagctgagg cgcatcgaga acaagatcaa caggcaggtc accttctcca 3240agcgccgcaa cggcctcctc aagaaggcct acgagctgtc cgttctctgc gacgccgagg 3300tcgcgctcat catcttctcc agccgcggca agctctacga gttcggcagc gccgggtata 3360attaatacag acacaacaac acacacaacc aacaaaccag catcaatttg aacctgcaga 3420tctgctgttt tctctgatca attgcttctt tttttttgtt cttttttgtt tcttttatct 3480gctgcaacgg cgtcctgctc ctctggggtt tctcgttttc tttttcattt atttttagca 3540ggtgccaagt agccgagcta ctatacttac ctggccatgt taattatttt attccgtctg 3600tctgtgtgtg tctgtgcata ctactatagg gacatggcgc ggtgttctta taaaccggga 3660ggccggatcc ctaactagca tgggaggata tcttttcagc ggatctatac aaaccctact 3720cctgctgacc tctttcttcc agtttctccg ggtcttcctt ggattattat tgcccatctt 3780ccgggttgtg cgtgtgtcag agacagctcg aacgataaat ttctcaaaac cagtactaga 3840gagggtgtgt tgtgtgtgag aactgagtgg agagttagca tgaaggctgc aaactagaaa 3900ggaaggtatg ttctttcctt tttgatccat caggggagcc ccttctggta ttaagatctt 3960tccggcacat tgattttcat actttgtgat gaccctggaa gaatcggcgt agcagcgtag 4020caccgctcca ttttggtctt accctcacct ccccatgcta tgaactgatc aatttcattg 4080ttcttcatca cccttctcct agctttccac ttccttcgga tctcatgcca tgtttctcag 4140catgaatcaa atttaattcg tgttttctac ttccatatat actggaagaa atttaattag 4200atctattttt gctcgggagg tcttcatact ttgagttctg atgccatcac cttatttccc 4260cccccccctt ctcttgttct atcttcttcc tcatcttggc ttgatca 4307274448DNAZea mays 27agttagcatg aaggctgcaa actagaaagg aaggtatgtt ctttcctttt tgatccatca 60ggggagcccc ttctggtatt aagatctttc cggcacattg attttcatac tttgtgatga 120ccctggaaga atcggcgtag cagcgtagca ccgctccatt ttggtcttac cctcacctcc 180ccatgctatg aactgatcaa tttcattgtt cttcatcacc cttctcctag ctttccactt 240ccttcggatc tcatgccatg tttctcagca tgaatcaaat ttaattcgtg ttttctactt 300ccatatatac tggaagaaat ttaattagat ctatttttgc tcgggaggtc ttcatacttt 360gagttctgat gccatcacct tatttccccc ccccccttct cttgttctat cttcttcctc 420atcttggctt gatcattttg atctgtcagt tatagcatga tgcattctca atttgactgt 480atgtaagttc aaccggaaat atgttgaatg gattttctat atatcaacac ttgatgtcag 540gcctgcatct gtttcgcttg tggtggtgtg gccaaaattg tctatatttg atctttgctc 600ttctttctcc tcatttcatg acgattccta ctacggctta aaccattctt tattctttac 660taatcatgga tgttgcttga ctcctagttg tttcgtacta gctcaacttg gagatctttt 720cattatttgc ctagttggtg ggtacgtttg tgacagatct aaaatggtgc acgaaaagtt 780ttacttatta tgaaaaaagg gagcttaaca gggtaatttc tctatttatt cgtgatgaca 840ttttttcctt gataaggggg attttttata atctgcactc acatgtttat atgtaaaatc 900tagctctttt gttttgtttt tggcatattt cccgctaagt atagagttta tgtggataac 960attataactt ttcaagatcc aatccacatc tttgattgtg aaaatcatac aatagggaaa 1020atcaactgaa gggttaatta gatgctatat gcatatatat atatatgtgc gcgcgcgcgc 1080gcctgaattt aactatgtat gcatccaact gtttcattga aaaagatttg atatttttca 1140gtctattctt tttcgagtat atatctaata tgtttcaatc tgttttgacc attataagat 1200aaagcctata ttcaccaggc atttgagatg atcttttcat gcatgaaaaa gctgttgtta 1260tcacttcaac taaccagacg atctaacatg tatttgtata agaaacagac cttgatttcc 1320ttctgtaaaa tcatgcatgt gttcgttttg aattggagtc ggcgcgcctg tgttttgacc 1380gtcaggaaag tctttttttt ccctgaatag tcaagggtct atacttcttg aagcaattgg 1440gacactaatc aattattgtt tatacctcgg accatctttt ccttcttcac accactaatc 1500agtttatgcc ttggaccatt aattgtgttg ttcacaagct tcttgtttat ggtttacaaa 1560gcattcgcct agatttgtgt gtgtctctac acatcgatca cttttaaata cttgtcgctt 1620tcagttattc ttttaacgtt tggttattta tcttatttaa aaaaattatc gtattattat 1680ttattttgtt tgtgatttac tttattatca aaagtatttc aaatatgact tatctttttt 1740tataagtgca ctaatttttc aaataagatg aatggtcaaa tgttacaaga aaaagttaaa 1800gcaaccacta atttagggcg gaggtagtaa aacctagtta ttgtaaccaa taattttatc 1860aatctataaa tgcaacacaa agtaacttcg tgatatctaa cacaaagcca cttcaacgat 1920gaaagctgac tgcatgtttt atcaaaacac atgtgatcag tttgttggat gaaaaaaatt 1980atctatgtca taaatcaaga gttataatat aagcttctgg ctctacaagt aacatttcta 2040tgtttttttt tacgttctta catactatgt tttgccaaaa aaaacatgat cattttgttg 2100gacgaaaaga aatagtaaat atagagtgac ctttgatatc attataatat aagcttctgc 2160ctctataaat aacatctatg cactttttac gtcgtagtaa tttgatatat gagaaattta 2220catataacat ttttgtgcag cataaccacc atggatttga gcaatagctc acctgtcatc 2280accgatccgg tggcgatcag ccagcagttg ttgggcggcc tgccttcaaa tctgatgcag 2340ttttcagtca tgcccggtgg ctactccagc tctggcatga acgttggtgt cagtaggctc 2400aaaatcgagg aagtccttgt caatggactg cttgatgcca tgaaatcctc gtcacctcgc 2460aggaggctga atgtagcatt tggcgaggac aattcatctg aagaagaaga ccctgcttac 2520agcgcttgga tggcaaaatg tccttctgct ttggcttcct tcaagcaaat tgtagccagt 2580gcacaaggga agaagattgc tgtgtttcta gactatgacg gcacactgtc gcctattgtg 2640gatgatcctg acaaagcagt gatgtctccc gtgatgagag ctgctgtgag aaatgttgcg 2700aagtacttcc ccactgcaat tgtcagcgga aggtcccgca ataaggtgtt tgaatttgta 2760aaactgaagg agctttatta tgctggaagt catggtatgg acataatggc accttcagca 2820aatcatgagc acagtgctga aaagagcaaa caggccaatc tcttccaacc tgcacacgac 2880tttctgccaa tgatcgatga ggttaccaag tccctcttgc aagttgtcag tggaattgaa 2940ggtgcaactg ttgagaacaa caaattctgc gtttctgtag attatcgcaa cgttgcagag 3000aaggattgga aactggtcgc acggctcgta aacgaagtgc tggaggcttt tcctcgtctc 3060aaagtaacca atggacgaat ggttttagag gttcgtccgg tgatcgactg ggacaaggga 3120aaggctgtgg agtttctgct ccagtcactc gggctaaatg actctgaaaa tgtgatcccc 3180atctacattg gagacgacag aactgacgaa gacgctttca aggtacttcg acagcgaaat 3240tgcggttatg gaatactagt ttcacaggtt cccaaggaaa ctgaagcctt ctactcgctg 3300agagatccat ctgaagtgat ggagttcctc aatttcttgg tgagatggaa gaagcactca 3360gtgtgagctc gctaagcagc catcgatcag ctgtcagaag ttggagctaa taataaaagg 3420gatgtggagt gggctacatg tatctcggat ctctctgcga gccacctaat ggtcttgcgt 3480ggccctttaa tctgtatgtt tttgtgtgta agctactgct agctgtttgc accttctgcg 3540tccgtggttg tgtttccgtg ctaccttttt atgttttgat ttggatcttg tttgaaaata 3600atcttaccag ctttgggtaa actgtttatt acgtactcta tatagcatat gtgaccgacg 3660acaacggttt cattttagat gatgtgtatg gatgatttct ttccaaaatc acatctttag 3720tataagagca attttaccat ccaataccaa attttatact agaaaatatt ttgggatatc 3780aaaatttatg gtacctccag taccaaatgt tgaatggtaa actttcataa tatacaagtc 3840actctaggat atttaagaca atttttagtt ttttcttatt gttgcccttg ttaaatacat 3900gagaaatttt acatcactta aaatgtatca agaggtatca aattttttta atacaaaatt 3960tagtactttc tccgtttata tatgaatgtg gacaatgctt gaaagtctta taacctgaaa 4020ctgaggtagt gtatcgagaa gtacaaaatt ttacactaaa atcccagtac ttactcaata 4080actgtaaaat tactctaaat atgtactccc tctatttcag attataagtc gttttaactt 4140tagtcaaagt taaactgttt caagtttaac caagtttgta gataaaagta gtaacatatt 4200caacacaaga caaatatatt ataaaaacat attgaattat agatttaatt aaattaattt 4260ggtattgcaa gtattactaa atttgtttat aaatttggtc gaatttaaaa tagtttgact 4320ttaaccaaag tcaaaacaaa ttataatcta aaacaaaggt aatacattgt atcactctca 4380tgaatggatt gtaacataca ttaatttaat tactatttta gttcttgtgc aaaagttgaa 4440aacgattt 4448284407DNAZea mays 28agacgacaga actgacgaag acgctttcaa ggtacttcga cagcgaaatt gcggttatgg 60aatactagtt tcacaggttc ccaaggaaac tgaagccttc tactcgctga gagatccatc 120tgaagtgatg gagttcctca atttcttggt gagatggaag aagcactcag tgtgagctcg 180ctaagcagcc atcgatcagc tgtcagaagt tggagctaat aataaaaggg atgtggagtg 240ggctacatgt atctcggatc tctctgcgag ccacctaatg gtcttgcgtg gccctttaat 300ctgtatgttt ttgtgtgtaa gctactgcta gctgtttgca ccttctgcgt ccgtggttgt 360gtttccgtgc taccttttta tgttttgatt tggatcttgt ttgaaaataa tcttaccagc 420tttgggtaaa ctgtttatta cgtactctat atagcatatg tgaccgacga caacggtttc 480attttagatg atgtgtatgg atgatttctt tccaaaatca catctttagt ataagagcaa 540ttttaccatc caataccaaa ttttatacta gaaaatattt tgggatatca aaatttatgg 600tacctccagt accaaatgtt gaatggtaaa ctttcataat atacaagtca ctctaggata 660tttaagacaa tttttagttt tttcttattg ttgcccttgt taaatacatg agaaatttta 720catcacttaa aatgtatcaa gaggtatcaa atttttttaa tacaaaattt agtactttct 780ccgtttatat atgaatgtgg acaatgcttg aaagtcttat aacctgaaac tgaggtagtg 840tatcgagaag tacaaaattt tacactaaaa tcccagtact tactcaataa ctgtaaaatt 900actctaaata tgtactccct ctatttcaga ttataagtcg ttttaacttt agtcaaagtt 960aaactgtttc aagtttaacc aagtttgtag ataaaagtag taacatattc aacacaagac 1020aaatatatta taaaaacata ttgaattata gatttaatta aattaatttg gtattgcaag 1080tattactaaa tttgtttata aatttggtcg aatttaaaat agtttgactt taaccaaagt 1140caaaacaaat tataatctaa aacaaaggta atacattgta tcactctcat gaatggattg 1200taacatacat taatttaatt actattttag ttcttgtgca aaagttgaaa acgatttatg 1260tttggaatct ttttgtggtg tatatatatg aaaccattcc tctaccatcc ttccccaacc 1320ataatcctca caaccgttag ccccattgtg atctcaccca

gttgctagcc tcttttgtca 1380ccttgtcaca gctctcctcc attcattaca caatggcatc ggaccgcgat cgcttaatta 1440agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc ctctctagag ataatgagca 1500ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt gtttgaagtg 1560cagtttatct atctttatac atatatttaa actttactct acgaataata taatctatag 1620tactacaata atatcagtgt tttagagaat catataaatg aacagttaga catggtctaa 1680aggacaattg agtattttga caacaggact ctacagtttt atctttttag tgtgcatgtg 1740ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt ttattagtac 1800atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt acatctattt 1860tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt ttttatttaa 1920taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa taccctttaa 1980gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc cagcctgtta 2040aacgccgccg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca 2100agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg agagttccgc 2160tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga gcggcagacg 2220tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct acgggggatt 2280cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct 2340ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc 2400ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc 2460ccctctctac cttctctaga tcggcgttcc ggtccatagt tagggcccgg tagttctact 2520tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgtta gcgttcgtac 2580acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg 2640gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg 2700tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg 2760tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg 2820ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt 2880ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga 2940aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga 3000tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct 3060agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat 3120gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga 3180taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc 3240tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat 3300tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt 3360agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc 3420tgttgtttgg tgttacttct gcagggatcc actagtccac catgtctccg gagaggagac 3480cagttgagat taggccagct acagcagctg atatggccgc ggtttgtgat atcgttaacc 3540attacattga gacgtctaca gtgaacttta ggacagagcc acaaacacca caagagtgga 3600ttgatgatct agagaggttg caagatagat acccttggtt ggttgctgag gttgagggtg 3660ttgtggctgg tattgcttac gctgggccct ggaaggctag gaacgcttac gattggacag 3720ttgagagtac tgtttacgtg tcacataggc atcaaaggtt gggcctagga tccacattgt 3780acacacattt gcttaagtct atggaggcgc aaggttttaa gtctgtggtt gctgttatag 3840gccttccaaa cgatccatct gttaggttgc atgaggcttt gggatacaca gcccggggta 3900cattgcgcgc agctggatac aagcatggtg gatggcatga tgttggtttt tggcaaaggg 3960attttgagtt gccagctcct ccaaggccag ttaggccagt tacccagatc tgaactagtg 4020atatcggcgc catgggtcga cctgcagatc gttcaaacat ttggcaataa agtttcttaa 4080gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg aattacgtta 4140agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt tttatgatta 4200gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc gcaaactagg 4260ataaattatc gcgcgcggtg tcatctatgt tactagatct gctagccctg caggaaattt 4320accggtgccc gggcggccag catggccgta tccgcaatgt gttattaagt cggggagggg 4380agcgtgagtt gtagtcgcgg tgtgatc 4407291116DNAOryza sativa 29atggatttga gcaatagctc acctgtcatc accgatccgg tggcgatcag ccagcagttg 60ttgggcggcc tgccttcaaa tctgatgcag ttttcagtca tgcccggtgg ctactccagc 120tctggcatga acgttggtgt cagtaggctc aaaatcgagg aagtccttgt caatggactg 180cttgatgcca tgaaatcctc gtcacctcgc aggaggctga atgtagcatt tggcgaggac 240aattcatctg aagaagaaga ccctgcttac agcgcttgga tggcaaaatg tccttctgct 300ttggcttcct tcaagcaaat tgtagccagt gcacaaggga agaagattgc tgtgtttcta 360gactatgacg gcacactgtc gcctattgtg gatgatcctg acaaagcagt gatgtctccc 420gtgatgagag ctgctgtgag aaatgttgcg aagtacttcc ccactgcaat tgtcagcgga 480aggtcccgca ataaggtgtt tgaatttgta aaactgaagg agctttatta tgctggaagt 540catggtatgg acataatggc accttcagca aatcatgagc acagtgctga aaagagcaaa 600caggccaatc tcttccaacc tgcacacgac tttctgccaa tgatcgatga ggttaccaag 660tccctcttgc aagttgtcag tggaattgaa ggtgcaactg ttgagaacaa caaattctgc 720gtttctgtac attatcgcaa cgttgcagag aaggattgga aactggtcgc acggctcgta 780aacgaagtgc tggaggcttt tcctcgtctc aaagtaacca atggacgaat ggttttagag 840gttcgtccgg tgatcgactg ggacaaggga aaggctgtgg agtttctgct ccagtcactc 900gggctaaatg actctgaaaa tgtgatcccc atctacattg gagacgacag aactgacgaa 960gacgctttca aggtacttcg acagcgaaat tgcggttatg gaatactagt ttcacaggtt 1020cccaaggaaa ctgaagcctt ctactcgctg agagatccat ctgaagtgat ggagttcctc 1080aatttcttgg tgagatggaa gaagcactca gtgtga 11163015810DNAArtificial Sequencebinary construct 15777 30aattcctgtg gttggcatgc acatacaaat ggacgaacgg ataaaccttt tcacgccctt 60ttaaatatcc gattattcta ataaacgctc ttttctctta ggtttacccg ccaatatatc 120ctgtcaaaca ctgatagttt aaactgaagg cgggaaacga caatctgatc atgagcggag 180aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt tttacgtttg 240gaactgacag aaccgcaacg ctgcaggaat tggccgcagc ggccatttaa atcaattggg 300cgcgccagct gcttgtgggg accagacaaa aaaggaatgg tgcagaattg ttaggcgcac 360ctaccaaaag catctttgcc tttattgcaa agataaagca gattcctcta gtacaagtgg 420ggaacaaaat aacgtggaaa agagctgtcc tgacagccca ctcactaatg cgtatgacga 480acgcagtgac gaccacaaaa ctcgagactt ttcaacaaag ggtaatatcc ggaaacctcc 540tcggattcca ttgcccagct atctgtcact ttattgtgaa gatagtggaa aaggaaggtg 600gctcctacaa atgccatcat tgcgataaag gaaaggctat cgttgaagat gcctctgccg 660acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa gaagacgttc 720caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta agggatgacg 780aacaatccca ctatccttcg gtaccggacc gctaggacga tggtgtgatg tgggaacacg 840aagaaaacat gaggaaaaaa tattaaaatg aatttcccac ttaaaatgca tcaaataaaa 900aaaataaaga aacgaccggg aatagacaca gggtttgtga actagctagg gcaaacatca 960tatggtccct tgctgatgca caagtacatt gagatgtcat ttcaattctg tgcatcatat 1020gcatgtggtc ccttgctgaa tattactctt gaaatatcta ccagtgccaa tctattgcat 1080gacttaatta attcacaggt tttgttgatt acattattag taagcttgag agcacaagct 1140caatggattt ttctataaat ggggatcatt ttgcaatttt ctttgtcgtg caaagttagc 1200cttctttatt actacttctg tttttaaata tacgatccta ttgacttttg gtcatatatt 1260taaccatgta tcttatttag atagtttgcg caaatatata taccttcaat gataaaatta 1320gttacaatga aacaaatgat atttacgcaa ttctttttac taaacaagtc acaagaagta 1380cctgcagcaa tatatgttgg aaccgtgcag tagatcgagc ctagctacgc aaaaaaacaa 1440aaagagaaaa aaagggaaag gaaaaacatt aatcatgcat gagcagtatg cccggcaact 1500ggaatttgtc aaagatatgg ggagaggaga ataatacaag tactactact acctagctct 1560accatgcata tgcacccaaa ggcaaactgg attattggat aaagcacaga tgctggcaaa 1620acaatcctta agcctcccct ccctgcttct ttatttttgg gcagcctcta ccggacggtg 1680ccgtggtcca ttggaccagt aggtggcgac atacatggtt tgggttaagt ctaggagagc 1740agtgtgtgtg cgcgcgcaag agagagagac tgtgagtctg ggagtagccc tctcccctcc 1800tttggccatc ttcctcgtgt atatgcatat atgcatcatc gcaacggtgt atatttgtgg 1860tgtggcgggt gtggcattgg attgccccca ttttggctcg tgcttcccag ttagggtaaa 1920acctgtggta aacttgctag ccccacgcca aagttaccct tctttattgt tgaaagggag 1980aggaggtgtg tgaattgtga tggagggaga gagagagaga tagaaagaga gatgtgtgtc 2040aaagcaagca agaaaccagt ttcacaaaga gctactacta gtactagtgt actactgtgg 2100tacagtgccc aatgtccttt ctccggactc gactccacta atattctcct cttctcgcgc 2160ggctcgttat attctcgtca tcattggagg ctttagcaag caagaagaga ggcagtggtg 2220gtggtggtgg aggaggagct agctagcctg tgcttgctga tcggtgctga gctgaggaat 2280cgttcgatcg atcgggcgag tcgacgaggg gaagagttga gctgaggcgc atcgagaaca 2340agatcaacag gcaggtcacc ttctccaagc gccgcaacgg cctcctcaag aaggcctacg 2400agctgtccgt tctctgcgac gccgaggtcg cgctcatcat cttctccagc cgcggcaagc 2460tctacgagtt cggcagcgcc gggtataatt aatacagaca caacaacaca cacaaccaac 2520aaaccagcat caatttgaac ctgcagatct gctgttttct ctgatcaatt gcttcttttt 2580ttttgttctt ttttgtttct tttatctgct gcaacggcgt cctgctcctc tggggtttct 2640cgttttcttt ttcatttatt tttagcaggt gccaagtagc cgagctacta tacttacctg 2700gccatgttaa ttattttatt ccgtctgtct gtgtgtgtct gtgcatacta ctatagggac 2760atggcgcggt gttcttataa accgggaggc cggatcccta actagcatgg gaggatatct 2820tttcagcgga tctatacaaa ccctactcct gctgacctct ttcttccagt ttctccgggt 2880cttccttgga ttattattgc ccatcttccg ggttgtgcgt gtgtcagaga cagctcgaac 2940gataaatttc tcaaaaccag tactagagag ggtgtgttgt gtgtgagaac tgagtggaga 3000gttagcatga aggctgcaaa ctagaaagga aggtatgttc tttccttttt gatccatcag 3060gggagcccct tctggtatta agatctttcc ggcacattga ttttcatact ttgtgatgac 3120cctggaagaa tcggcgtagc agcgtagcac cgctccattt tggtcttacc ctcacctccc 3180catgctatga actgatcaat ttcattgttc ttcatcaccc ttctcctagc tttccacttc 3240cttcggatct catgccatgt ttctcagcat gaatcaaatt taattcgtgt tttctacttc 3300catatatact ggaagaaatt taattagatc tatttttgct cgggaggtct tcatactttg 3360agttctgatg ccatcacctt atttcccccc cccccttctc ttgttctatc ttcttcctca 3420tcttggcttg atcattttga tctgtcagtt atagcatgat gcattctcaa tttgactgta 3480tgtaagttca accggaaata tgttgaatgg attttctata tatcaacact tgatgtcagg 3540cctgcatctg tttcgcttgt ggtggtgtgg ccaaaattgt ctatatttga tctttgctct 3600tctttctcct catttcatga cgattcctac tacggcttaa accattcttt attctttact 3660aatcatggat gttgcttgac tcctagttgt ttcgtactag ctcaacttgg agatcttttc 3720attatttgcc tagttggtgg gtacgtttgt gacagatcta aaatggtgca cgaaaagttt 3780tacttattat gaaaaaaggg agcttaacag ggtaatttct ctatttattc gtgatgacat 3840tttttccttg ataaggggga ttttttataa tctgcactca catgtttata tgtaaaatct 3900agctcttttg ttttgttttt ggcatatttc ccgctaagta tagagtttat gtggataaca 3960ttataacttt tcaagatcca atccacatct ttgattgtga aaatcataca atagggaaaa 4020tcaactgaag ggttaattag atgctatatg catatatata tatatgtgcg cgcgcgcgcg 4080cctgaattta actatgtatg catccaactg tttcattgaa aaagatttga tatttttcag 4140tctattcttt ttcgagtata tatttaatat gtttcaatct gttttgacca ttataagata 4200aagcctatat tcaccaggca tttgagatga tcttttcatg catgaaaaag ctgttgttat 4260cacttcaact aaccagacga tctaacatgt atttgtataa gaaacagacc ttgatttcct 4320tctgtaaaat catgcatgtg ttcgttttga attggagtcg gcgcgcctgt gttttgaccg 4380tcaggaaagt cttttttttc cctgaatagt caagggtcta tacttcttga agcaattggg 4440acactaatca attattgttt atacctcgga ccatcttttc cttcttcaca ccactaatca 4500gtttatgcct tggaccatta attgtgttgt tcacaagctt cttgtttatg gtttacaaag 4560cattcgccta gatttgtgtg tgtctctaca catcgatcac ttttaaatac ttgtcgcttt 4620cagttattct tttaacgttt ggttatttat cttatttaaa aaaattatcg tattattatt 4680tattttgttt gtgatttact ttattatcaa aagtatttca aatatgactt atcttttttt 4740ataagtgcac taatttttca aataagatga atggtcaaat gttacaagaa aaagttaaag 4800caaccactaa tttagggcgg aggtagtaaa acctagttat tgtaaccaat aattttatca 4860atctataaat gcaacacaaa gtcacttcgt gatatctcac acaaagccac ttcaacgatg 4920aaagctgact gcatgtttta tcaaaacaca tgtgatcagt ttgttggatg aaaaaaatta 4980tctatgtcat aaatcaagag ttataatata agcttctggc tctacaagta acatttctat 5040gttttttttt acgttcttac atactatgtt ttgccaaaaa aaacatgatc attttgttgg 5100acgaaaagaa atagtaaata tagagtgacc tttgatatca ttataatata agcttctgcc 5160tctataaata acatctatgc actttttacg tcgtagtaat ttgatatatg agaaatttac 5220atataacatt tttgtgcagc ataaccacca tggatttgag caatagctca cctgtcatca 5280ccgatccggt ggcgatcagc cagcagttgt tgggcggcct gccttcaaat ctgatgcagt 5340tttcagtcat gcccggtggc tactccagct ctggcatgaa cgttggtgtc agtaggctca 5400aaatcgagga agtccttgtc aatggactgc ttgatgccat gaaatcctcg tcacctcgca 5460ggaggctgaa tgtagcattt ggcgaggaca attcatctga agaagaagac cctgcttaca 5520gcgcttggat ggcaaaatgt ccttctgctt tggcttcctt caagcaaatt gtagccagtg 5580cacaagggaa gaagattgct gtgtttctag actatgacgg cacactgtcg cctattgtgg 5640atgatcctga caaagcagtg atgtctcccg tgatgagagc tgctgtgaga aatgttgcga 5700agtacttccc cactgcaatt gtcagcggaa ggtcccgcaa taaggtgttt gaatttgtaa 5760aactgaagga gctttattat gctggaagtc atggtatgga cataatggca ccttcagcaa 5820atcatgagca cagtgctgaa aagagcaaac aggccaatct cttccaacct gcacacgact 5880ttctgccaat gatcgatgag gttaccaagt ccctcttgca agttgtcagt ggaattgaag 5940gtgcaactgt tgagaacaac aaattctgcg tttctgtaca ttatcgcaac gttgcagaga 6000aggattggaa actggtcgca cggctcgtaa acgaagtgct ggaggctttt cctcgtctca 6060aagtaaccaa tggacgaatg gttttagagg ttcgtccggt gatcgactgg gacaagggaa 6120aggctgtgga gtttctgctc cagtcactcg ggctaaatga ctctgaaaat gtgatcccca 6180tctacattgg agacgacaga actgacgaag acgctttcaa ggtacttcga cagcgaaatt 6240gcggttatgg aatactagtt tcacaggttc ccaaggaaac tgaagccttc tactcgctga 6300gagatccatc tgaagtgatg gagttcctca atttcttggt gagatggaag aagcactcag 6360tgtgagctcg ctaagcagcc atcgatcagc tgtcagaagt tggagctaat aataaaaggg 6420atgtggagtg ggctacatgt atctcggatc tctctgcgag ccacctaatg gtcttgcgtg 6480gccctttaat ctgtatgttt ttgtgtgtaa gctactgcta gctgtttgca ccttctgcgt 6540ccgtggttgt gtttccgtgc taccttttta tgttttgatt tggatcttgt ttgaaaataa 6600tcttaccagc tttgggtaaa ctgtttatta cgtactctat atagcatatg tgaccgacga 6660caacggtttc attttagatg atgtgtatgg atgatttctt tccaaaatca catctttagt 6720ataagagcaa ttttaccatc caataccaaa ttttatacta gaaaatattt tgggatatca 6780aaatttatgg tacctccagt accaaatgtt gaatggtaaa ctttcataat atacaagtca 6840ctctaggata tttaagacaa tttttagttt tttcttattg ttgcccttgt taaatacatg 6900agaaatttta catcacttaa aatgtatcaa gaggtatcaa atttttttaa tacaaaattt 6960agtactttct ccgtttatat atgaatgtgg acaatgcttg aaagtcttat aacctgaaac 7020tgaggtagtg tatcgagaag tacaaaattt tacactaaaa tcccagtact tactcaataa 7080ctgtaaaatt actctaaata tgtactccct ctatttcaga ttataagtcg ttttaacttt 7140agtcaaagtt aaactgtttc aagtttaacc aagtttgtag ataaaagtag taacatattc 7200aacacaagac aaatatatta taaaaacata ttgaattata gatttaatta aattaatttg 7260gtattgcaag tattactaaa tttgtttata aatttggtcg aatttaaaat agtttgactt 7320taaccaaagt caaaacaaat tataatctaa aacaaaggta atacattgta tcactctcat 7380gaatggattg taacatacat taatttaatt actattttag ttcttgtgca aaagttgaaa 7440acgatttatg tttggaatct ttttgtggtg tatatatatg aaaccattcc tctaccatcc 7500ttccccaacc ataatcctca caaccgttag ccccattgtg atctcaccca gttgctagcc 7560tcttttgtca ccttgtcaca gctctcctcc attcattaca caatggcatc ggaccgcgat 7620cgcttaatta agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc ctctctagag 7680ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt 7740gtttgaagtg cagtttatct atctttatac atatatttaa actttactct acgaataata 7800taatctatag tactacaata atatcagtgt tttagagaat catataaatg aacagttaga 7860catggtctaa aggacaattg agtattttga caacaggact ctacagtttt atctttttag 7920tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt 7980ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt 8040acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt 8100ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa 8160taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc 8220cagcctgtta aacgccgccg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg 8280cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg 8340agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga 8400gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct 8460acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata 8520gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca 8580accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct 8640cccccccccc ccctctctac cttctctaga tcggcgttcc ggtccatagt tagggcccgg 8700tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgtta 8760gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg 8820tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg 8880attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc 8940cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt 9000ggtctggttg ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga 9060tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat 9120gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca 9180tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt 9240cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt 9300ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat 9360cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat 9420atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat 9480gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt 9540ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg 9600atgctcaccc tgttgtttgg tgttacttct gcagggatcc actagtccac catgtctccg 9660gagaggagac cagttgagat taggccagct acagcagctg atatggccgc ggtttgtgat 9720atcgttaacc attacattga gacgtctaca gtgaacttta ggacagagcc acaaacacca 9780caagagtgga ttgatgatct agagaggttg caagatagat acccttggtt ggttgctgag 9840gttgagggtg ttgtggctgg tattgcttac gctgggccct ggaaggctag gaacgcttac 9900gattggacag ttgagagtac tgtttacgtg tcacataggc atcaaaggtt gggcctagga 9960tccacattgt acacacattt gcttaagtct atggaggcgc aaggttttaa gtctgtggtt 10020gctgttatag gccttccaaa cgatccatct gttaggttgc atgaggcttt gggatacaca 10080gcccggggta cattgcgcgc agctggatac aagcatggtg gatggcatga tgttggtttt 10140tggcaaaggg attttgagtt gccagctcct ccaaggccag ttaggccagt tacccagatc 10200tgaactagtg atatcggcgc catgggtcga cctgcagatc gttcaaacat ttggcaataa 10260agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 10320aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 10380tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 10440gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatct gctagccctg 10500caggaaattt accggtgccc gggcggccag catggccgta tccgcaatgt gttattaagt 10560tgtctaagcg tcaatttgtt tacaccacaa tatatcctgc caccagccag ccaacagctc 10620cccgaccggc agctcggcac aaaatcacca ctcgatacag gcagcccatc agaattaatt 10680ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag 10740ccatcggaag ctgtggtatg gctgtgcagg

tcgtaaatca ctgcataatt cgtgtcgctc 10800aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa 10860tattctgaaa tgagctgttg acaattaatc atccggctcg tataatgtgt ggaattgtga 10920gcggataaca atttcacaca ggaaacagac catgagggaa gcgttgatcg ccgaagtatc 10980gactcaacta tcagaggtag ttggcgtcat cgagcgccat ctcgaaccga cgttgctggc 11040cgtacatttg tacggctccg cagtggatgg cggcctgaag ccacacagtg atattgattt 11100gctggttacg gtgaccgtaa ggcttgatga aacaacgcgg cgagctttga tcaacgacct 11160tttggaaact tcggcttccc ctggagagag cgagattctc cgcgctgtag aagtcaccat 11220tgttgtgcac gacgacatca ttccgtggcg ttatccagct aagcgcgaac tgcaatttgg 11280agaatggcag cgcaatgaca ttcttgcagg tatcttcgag ccagccacga tcgacattga 11340tctggctatc ttgctgacaa aagcaagaga acatagcgtt gccttggtag gtccagcggc 11400ggaggaactc tttgatccgg ttcctgaaca ggatctattt gaggcgctaa atgaaacctt 11460aacgctatgg aactcgccgc ccgactgggc tggcgatgag cgaaatgtag tgcttacgtt 11520gtcccgcatt tggtacagcg cagtaaccgg caaaatcgcg ccgaaggatg tcgctgccga 11580ctgggcaatg gagcgcctgc cggcccagta tcagcccgtc atacttgaag ctaggcaggc 11640ttatcttgga caagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgttca 11700ctacgtgaaa ggcgagatca ccaaagtagt cggcaaataa agctctagtg gatctccgta 11760cccagggatc tggctcgcgg cggacgcacg acgccggggc gagaccatag gcgatctcct 11820aaatcaatag tagctgtaac ctcgaagcgt ttcacttgta acaacgattg agaatttttg 11880tcataaaatt gaaatacttg gttcgcattt ttgtcatccg cggtcagccg caattctgac 11940gaactgccca tttagctgga gatgattgta catccttcac gtgaaaattt ctcaagcgct 12000gtgaacaagg gttcagattt tagattgaaa ggtgagccgt tgaaacacgt tcttcttgtc 12060gatgacgacg tcgctatgcg gcatcttatt attgaatacc ttacgatcca cgccttcaaa 12120gtgaccgcgg tagccgacag cacccagttc acaagagtac tctcttccgc gacggtcgat 12180gtcgtggttg ttgatctaga tttaggtcgt gaagatgggc tcgagatcgt tcgtaatctg 12240gcggcaaagt ctgatattcc aatcataatt atcagtggcg accgccttga ggagacggat 12300aaagttgttg cactcgagct aggagcaagt gattttatcg ctaagccgtt cagtatcaga 12360gagtttctag cacgcattcg ggttgccttg cgcgtgcgcc ccaacgttgt ccgctccaaa 12420gaccgacggt ctttttgttt tactgactgg acacttaatc tcaggcaacg tcgcttgatg 12480tccgaagctg gcggtgaggt gaaacttacg gcaggtgagt tcaatcttct cctcgcgttt 12540ttagagaaac cccgcgacgt tctatcgcgc gagcaacttc tcattgccag tcgagtacgc 12600gacgaggagg tttatgacag gagtatagat gttctcattt tgaggctgcg ccgcaaactt 12660gaggcagatc cgtcaagccc tcaactgata aaaacagcaa gaggtgccgg ttatttcttt 12720gacgcggacg tgcaggtttc gcacgggggg acgatggcag cctgagccaa ttcccagatc 12780cccgaggaat cggcgtgagc ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc 12840tgggtgatga cctggtggag aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg 12900aggcagaagc acgccccggt gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat 12960cccggcaacc gccggcagcc ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc 13020aaccagattt tttcgttccg atgctctatg acgtgggcac ccgcgatagt cgcagcatca 13080tggacgtggc cgttttccgt ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct 13140acgagcttcc agacgggcac gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt 13200gggattacga cctggtactg atggcggttt cccatctaac cgaatccatg aaccgatacc 13260gggaagggaa gggagacaag cccggccgcg tgttccgtcc acacgttgcg gacgtactca 13320agttctgccg gcgagccgat ggcggaaagc agaaagacga cctggtagaa acctgcattc 13380ggttaaacac cacgcacgtt gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg 13440tgacggtatc cgagggtgaa gccttgatta gccgctacaa gatcgtaaag agcgaaaccg 13500ggcggccgga gtacatcgag atcgagctgg ctgattggat gtaccgcgag atcacagaag 13560gcaagaaccc ggacgtgctg acggttcacc ccgattactt tttgatcgat cccggcatcg 13620gccgttttct ctaccgcctg gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt 13680tcaagacgat ctacgaacgc agtggcagcg ccggagagtt caagaagttc tgtttcaccg 13740tgcgcaagct gatcgggtca aatgacctgc cggagtacga tttgaaggag gaggcggggc 13800aggctggccc gatcctagtc atgcgctacc gcaacctgat cgagggcgaa gcatccgccg 13860gttcctaatg tacggagcag atgctagggc aaattgccct agcaggggaa aaaggtcgaa 13920aaggtctctt tcctgtggat agcacgtaca ttgggaaccc aaagccgtac attgggaacc 13980ggaacccgta cattgggaac ccaaagccgt acattgggaa ccggtcacac atgtaagtga 14040ctgatataaa agagaaaaaa ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa 14100ctcttaaaac ccgcctggcc tgtgcataac tgtctggcca gcgcacagcc gaagagctgc 14160aaaaagcgcc tacccttcgg tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta 14220tcgcggccgc tggccgctca aaaatggctg gcctacggcc aggcaatcta ccagggcgcg 14280gacaagccgc gccgtcgcca ctcgaccgcc ggcgctgagg tctgcctcgt gaagaaggtg 14340ttgctgactc ataccaggcc tgaatcgccc catcatccag ccagaaagtg agggagccac 14400ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt tgctttgcca 14460cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc cttcaactca gcaaaagttc 14520gatttattca acaaagccgc cgtcccgtca agtcagcgta atgctctgcc agtgttacaa 14580ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt 14640catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa 14700ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg 14760tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa 14820atcaccatga gtgacgactg aatccggtga gaatggcaaa agctctgcat taatgaatcg 14880gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 14940actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 15000tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 15060aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 15120ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 15180aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 15240cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 15300cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 15360aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 15420cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 15480ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 15540gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 15600gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 15660agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 15720acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 15780tcttcaccta gatccttttg atccggaatt 158103115329DNAArtificial Sequencebinary construct 15769 31attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 60taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 120tgtcaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 180attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 240aactgacaga accgcaacgc tgcaggaatt ggccgcagcg gccatttaaa tcaattgggc 300gcgccgaatt cgagctcggt accggaccgc taggacgatg gtgtgatgtg ggaacacgaa 360gaaaacatga ggaaaaaata ttaaaatgaa tttcccactt aaaatgcatc aaataaaaaa 420aataaagaaa cgaccgggaa tagacacagg gtttgtgaac tagctagggc aaacatcata 480tggtcccttg ctgatgcaca agtacattga gatgtcattt caattctgtg catcatatgc 540atgtggtccc ttgctgaata ttactcttga aatatctacc agtgccaatc tattgcatga 600cttaattaat tcacaggttt tgttgattac attattagta agcttgagag cacaagctca 660atggattttt ctataaatgg ggatcatttt gcaattttct ttgtcgtgca aagttagcct 720tctttattac tacttctgtt tttaaatata cgatcctatt gacttttggt catatattta 780accatgtatc ttatttagat agtttgcgca aatatatata ccttcaatga taaaattagt 840tacaatgaaa caaatgatat ttacgcaatt ctttttacta aacaagtcac aagaagtacc 900tgcagcaata tatgttggaa ccgtgcagta gatcgagcct agctacgcaa aaaaacaaaa 960agagaaaaaa agggaaagga aaaacattaa tcatgcatga gcagtatgcc cggcaactgg 1020aatttgtcaa agatatgggg agaggagaat aatacaagta ctactactac ctagctctac 1080catgcatatg cacccaaagg caaactggat tattggataa agcacagatg ctggcaaaac 1140aatccttaag cctcccctcc ctgcttcttt atttttgggc agcctctacc ggacggtgcc 1200gtggtccatt ggaccagtag gtggcgacat acatggtttg ggttaagtct aggagagcag 1260tgtgtgtgcg cgcgcaagag agagagactg tgagtctggg agtagccctc tcccctcctt 1320tggccatctt cctcgtgtat atgcatatat gcatcatcgc aacggtgtat atttgtggtg 1380tggcgggtgt ggcattggat tgcccccatt ttggctcgtg cttcccagtt agggtaaaac 1440ctgtggtaaa cttgctagcc ccacgccaaa gttacccttc tttattgttg aaagggagag 1500gaggtgtgtg aattgtgatg gagggagaga gagagagata gaaagagaga tgtgtgtcaa 1560agcaagcaag aaaccagttt cacaaagagc tactactagt actagtgtac tactgtggta 1620cagtgcccaa tgtcctttct ccggactcga ctccactaat attctcctct tctcgcgcgg 1680ctcgttatat tctcgtcatc attggaggct ttagcaagca agaagagagg cagtggtggt 1740ggtggtggag gaggagctag ctagcctgtg cttgctgatc ggtgctgagc tgaggaatcg 1800ttcgatcgat cgggcgagtc gacgagggga agagttgagc tgaggcgcat cgagaacaag 1860atcaacaggc aggtcacctt ctccaagcgc cgcaacggcc tcctcaagaa ggcctacgag 1920ctgtccgttc tctgcgacgc cgaggtcgcg ctcatcatct tctccagccg cggcaagctc 1980tacgagttcg gcagcgccgg gtataattaa tacagacaca acaacacaca caaccaacaa 2040accagcatca atttgaacct gcagatctgc tgttttctct gatcaattgc ttcttttttt 2100ttgttctttt ttgtttcttt tatctgctgc aacggcgtcc tgctcctctg gggtttctcg 2160ttttcttttt catttatttt tagcaggtgc caagtagccg agctactata cttacctggc 2220catgttaatt attttattcc gtctgtctgt gtgtgtctgt gcatactact atagggacat 2280ggcgcggtgt tcttataaac cgggaggccg gatccctaac tagcatggga ggatatcttt 2340tcagcggatc tatacaaacc ctactcctgc tgacctcttt cttccagttt ctccgggtct 2400tccttggatt attattgccc atcttccggg ttgtgcgtgt gtcagagaca gctcgaacga 2460taaatttctc aaaaccagta ctagagaggg tgtgttgtgt gtgagaactg agtggagagt 2520tagcatgaag gctgcaaact agaaaggaag gtatgttctt tcctttttga tccatcaggg 2580gagccccttc tggtattaag atctttccgg cacattgatt ttcatacttt gtgatgaccc 2640tggaagaatc ggcgtagcag cgtagcaccg ctccattttg gtcttaccct cacctcccca 2700tgctatgaac tgatcaattt cattgttctt catcaccctt ctcctagctt tccacttcct 2760tcggatctca tgccatgttt ctcagcatga atcaaattta attcgtgttt tctacttcca 2820tatatactgg aagaaattta attagatcta tttttgctcg ggaggtcttc atactttgag 2880ttctgatgcc atcaccttat ttcccccccc cccttctctt gttctatctt cttcctcatc 2940ttggcttgat cattttgatc tgtcagttat agcatgatgc attctcaatt tgactgtatg 3000taagttcaac cggaaatatg ttgaatggat tttctatata tcaacacttg atgtcaggcc 3060tgcatctgtt tcgcttgtgg tggtgtggcc aaaattgtct atatttgatc tttgctcttc 3120tttctcctca tttcatgacg attcctacta cggcttaaac cattctttat tctttactaa 3180tcatggatgt tgcttgactc ctagttgttt cgtactagct caacttggag atcttttcat 3240tatttgccta gttggtgggt acgtttgtga cagatctaaa atggtgcacg aaaagtttta 3300cttattatga aaaaagggag cttaacaggg taatttctct atttattcgt gatgacattt 3360tttccttgat aagggggatt ttttataatc tgcactcaca tgtttatatg taaaatctag 3420ctcttttgtt ttgtttttgg catatttccc gctaagtata gagtttatgt ggataacatt 3480ataacttttc aagatccaat ccacatcttt gattgtgaaa atcatacaat agggaaaatc 3540aactgaaggg ttaattagat gctatatgca tatatatata tatgtgcgcg cgcgcgcgcc 3600tgaatttaac tatgtatgca tccaactgtt tcattgaaaa agatttgata tttttcagtc 3660tattcttttt cgagtatata tttaatatgt ttcaatctgt tttgaccatt ataagataaa 3720gcctatattc accaggcatt tgagatgatc ttttcatgca tgaaaaagct gttgttatca 3780cttcaactaa ccagacgatc taacatgtat ttgtataaga aacagacctt gatttccttc 3840tgtaaaatca tgcatgtgtt cgttttgaat tggagtcggc gcgcctgtgt tttgaccgtc 3900aggaaagtct tttttttccc tgaatagtca agggtctata cttcttgaag caattgggac 3960actaatcaat tattgtttat acctcggacc atcttttcct tcttcacacc actaatcagt 4020ttatgccttg gaccattaat tgtgttgttc acaagcttct tgtttatggt ttacaaagca 4080ttcgcctaga tttgtgtgtg tctctacaca tcgatcactt ttaaatactt gtcgctttca 4140gttattcttt taacgtttgg ttatttatct tatttaaaaa aattatcgta ttattattta 4200ttttgtttgt gatttacttt attatcaaaa gtatttcaaa tatgacttat ctttttttat 4260aagtgcacta atttttcaaa taagatgaat ggtcaaatgt tacaagaaaa agttaaagca 4320accactaatt tagggcggag gtagtaaaac ctagttattg taaccaataa ttttatcaat 4380ctataaatgc aacacaaagt cacttcgtga tatctcacac aaagccactt caacgatgaa 4440agctgactgc atgttttatc aaaacacatg tgatcagttt gttggatgaa aaaaattatc 4500tatgtcataa atcaagagtt ataatataag cttctggctc tacaagtaac atttctatgt 4560ttttttttac gttcttacat actatgtttt gccaaaaaaa acatgatcat tttgttggac 4620gaaaagaaat agtaaatata gagtgacctt tgatatcatt ataatataag cttctgcctc 4680tataaataac atctatgcac tttttacgtc gtagtaattt gatatatgag aaatttacat 4740ataacatttt tgtgcagcat aaccaccatg gatttgagca atagctcacc tgtcatcacc 4800gatccggtgg cgatcagcca gcagttgttg ggcggcctgc cttcaaatct gatgcagttt 4860tcagtcatgc ccggtggcta ctccagctct ggcatgaacg ttggtgtcag taggctcaaa 4920atcgaggaag tccttgtcaa tggactgctt gatgccatga aatcctcgtc acctcgcagg 4980aggctgaatg tagcatttgg cgaggacaat tcatctgaag aagaagaccc tgcttacagc 5040gcttggatgg caaaatgtcc ttctgctttg gcttccttca agcaaattgt agccagtgca 5100caagggaaga agattgctgt gtttctagac tatgacggca cactgtcgcc tattgtggat 5160gatcctgaca aagcagtgat gtctcccgtg atgagagctg ctgtgagaaa tgttgcgaag 5220tacttcccca ctgcaattgt cagcggaagg tcccgcaata aggtgtttga atttgtaaaa 5280ctgaaggagc tttattatgc tggaagtcat ggtatggaca taatggcacc ttcagcaaat 5340catgagcaca gtgctgaaaa gagcaaacag gccaatctct tccaacctgc acacgacttt 5400ctgccaatga tcgatgaggt taccaagtcc ctcttgcaag ttgtcagtgg aattgaaggt 5460gcaactgttg agaacaacaa attctgcgtt tctgtacatt atcgcaacgt tgcagagaag 5520gattggaaac tggtcgcacg gctcgtaaac gaagtgctgg aggcttttcc tcgtctcaaa 5580gtaaccaatg gacgaatggt tttagaggtt cgtccggtga tcgactggga caagggaaag 5640gctgtggagt ttctgctcca gtcactcggg ctaaatgact ctgaaaatgt gatccccatc 5700tacattggag acgacagaac tgacgaagac gctttcaagg tacttcgaca gcgaaattgc 5760ggttatggaa tactagtttc acaggttccc aaggaaactg aagccttcta ctcgctgaga 5820gatccatctg aagtgatgga gttcctcaat ttcttggtga gatggaagaa gcactcagtg 5880tgagctcgct aagcagccat cgatcagctg tcagaagttg gagctaataa taaaagggat 5940gtggagtggg ctacatgtat ctcggatctc tctgcgagcc acctaatggt cttgcgtggc 6000cctttaatct gtatgttttt gtgtgtaagc tactgctagc tgtttgcacc ttctgcgtcc 6060gtggttgtgt ttccgtgcta cctttttatg ttttgatttg gatcttgttt gaaaataatc 6120ttaccagctt tgggtaaact gtttattacg tactctatat agcatatgtg accgacgaca 6180acggtttcat tttagatgat gtgtatggat gatttctttc caaaatcaca tctttagtat 6240aagagcaatt ttaccatcca ataccaaatt ttatactaga aaatattttg ggatatcaaa 6300atttatggta cctccagtac caaatgttga atggtaaact ttcataatat acaagtcact 6360ctaggatatt taagacaatt tttagttttt tcttattgtt gcccttgtta aatacatgag 6420aaattttaca tcacttaaaa tgtatcaaga ggtatcaaat ttttttaata caaaatttag 6480tactttctcc gtttatatat gaatgtggac aatgcttgaa agtcttataa cctgaaactg 6540aggtagtgta tcgagaagta caaaatttta cactaaaatc ccagtactta ctcaataact 6600gtaaaattac tctaaatatg tactccctct atttcagatt ataagtcgtt ttaactttag 6660tcaaagttaa actgtttcaa gtttaaccaa gtttgtagat aaaagtagta acatattcaa 6720cacaagacaa atatattata aaaacatatt gaattataga tttaattaaa ttaatttggt 6780attgcaagta ttactaaatt tgtttataaa tttggtcgaa tttaaaatag tttgacttta 6840accaaagtca aaacaaatta taatctaaaa caaaggtaat acattgtatc actctcatga 6900atggattgta acatacatta atttaattac tattttagtt cttgtgcaaa agttgaaaac 6960gatttatgtt tggaatcttt ttgtggtgta tatatatgaa accattcctc taccatcctt 7020ccccaaccat aatcctcaca accgttagcc ccattgtgat ctcacccagt tgctagcctc 7080ttttgtcacc ttgtcacagc tctcctccat tcattacaca atggcatcgg accgcgatcg 7140cttaattaag cttgcatgcc tgcagtgcag cgtgacccgg tcgtgcccct ctctagagat 7200aatgagcatt gcatgtctaa gttataaaaa attaccacat attttttttg tcacacttgt 7260ttgaagtgca gtttatctat ctttatacat atatttaaac tttactctac gaataatata 7320atctatagta ctacaataat atcagtgttt tagagaatca tataaatgaa cagttagaca 7380tggtctaaag gacaattgag tattttgaca acaggactct acagttttat ctttttagtg 7440tgcatgtgtt ctcctttttt tttgcaaata gcttcaccta tataatactt catccatttt 7500attagtacat ccatttaggg tttagggtta atggttttta tagactaatt tttttagtac 7560atctatttta ttctatttta gcctctaaat taagaaaact aaaactctat tttagttttt 7620ttatttaata atttagatat aaaatagaat aaaataaagt gactaaaaat taaacaaata 7680ccctttaaga aattaaaaaa actaaggaaa catttttctt gtttcgagta gataatgcca 7740gcctgttaaa cgccgccgac gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg 7800tcgggccaag cgaagcagac ggcacggcat ctctgtcgct gcctctggac ccctctcgag 7860agttccgctc caccgttgga cttgctccgc tgtcggcatc cagaaattgc gtggcggagc 7920ggcagacgtg agccggcacg gcaggcggcc tcctcctcct ctcacggcac cggcagctac 7980gggggattcc tttcccaccg ctccttcgct ttcccttcct cgcccgccgt aataaataga 8040caccccctcc acaccctctt tccccaacct cgtgttgttc ggagcgcaca cacacacaac 8100cagatctccc ccaaatccac ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc 8160cccccccccc ctctctacct tctctagatc ggcgttccgg tccatagtta gggcccggta 8220gttctacttc tgttcatgtt tgtgttagat ccgtgtttgt gttagatccg tgctgttagc 8280gttcgtacac ggatgcgacc tgtacgtcag acacgttctg attgctaact tgccagtgtt 8340tctctttggg gaatcctggg atggctctag ccgttccgca gacgggatcg atttcatgat 8400tttttttgtt tcgttgcata gggtttggtt tgcccttttc ctttatttca atatatgccg 8460tgcacttgtt tgtcgggtca tcttttcatg cttttttttg tcttggttgt gatgatgtgg 8520tctggttggg cggtcgttct agatcggagt agaattctgt ttcaaactac ctggtggatt 8580tattaatttt ggatctgtat gtgtgtgcca tacatattca tagttacgaa ttgaagatga 8640tggatggaaa tatcgatcta ggataggtat acatgttgat gcgggtttta ctgatgcata 8700tacagagatg ctttttgttc gcttggttgt gatgatgtgg tgtggttggg cggtcgttca 8760ttcgttctag atcggagtag aatactgttt caaactacct ggtgtattta ttaattttgg 8820aactgtatgt gtgtgtcata catcttcata gttacgagtt taagatggat ggaaatatcg 8880atctaggata ggtatacatg ttgatgtggg ttttactgat gcatatacat gatggcatat 8940gcagcatcta ttcatatgct ctaaccttga gtacctatct attataataa acaagtatgt 9000tttataatta ttttgatctt gatatacttg gatgatggca tatgcagcag ctatatgtgg 9060atttttttag ccctgccttc atacgctatt tatttgcttg gtactgtttc ttttgtcgat 9120gctcaccctg ttgtttggtg ttacttctgc agggatccac tagtccacca tgtctccgga 9180gaggagacca gttgagatta ggccagctac agcagctgat atggccgcgg tttgtgatat 9240cgttaaccat tacattgaga cgtctacagt gaactttagg acagagccac aaacaccaca 9300agagtggatt gatgatctag agaggttgca agatagatac ccttggttgg ttgctgaggt 9360tgagggtgtt gtggctggta ttgcttacgc tgggccctgg aaggctagga acgcttacga 9420ttggacagtt gagagtactg tttacgtgtc acataggcat caaaggttgg gcctaggatc 9480cacattgtac acacatttgc ttaagtctat ggaggcgcaa ggttttaagt ctgtggttgc 9540tgttataggc cttccaaacg atccatctgt taggttgcat gaggctttgg gatacacagc 9600ccggggtaca ttgcgcgcag ctggatacaa gcatggtgga tggcatgatg ttggtttttg 9660gcaaagggat tttgagttgc cagctcctcc aaggccagtt aggccagtta cccagatctg 9720aactagtgat atcggcgcca tgggtcgacc tgcagatcgt tcaaacattt ggcaataaag 9780tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa 9840ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 9900tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa

tatagcgcgc 9960aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatctgc tagccctgca 10020ggaaatttac cggtgcccgg gcggccagca tggccgtatc cgcaatgtgt tattaagttg 10080tctaagcgtc aatttgttta caccacaata tatcctgcca ccagccagcc aacagctccc 10140cgaccggcag ctcggcacaa aatcaccact cgatacaggc agcccatcag aattaattct 10200catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc 10260atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa 10320ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt ctggcaaata 10380ttctgaaatg agctgttgac aattaatcat ccggctcgta taatgtgtgg aattgtgagc 10440ggataacaat ttcacacagg aaacagacca tgagggaagc gttgatcgcc gaagtatcga 10500ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg 10560tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc 10620tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt 10680tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg 10740ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag 10800aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc gacattgatc 10860tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg 10920aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa 10980cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt 11040cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact 11100gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct aggcaggctt 11160atcttggaca agaagatcgc ttggcctcgc gcgcagatca gttggaagaa tttgttcact 11220acgtgaaagg cgagatcacc aaagtagtcg gcaaataaag ctctagtgga tctccgtacc 11280cagggatctg gctcgcggcg gacgcacgac gccggggcga gaccataggc gatctcctaa 11340atcaatagta gctgtaacct cgaagcgttt cacttgtaac aacgattgag aatttttgtc 11400ataaaattga aatacttggt tcgcattttt gtcatccgcg gtcagccgca attctgacga 11460actgcccatt tagctggaga tgattgtaca tccttcacgt gaaaatttct caagcgctgt 11520gaacaagggt tcagatttta gattgaaagg tgagccgttg aaacacgttc ttcttgtcga 11580tgacgacgtc gctatgcggc atcttattat tgaatacctt acgatccacg ccttcaaagt 11640gaccgcggta gccgacagca cccagttcac aagagtactc tcttccgcga cggtcgatgt 11700cgtggttgtt gatctagatt taggtcgtga agatgggctc gagatcgttc gtaatctggc 11760ggcaaagtct gatattccaa tcataattat cagtggcgac cgccttgagg agacggataa 11820agttgttgca ctcgagctag gagcaagtga ttttatcgct aagccgttca gtatcagaga 11880gtttctagca cgcattcggg ttgccttgcg cgtgcgcccc aacgttgtcc gctccaaaga 11940ccgacggtct ttttgtttta ctgactggac acttaatctc aggcaacgtc gcttgatgtc 12000cgaagctggc ggtgaggtga aacttacggc aggtgagttc aatcttctcc tcgcgttttt 12060agagaaaccc cgcgacgttc tatcgcgcga gcaacttctc attgccagtc gagtacgcga 12120cgaggaggtt tatgacagga gtatagatgt tctcattttg aggctgcgcc gcaaacttga 12180ggcagatccg tcaagccctc aactgataaa aacagcaaga ggtgccggtt atttctttga 12240cgcggacgtg caggtttcgc acggggggac gatggcagcc tgagccaatt cccagatccc 12300cgaggaatcg gcgtgagcgg tcgcaaacca tccggcccgg tacaaatcgg cgcggcgctg 12360ggtgatgacc tggtggagaa gttgaaggcc gcgcaggccg cccagcggca acgcatcgag 12420gcagaagcac gccccggtga atcgtggcaa gcggccgctg atcgaatccg caaagaatcc 12480cggcaaccgc cggcagccgg tgcgccgtcg attaggaagc cgcccaaggg cgacgagcaa 12540ccagattttt tcgttccgat gctctatgac gtgggcaccc gcgatagtcg cagcatcatg 12600gacgtggccg ttttccgtct gtcgaagcgt gaccgacgag ctggcgaggt gatccgctac 12660gagcttccag acgggcacgt agaggtttcc gcagggccgg ccggcatggc cagtgtgtgg 12720gattacgacc tggtactgat ggcggtttcc catctaaccg aatccatgaa ccgataccgg 12780gaagggaagg gagacaagcc cggccgcgtg ttccgtccac acgttgcgga cgtactcaag 12840ttctgccggc gagccgatgg cggaaagcag aaagacgacc tggtagaaac ctgcattcgg 12900ttaaacacca cgcacgttgc catgcagcgt acgaagaagg ccaagaacgg ccgcctggtg 12960acggtatccg agggtgaagc cttgattagc cgctacaaga tcgtaaagag cgaaaccggg 13020cggccggagt acatcgagat cgagctggct gattggatgt accgcgagat cacagaaggc 13080aagaacccgg acgtgctgac ggttcacccc gattactttt tgatcgatcc cggcatcggc 13140cgttttctct accgcctggc acgccgcgcc gcaggcaagg cagaagccag atggttgttc 13200aagacgatct acgaacgcag tggcagcgcc ggagagttca agaagttctg tttcaccgtg 13260cgcaagctga tcgggtcaaa tgacctgccg gagtacgatt tgaaggagga ggcggggcag 13320gctggcccga tcctagtcat gcgctaccgc aacctgatcg agggcgaagc atccgccggt 13380tcctaatgta cggagcagat gctagggcaa attgccctag caggggaaaa aggtcgaaaa 13440ggtctctttc ctgtggatag cacgtacatt gggaacccaa agccgtacat tgggaaccgg 13500aacccgtaca ttgggaaccc aaagccgtac attgggaacc ggtcacacat gtaagtgact 13560gatataaaag agaaaaaagg cgatttttcc gcctaaaact ctttaaaact tattaaaact 13620cttaaaaccc gcctggcctg tgcataactg tctggccagc gcacagccga agagctgcaa 13680aaagcgccta cccttcggtc gctgcgctcc ctacgccccg ccgcttcgcg tcggcctatc 13740gcggccgctg gccgctcaaa aatggctggc ctacggccag gcaatctacc agggcgcgga 13800caagccgcgc cgtcgccact cgaccgccgg cgctgaggtc tgcctcgtga agaaggtgtt 13860gctgactcat accaggcctg aatcgcccca tcatccagcc agaaagtgag ggagccacgg 13920ttgatgagag ctttgttgta ggtggaccag ttggtgattt tgaacttttg ctttgccacg 13980gaacggtctg cgttgtcggg aagatgcgtg atctgatcct tcaactcagc aaaagttcga 14040tttattcaac aaagccgccg tcccgtcaag tcagcgtaat gctctgccag tgttacaacc 14100aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca 14160tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact 14220caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc 14280caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat 14340caccatgagt gacgactgaa tccggtgaga atggcaaaag ctctgcatta atgaatcggc 14400caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 14460tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 14520cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 14580aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 14640gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 14700agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 14760cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 14820cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 14880ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 14940gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 15000tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 15060acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 15120tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 15180attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 15240gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 15300ttcacctaga tccttttgat ccggaatta 153293219DNAArtificial SequenceTaqMan forward primer P23198 32gcgtgagttg gagtcgtgg 193323DNAArtificial SequenceTaqMan reverse primer P23352 33attctccgct catgatcaga ttg 233431DNAArtificial SequenceTaqMan probe labeled with FAM on 5'-terminus and labeled with BHQ1 on 3'-terminus 34cactgatagt ttaaactgaa ggcgggaaac g 313580DNAArtificial SequenceTaqMan amplicon 35gcgtgagttg gagtcgtgga ggaaacactg atagtttaaa ctgaaggcgg gaaacgacaa 60tctgatcatg agcggagaat 80

Claims

1. A nucleic acid molecule, preferably isolated, comprising a nucleotide sequence that is unique to event MZDT09Y, wherein the nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and the complements thereof.

2. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is comprised in a corn seed deposited at the American Type Culture Collection under the accession number PTA-13025.

3. A pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer which function together in the presence of an event MZDT09Y DNA template in a sample to produce an amplicon diagnostic for event MZDT09Y.

4. The pair of polynucleotide primers according to claim 3, wherein a. a sequence of the first polynucleotide primer or a sequence of the second polynucleotide primer is chosen from SEQ ID NO: 7, or the complement thereof; or b. a sequence of the first polynucleotide primer is or is complementary to a corn plant genome sequence flanking the point of insertion of a heterologous DNA sequence inserted into the corn plant genome of event MZDT09Y, and a sequence of the second polynucleotide primer is or is complementary to the heterologous DNA sequence inserted into the genome of event MZDT09Y.

5. The pair of polynucleotide primers according to claim 4, wherein a. the first polynucleotide primer comprises at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and the complements thereof; and b. the second polynucleotide primer comprises at least 10 contiguous nucleotides from SEQ ID NO: 7, or the complements thereof.

6. The pair of polynucleotide primer according for claim 5, wherein a. the first polynucleotide primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 32, and the complements thereof; and b. the second polynucleotide primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 33, and the complements thereof.

7. The pair of polynucleotide primers according to claim 6, wherein a. the first polynucleotide primer consists of SEQ ID NO: 12 and the second polynucleotide primer consists of SEQ ID NO: 13; or b. the first polynucleotide primer consists of SEQ ID NO: 14 and the second polynucleotide primer consists of SEQ ID NO: 15; or c. the first polynucleotide primer consists of SEQ ID NO: 32 and the second polynucleotide primer consists of SEQ ID NO: 33.

8. A method of detecting the presence of a nucleic acid molecule that is unique to event MZDT09Y in a sample comprising corn nucleic acids, the method comprising: a. contacting the sample with a pair of primers that, when used in a nucleic acid amplification reaction with genomic DNA from event MZDT09Y produces an amplicon that is diagnostic for event MZDT09Y; b. performing a nucleic acid amplification reaction, thereby producing the amplicon; and c. detecting the amplicon.

9. A method of detecting the presence of a nucleic acid molecule that is unique to event MZDT09Y in a sample comprising corn nucleic acids, the method comprising: a. contacting the sample with a probe that hybridizes under high stringency conditions with genomic DNA from event MZDT09Y and does not hybridize under high stringency conditions with DNA of a control corn plant; b. subjecting the sample and probe to high stringency hybridization conditions; and c. detecting hybridization of the probe to the nucleic acid molecule.

10. A kit for detecting nucleic acids that are unique to event MZDT09Y comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event MZDT09Y in the sample.

11. The kit according to claim 10, wherein the nucleic acid molecule of sufficient length of continuous polynucleotides comprises a nucleotide sequence selected from the group consisting SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, and the complements thereof.

12. The kit according to claim 11, wherein the nucleic acid molecule is selected from the group consisting of SEQ ID NOs: 12-15, SEQ ID NOs: 32-34, and the complements thereof.

13. A transgenic corn plant, or cells or tissues thereof, comprising a nucleic acid molecule according to claim 1.

14. A corn seed comprising a nucleic acid molecule according to claim 1, an example of the seed being deposited at the American Type Culture Collection under the accession number PTA-13025.

15. A biological sample derived from an event MZDT09Y corn plant, tissue, or seed, wherein the sample comprises a nucleotide sequence which is or is complementary to a nucleotide sequence of claim 1 and wherein the sequence is detectable in the sample using a nucleic acid amplification or nucleic acid hybridization method.

16. The biological sample of claim 15 wherein the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products.

17. An extract derived from the biological sample according to claim 15.

18. The extract of claim 17 wherein the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, corn starch, and cereals manufactured in whole or in part to contain corn by-products.

19. A method for producing a corn plant with increased yield comprising: a. sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises event MZDT09Y DNA, thereby producing a plurality of first generation progeny plants; b. selecting a first generation progeny plant with increased yield; c. selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and d. selecting from the second generation progeny plants, a plant with increased yield; wherein the second generation progeny plants comprise the nucleic acid molecule according to claim 1.

20. The method of claim 19, wherein increased yield is indicated by an increase as compared to a control plant of any one of the following: increased grain yield, increased seed, increased seed weight, increased biomass, increased sugar, increased oil, increased plant vigor, increased yield under non-optimal conditions, increased yield under stress conditions and increased yield under water stress conditions.

21. A method for producing a corn plant with increased tolerance to abiotic stress comprising: a. sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises event MZDT09Y DNA, thereby producing a plurality of first generation progeny plants; b. selecting a first generation progeny plant with increased tolerance to abiotic stress; c. selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and d. selecting from the second generation progeny plants, a plant with increase tolerance to abiotic stress; wherein the second generation progeny plants comprise the nucleic acid molecule according to claim 1.

22. The method of claim 21, wherein said abiotic stress comprises stress selected from the group consisting of water stress, heat stress or cold stress.

23. The method of claim 22, wherein water stress is caused by drought.

24. A method of producing hybrid corn seeds comprising: a. planting seeds of a first inbred corn line comprising event MZDT09Y and seeds of a second inbred line having a genotype different from the first inbred corn line; b. cultivating corn plants resulting from said planting until time of flowering; c. emasculating said flowers of plants of one of the corn inbred lines; d. sexually crossing the two different inbred lines with each other; and e. harvesting the hybrid seed produced thereby; wherein the hybrid seed comprise the nucleic acid molecule according to claim 1.

25. The method according to claim 24, wherein the plants of the first inbred corn line are the female parents or male parents.

26. Hybrid seed produced by the method of claim 24.