Glucanase Production And Methods Of Using The Same

Abstract

Methods and compositions are described for producing a glucanase in transgenic plants and then incorporating parts of the transgenic plants in animal feed. The feed glucanase enzyme displays activity across a broad pH range, and tolerance to temperatures that are often encountered during the process of preparing animal feeds. Producing the enzyme in the transgenic plant enhances the thermal stability of the enzyme.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 16/599,543, which was filed on Oct. 11, 2019. U.S. patent application Ser. No. 16/599,543 is a continuation of U.S. patent application Ser. No. 15/569,592, which was filed on Oct. 26, 2017 as 35 U.S.C. .sctn. 371 national phase application of PCT/US2016/032418, which was filed May 13, 2016, and claimed the benefit of U.S. Provisional Patent Application No. 62/161,482 filed May 14, 2015, all of which are incorporated herein by reference as if fully set forth.

[0002] The sequence listing electronically filed with this application titled "Sequence Listing," which was created on Jun. 23, 2021 and has a size of 146,050 bytes, is incorporated by reference herein as if fully set forth.

FIELD

[0003] This disclosure relates to transgenic plants expressing glucanases with improved thermal stability, nucleic acids encoding the same, as well as methods of processing transgenic plants and tissues, and producing and utilizing animal feed. This disclosure also relates to feed additives and grain and fiber processing additives that include glucanases.

BACKGROUND

[0004] The abundance of non-starch polysaccharides (NSPs) in the diets of monogastric and ruminant animals can adversely affect the nutritional value of feed, and also present an opportunity to improve nutritional content if they can be degraded in the diet or converted into beneficial nutritional components. NSPs are among the primary structural components of plant cell wall (cellulose, hemicellulose, xyloglucans, arabionxylans, galactans, arabinogalactans, etc.) and can also serve as carbohydrate storage reserves in some plants. Additionally, pectins and gums are considered non-cell wall NSP. Because of their various structural and biological roles, NSPs often bind or encase the starch, proteins, fats and other nutrients that are present in plant-based feed ingredients (such as cereals, legumes, silage etc.) and other ingredients, inhibiting the animal's ability to digest nutrients efficiently. Increased levels of NSPs in the diet may increase viscosity of intestinal contents, which can interfere with digestive enzymes and reduce the digestibility of nutrients, thereby increasing feed conversion (mass of feed divided by the mass of meat produced) and reducing body weight gain (Iji, P. A. 1999. The impacts of cereal non-starch polysaccharides on intestinal development and function in the broiler chickens. Worlds Poult. Sci. J. 55:375-387, which is incorporated herein by reference as if fully set forth). For example, feeding increasing levels of guar meal germ (0, 5, or 7.5%) or guar meal hulls (0, 2.5, or 5%) to broilers resulted in increasing digesta viscosity (Lee, J. T., C. A. Bailey, and A. L. Cartwright. 2003. .beta.-Mannanase ameliorates viscosity-associated depression of growth in broiler chickens fed guar germ and hull fractions. Poult. Sci. 82:1925-1931, which is incorporated herein by reference as if fully set forth). In addition to increasing the viscosity, body weight gain and feed conversion was also worse with increasing guar meal hull, demonstrating the negative effects of high viscosity on animal performance.

[0005] NSPs have also been known to inadvertently trigger immune responses in the gut, which may further detract from efficiency of feed utilization and have implications for animal health.

[0006] In addition to the cereal components, diets now also routinely contain DDGS (dried distillers grains and solubles) that is also not easily digested. Multiple studies have shown that enzyme supplementation can increase diet metabolizable energy (ME), and, or, decrease the viscosity of diets containing high levels of wheat, barley, DDGSs, or other fibrous components. The addition of carbohydrases to corn-soybean meal-based broiler diets, when formulated to have a 3% reduction in dietary ME, has been accomplished without compromising the feed conversions of broilers reared under either hot or cool seasons. It has been determined that the hydrolyzed .beta.-d-glucan is responsible for improved growth.

SUMMARY

[0007] In an aspect, the invention relates to a method of identifying maize event 4588.259, 4588.757 or 4588.652 in a sample. The method comprises contacting a sample with a first primer and a second primer. The method comprises amplifying a nucleic acid in the sample to obtain an amplified product. The method also comprises detecting the amplified product specific to a target sequence in maize event 4588.259, 4588.757 or 4588.652.

[0008] In an aspect, the invention relates to an animal feedstock comprising a transgenic plant or part thereof. The transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase. The glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.

[0009] In an aspect, the invention relates to a method of producing an animal feedstock. The method includes mixing a transgenic plant or part thereof with plant material to form a mixture. The transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase. The glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.

[0010] In an aspect, the invention relates to a method of increasing metabolizable energy of a diet. The method includes mixing a transgenic plant or part thereof with a feed ingredient. The transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase comprising an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides.

[0011] In an aspect, the invention relates to a method of enhancing production of fermentable sugars from grains. The method includes mixing grains derived from any one of the transgenic plants described herein with grains derived from a different plant to form mixed grains. The method also includes processing mixed grains into fermentable sugars. The fermentable sugars are subsequently converted into ethanol or a similar fermentation product, which may include butanol, lactic acid, citric acid, acetic acid, or other fuels or chemicals.

[0012] In an aspect, the invention relates to a transgenic plant, transgenic grain, or transgenic biomass comprising a synthetic nucleic acid encoding a glucanase. The glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6. The glucanase is capable of degrading one or more polysaccharides.

[0013] In an aspect the invention relates to a synthetic polypeptide or a fragment thereof. The synthetic polypeptide or a fragment thereof comprises an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6. The glucanase is capable of degrading one or more polysaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings particular embodiments. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0015] FIG. 1 illustrates the expression vector pAG4258 carrying a single feed glucanase expression unit.

[0016] FIG. 2 illustrates the expression vector pAG4588 carrying a single feed glucanase expression unit.

[0017] FIG. 3 illustrates the expression vector pAG4597 carrying a single feed glucanase expression unit.

[0018] FIG. 4 illustrates the expression vector pAG4708 carrying a single feed glucanase expression unit.

[0019] FIG. 5 illustrates the expression vector pAG4766 carrying two feed glucanase expression units.

[0020] FIG. 6 illustrated the expression vector pAG4767 carrying two feed glucanase expression units.

[0021] FIG. 7 illustrates the expression vector pAG4770 carrying three feed glucanase expression units.

[0022] FIG. 8 illustrates the expression vector pAG4771 carrying three feed glucanase expression units.

[0023] FIG. 9 is a chart illustrating the range of glucanase activity recovered from ears of the maize plants that carried pAG4588 construct.

[0024] FIG. 10 is a chart illustrating the range of glucanase activity recovered from ears of the maize plants that carried pAG4597 construct.

[0025] FIG. 11 is a diagram showing the T-DNA integration site in chromosome 7 of maize event 4588.652.

[0026] FIG. 12 is a chart illustrating glucanase activity observed in T1 plants.

[0027] FIG. 13 is a chart illustrating the glucanase activity in the seeds of hemizygous, homozygous, and hybrid plants.

[0028] FIG. 14 illustrates general design of the real-time PCR assay used to determine presence of the T-DNA locus (standard and real-time PCR) and zygosity (real-time PCR only) in transgenic events. Letters A, B, X and Y with arrows indicate primer binding sites. Rectangular boxes A+B and X+Y represent PCR products amplified from respective primer pairs.

[0029] FIG. 15 illustrates general design of the standard PCR assay used to determine presence of the T-DNA locus and zygosity in transgenic events. Letters A, B, and C with arrows indicate primer binding sites. Rectangular boxes A+B and A+C represent PCR products amplified from respective primer pairs.

[0030] FIG. 16 illustrates the standard multiplex PCR analysis of the selfed segregating 4588.652 plants.

[0031] FIG. 17 is a graph illustrating real-time PCR data to determine presence of the T-DNA locus and zygosity for maize event 4588.259 (FG259).

[0032] FIGS. 18A and 18B are charts illustrating glucanase activity in the Grower Diet (FIG. 18A) and the Starter Diet (FIG. 18B) before and after pelleting.

[0033] FIGS. 19A and 19B are charts illustrating glucanase activity in wild type (WT) flour mixed with microbial glucanase and transgenic flour producing glucanase after heat treatment at temperatures of 130.degree. C. (FIG. 19A) and 94.degree. C. (FIG. 19B).

[0034] FIGS. 20 and 21 are graphs illustrating the optimum pH for measuring AGR2314 activity in an assay at 37.degree. C. (FIG. 20) and 80.degree. C. (FIG. 21).

[0035] FIG. 22 is a graph illustrating an example of the optimum pH of the feed glucanase that is produced in transgenic flour.

[0036] FIGS. 23A and 23B are charts illustrating glucanase activity on multiple substrates at 37.degree. C. (FIG. 23A) and 80.degree. C. (FIG. 23B).

[0037] FIGS. 24A and 24B are charts illustrating enzymatic hydrolysis of untreated seeds fiber of transgenic maize plants expressing AGR2314. FIG. 24A shows glucose yield and FIG. 24B shows xylose yield.

[0038] FIGS. 25A and 25B are charts illustrating enzymatic hydrolysis of seed fiber of transgenic maize plants expressing AGR2314 pretreated with the dilute acid. FIG. 25A shows glucose yield and FIG. 25B shows xylose yield.

[0039] FIG. 26 is a chart illustrating the body weight gain (BWG) during the 28-day poultry feeding trial.

[0040] FIG. 27 is a chart illustrating the changes in poultry BWG per time interval during 28 day feeding trial.

[0041] FIG. 28 is a chart illustrating feed consumption during the 28-day poultry feeding trial using two different diets (corn/barley based and corn/LF-DDGS based) with (+) or without (-) a glucanase.

[0042] FIG. 29 is a chart illustrating feed conversion rate (FCR) during the 28-day poultry feeding trial with two different diets (corn/barley based and corn/LF-DDGS based diets) with (+) or without (-) a glucanase.

[0043] FIG. 30 is a chart illustrating the effect of glucanase on poultry BWF in experimental treatment.

DETAILED DESCRIPTION OF EMBODIMENTS

[0044] Certain terminology is used in the following description for convenience only and is not limiting.

[0045] "Synthetic nucleic acid sequence," "synthetic polynucleotide," "synthetic oligonucleotide," "synthetic DNA," or "synthetic RNA" as used herein refers to a nucleic acid sequence, a polynucleotide, an oligonucleotide, DNA, or RNA that differs from one found in nature by having a different sequence than one found in nature or a chemical modification not found in nature. This can include, but is not limited to, a DNA sequence created using biotechnology tools. Such tools include but are not limited to recombinant DNA technology, polymerase chain reaction (PCR), biotechnology mutagenesis techniques using PCR or recombination techniques including digestion and ligation of DNA, chemical mutagenesis techniques, chemical synthesis, or directed use of nucleases (so called "genome editing" or "gene optimizing" technologies).

[0046] "Synthetic protein," "synthetic polypeptide," "synthetic oligopeptide," or "synthetic peptide" as used herein refers to a protein, polypeptide, oligopeptide or peptide that was made through a synthetic process. The synthetic process can include, but is not limited, to chemical synthesis or recombinant technology. The synthetic process may include production of a protein, polypeptide, oligopeptide or peptide by expression of a synthetic nucleic acid sequence in a living cell or by in vitro expression using a cell-free extract.

[0047] As used herein, "variant" refers to a molecule that retains a biological activity that is the same or substantially similar to that of the original sequence. The variant may be from the same or different species or be a synthetic sequence based on a natural or prior molecule.

[0048] As used herein, "alignment" refers to a plurality of nucleic acid or amino acid sequences aligned lengthwise for visual identification of commonly shared nucleotides or amino acids. The percentage of commonly shared nucleotides or amino acid is related to homology or identity between sequences. An alignment may be determined by or used to identify conserved domains and relatedness between the sequences. An alignment may be determined by computer programs such as CLUSTAL 0 (1.2.1) (Sievers et al. (2011) Molecular Systems Biology 7: 539 doi: 10. 1038/msb.2011.75).

[0049] The words "a" and "one," as used in the claims and in the corresponding portions of the specification, are defined as including one or more of the referenced item unless specifically stated otherwise.

[0050] In an embodiment, a synthetic nucleic acid encoding a glucanase is provided. The synthetic nucleic acid may include a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 1 [AGR2314], SEQ ID NO: 2 [AGR2414], and SEQ ID NO: 3 [AGR2514]. The encoded glucanase may be capable of degrading one or more polysaccharides.

[0051] An embodiment includes a glucanase that includes a synthetic polypeptide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414], and SEQ ID NO: 6 [AGR2514]. The glucanase may be capable of degrading one or more polysaccharides. The glucanase may be modified for improved thermal stability.

[0052] A glucanase modified for thermal stability can be produced by standard molecular biological techniques and then screened. The glucanase can be subjected to mutation and then screened for thermal stability. Screening systems that can be utilized include lambda phage, yeast, or other expression systems that allow production of the protein and/or testing of its physical and/or functional characteristics. From a population of modified proteins, candidates can be isolated and analyzed further. Further analysis may include DNA sequencing, functional assays, structural assays, enzyme activity assays, and monitoring changes in thermal stability, or structure in response to elevated temperature conditions.

[0053] In an embodiment, a glucanase may be produced in a plant or plant tissue. The glucanase may be isolated from the plant or plant tissue.

[0054] An embodiment includes a composition comprising, consisting essentially of, or consisting of one or more glucanases. The composition may be, but is not limited, to a transgenic plant including the one or more glucanases, an animal feedstock or animal feed additive including the one or more glucanases or an enzyme mixture including the one or more glucanases. A glucanase in the composition may be encoded by any one of the synthetic nucleic acids described herein. As used herein, the term "glucanase" refers to an enzyme capable of catalyzing the degradation or depolymerization of complex carbohydrates.

[0055] A glucanase in the composition may be capable of degrading one or more of disaccharides, trisaccharides, and oligosaccharides into lower molecular weight saccharides. A glucanase in the composition may be capable of degrading one or more of cellooligosaccharide, lignocellulose, cellulose, hemicellulose, and pectin. A glucanase of the composition may act upon cellulose or mixed linkage beta glucans Some glucanases may have broader substrate specificities and may act on a wide range of carbohydrate polymers. A glucanase of the composition may have enzymatic activity on a range of carbohydrate polymers. Such enzymatic activit may be, but is not limited to, endoglucanase, exoglucanase, .beta.-glucosidase, cellobiohydrolase, endo-1,4-.beta.-xylanase, .beta.-xylosidase, .alpha.-glucuronidase, .alpha.-L-arabinofuranosidase, acetylesterase, acetylxylanesterase, .alpha.-amylase, .beta.-amylase, glucoamylase, pullulanase, .beta.-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase, or pectate lyase activities. The glucanase of the composition may be capable of degrading one or more of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. Assays for determining activity of a glucanase for degrading various substrates are known in the art. The beta-glucosidase assay, endocellulase assay, exocellulase (cellobiohydrolase) assay, amylase assay, endoxylanase assay, pectinase assay, 1,3-beta-glucosidase assay, 1,4-beta-glucosidase assay are described herein in Example 13.

[0056] A glucanase of the composition may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514].

[0057] An embodiment includes a composition comprising, consisting essentially of, or consisting of an individual glucanase or a combination of two or more glucanases herein.

[0058] In an embodiment, a glucanase of the composition may be a variant. Variants may include conservative amino acid substitutions; i.e., substitutions with amino acids having similar properties. Conservative substitutions may be a polar for polar amino acid (Glycine (G, Gly), Serine (S, Ser), Threonine (T, Thr), Tyrosine (Y, Tyr), Cysteine (C, Cys), Asparagine (N, Asn) and Glutamine (Q, Gln)); a non-polar for non-polar amino acid (Alanine (A, Ala), Valine (V, Val), Thyptophan (W, Trp), Leucine (L, Leu), Proline (P, Pro), Methionine (M, Met), Phenilalanine (F, Phe)); acidic for acidic amino acid (Aspartic acid (D, Asp), Glutamic acid (E, Glu)); basic for basic amino acid (Arginine (R, Arg), Histidine (H, His), Lysine (K, Lys)); charged for charged amino acids (Aspartic acid (D, Asp), Glutamic acid (E, Glu), Histidine (H, His), Lysine (K, Lys) and Arginine (R, Arg)); and a hydrophobic for hydrophobic amino acid (Alanine (A, Ala), Leucine (L, Leu), Isoleucine (I, Ile), Valine (V, Val), Proline (P, Pro), Phenylalanine (F, Phe), Tryptophan (W, Trp) and Methionine (M, Met)). Conservative nucleotide substitutions may be made in a nucleic acid sequence by substituting a codon for an amino acid with a different codon for the same amino acid. Variants may include non-conservative substitutions. A variant may have 40% glucanase activity in comparison to the unchanged glucanase. A variant may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% activity, or an integer between any of the two values herein, in comparison to the unchanged glucanase.

[0059] In an embodiment, the one or more proteins having less than 100% identity to its corresponding amino acid sequence of SEQ ID NOS: 4-6 is a variant of the referenced protein or amino acid. In an embodiment, an isolated protein, polypeptide, oligopeptide, or peptide having a sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a protein having the sequence of any one of SEQ ID NOS: 4-6 may be a less than full length protein having the sequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity any one of SEQ ID NO: 4-6 along 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to 700, 10 to 800, 10 to 900, or 10 to all amino acids of a protein. This list of sequence lengths encompasses every full length protein in SEQ ID NOS: 4-6 and every smaller length within the list, even for proteins that do not include over 400 amino acids. For example, the lengths of 6, 10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, and 10 to all amino acids would apply to a sequence with 322 amino acids. A range of amino acid sequence lengths recited herein includes every length of amino acid sequence within the range, endpoints inclusive. The recited length of amino acids may start at any single position within a reference sequence where enough amino acids follow the single position to accommodate the recited length. The range of sequence lengths can be extended by increments of 10 to 100N amino acids, where N=an integer of ten or greater, for sequences of 1000 amino acids or larger. The fragment of the glucanase may be a subsequence of the polypeptides herein that retain at least 40% activity of the glucanase. The fragment may have 316, 317, or 322 amino acids. The fragments may include 20, 30, 40, 50, 100, 150, 200, or 300 contiguous amino acids. Embodiments also include nucleic acids encoding said amino acid sequences, and antibodies recognizing epitopes on said amino acid sequences.

[0060] A less than full length amino acid sequence may be selected from any portion of one of the sequences of SEQ ID NOS: 4-6 corresponding to the recited length of amino acids. A less than full length amino acid sequence may be selected from a portion of any one of SEQ ID NOS: 4-6 having a catalytic domain. The fragment may include a catalytic domain of a glucanase. For example, the catalytic domain of the glucanase of SEQ ID NO: 4 [AGR2314] includes the following sequence:

TABLE-US-00001 (SEQ ID NO: 21) 10 20 30 40 -GVDPFERNKILGRGINIG ALEAPNEGDWGVVIKDEFFD 50 60 70 80 IIKEAGFSHVRIPIRWSTHAAFPPYKIEPSFFKRVDEVIN 90 100 110 120 GALKRGLAVVINI YEELMNDPEEHKERFLALWKQIADR 130 140 150 160 YKDYPETLFFEIL PHGNLTPEKWNELLEEALKVIRSID 170 180 190 200 KKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHY NPF 210 220 230 240 EFT QGAEWVPGSEKWLGRKWGSPDDQKHLIEEFNFIEEW 250 260 270 280 SKKNKRPIYIG FGAYRKADLESRIKWTSFVVREAEKRGW 290 300 310 SWAY EFCSGFGVYDPLRK WNKDLLEALIGGDSIE

[0061] In the sequence of SEQ ID NO: 21, catalytic residues in active site are shown by enlarged characters in bold. Other active site residues that interact with the substrate are italicized, bold and underlined.

[0062] For example, positions 136 and 253 in SEQ ID NO: 21 are catalytic residues in the active site, and a less than full length amino acid sequence selected from SEQ ID NO: 21 may include residues 134 and 135 at any two respective, consecutive positions within the recited length. A less than full length amino acid sequence may be selected from a portion of any one of SEQ ID NO: 21 may have other active site residues that interact with the substrate. For example, positions 20, 35, 36, 135, 198, 205, 210 and 286 of SEQ ID NO: 21 are the active site residues that interact with the substrate, and a less than full length amino acid sequence selected from SEQ ID NO: 21 may include residues 20, 35, 36, 135, 198, 205, 210 and 286 at any respective, consecutive positions within the recited length.

[0063] A less than full length amino acid sequence may be selected from a portion of any one of SEQ ID NO: 21 may include amino acids 136-253. A less than full length amino acid sequence may possess the glucanase activity. A less than full length amino acid sequence may be capable of degrading polysaccharides. A less than full length amino acid sequence may contain those amino acids would contain the active site residues.

[0064] A catalytic domain may be a conserved domain. A "conserved domain" herein refers to a region in a heterologous polynucleotide or polypeptide sequences where there is a relatively high degree of sequence identity between the distinct sequences. With respect to polynucleotides encoding a conserved domain is preferably at least 10 base pairs (bp) in length.

[0065] A conserved domain of any one of polypeptides described herein refers to a domain within a glucanase that exhibits a higher degree of sequence identity High degree of sequence identity may be at least 50% identity, at least 55% identity, at least 60% identity, at least 65%, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or at least 100% identity to consecutive amino acid residues of a polypeptide described herein. Conserved domains may be identified as domains of identity to a specific consensus sequence. Conserved domains may be identified by using alignment methods. Conserved domain may be identified with multiple sequence alignments of related proteins. These alignments reveal sequence regions containing the same, or similar, patterns of amino acids. Multiple sequence alignments, three-dimensional structure and three-dimensional structure superposition of conserved domains can be used to infer sequence, structure, and functional relationships. Since the presence of a particular conserved domain within a polypeptide is highly correlated with an evolutionarily conserved function, a conserved domain database may be used to identify the amino acids in a protein sequence that are putatively involved in functions such as degrading polysaccharides, as mapped from conserved domain annotations to the query sequence. For example, the presence in a protein of a sequence of SEQ ID NO: 21 that is structurally and phylogenetically similar to one or more domains in the polypeptides of the accompanying Sequence Listing is a strong indicator of a related function in plants. Sequences herein referred to as functionally-related and/or closely-related to the sequences or domains of polypeptides having sequences of SEQ ID NOS: 4-6 may have conserved domains that share at least at least ten amino acids in length and at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about 100% amino acid identity to the sequences of AGR2314, AGR2414 and AGR2514, and have similar functions that the polypeptides of the instant description.

[0066] In an example, sequences of AGR2314, AGR2414, and AGR2514 may be aligned as shown below.

[0067] AGR2414 sequence is a sequence of Thermotoga maritima Cel5A (3AMC in Protein Data Bank and in PubMed protein sequence database). Residues that interact with the substrate are underlined and are shown in enlarged bold characters: N20, H95, H96, N135, E136 (catalytic residue), Y198, H205, W210, E253 (catalytic residue), W286. (T. Wu et al. (2011), Biochim. Biophys. Acta 1814, 1.832-1840, which is incorporated herein by reference as if fully set forth). The numbering of these residues in AGR2514 is one higher because of the presence of one additional residue at the N-terminus in this sequence.

[0068] In an example, the sequences of AGR2314 and AGR2414 have 305 residues conserved out of 317 residues and have 96% identity.

TABLE-US-00002 CLUSTAL O(1.2.1) multiple sequence alignment AGR2414 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHA AGR2314 MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHA ************************************************************ AGR2414 YAFPPYKIMDRFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIAD AGR2314 QAFPPYKIEPSFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIAD ******* ************************************************* AGR2414 RYKDYPETLFFEILNEPHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISALE AGR2314 RYKDYPETLFFEILNEPHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISALE ********************************************:*************** AGR2414 KLSVPKWEKNSIVTIHYYNPFEFTHQGAEWVEGSEKWLGRKWGSPDDQKHLIEEFNFIEE AGR2314 KLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVPGSEKWLGRKWGSPDDQKHLIEEFNFIEE ** *******:******************* ***************************** AGR2414 WSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREMEKRRWSWAYWEFCSGFGVYDTLRK AGR2314 WSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYWEFCSGFGVYDPLRK *********************************** *** *************** *** AGR2414 TWNKDLLEALIGGDSIE (SEQ ID NO: 5) AGR2314 QWNKDLLEALIGGDSIE (SEQ ID NO: 4) ****************

[0069] In an example, the sequences of AGR2414 and AGR2514 below have 310 residues conserved out of 318 residues and have 97% identity.

TABLE-US-00003 CLUSTAL O(1.2.1) multiple sequence alignment AGR2414 -MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTH AGR2514 MSGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTH ************************************:********************* AGR2414 AYAFPPYKIMDRFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIA AGR2514 AQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQHHYEELMNDPEEHKERFLALWKQIA * ****** ************************ ************************* AGR2414 DRYKDYPETLFFEILNEPHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL AGR2514 DRYKDYPETLFFEILNEPHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL ************************************************************ AGR2414 EKLSVPKWEKNSIVTIHYYNPFEFTHQGAEWVEGSEKWLGRKWGSPDDQKHLIEEFNFIE AGR2514 EKLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVEGSEKWLGRKWGSPDDQKHLIEEFNFIE *** *******:*********************************************** AGR2414 EWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREMEKRRWSWAYWEFCSGFGVYDTLR AGR2514 EWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRRWSWAYWEFCSGFGVYDTLR ************************************ *********************** AGR2414 KTWNKDLLEALIGGDSIW (SEQ ID NO: 5) AGR2514 KTWNKDLLEALIGGDSIE (SEQ ID NO: 6) ******************

[0070] In an example, the sequences of AGR2314 and AGR2514 have 308 residues conserved out of 318 residues and have 97% identity.

TABLE-US-00004 CLUSTAL O(1.2.1) multiple sequence alignment AGR2314 -MGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTH AGR2514 MSGVDPFERNKILGRGINIGNALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTH ************************************:********************* AGR2314 AQAFPPYKEIPSFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIA AGR2514 AQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQHHYEELMNDPEEHKERFLALWKQIA ********** ********************** ************************ AGR2314 DRYKDYPETLFFEILNEPHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISAL AGR2514 DRYKDYPETLFFEILNEPHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL *********************************************:************* AGR2314 EKLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVPGSEKWLGRKWGSPDDQKHLIEEFNFIE AGR2514 EKLRVPKWEKNAIVTIHYYNPFEFTHQGAEWVEGSEKWLGRKWGSPDDQKHLIEEFNFIE ******************************* *************************** AGR2314 EWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRGWSWAYWEFCSGFGVYDPLR AGR2514 EWSKKNKRPIYIGEFGAYRKADLESRIKWTSFVVREAEKRRWSWAYWEFCSGFGVYDTLR **************************************** *************** ** AGR2314 KQWNKDLLEALIGGDSIE (SEQ ID NO: 4) AGR2514 KTWNKDLLEALIGGDSIE (SEQ ID NO: 6) * ****************

[0071] Sequences that possess or encode for conserved domains that meet these criteria of percentage identity, and that have comparable biological and regulatory activity to the present polypeptide sequences, thus being glucanases, described herein. Sequences having lesser degrees of identity, but comparable biological activity, are considered to be equivalents.

[0072] The functionality of a glucanase, variants, or fragments thereof, may be determined using any methods. The functionality of a glucanase may be measured by any one of the assays described in Example 3.

[0073] Any one or more glucanases herein may be expressed in a plant upon introduction into the plant genome of any one more of synthetic nucleic acids described herein. The methods of introduction of synthetic nucleic acids into the plants are known in the art. The method may be transformation of the plant with a vector that includes synthetic nucleic acids.

[0074] In an embodiment, a synthetic polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of SEQ ID NO: 7 [pAG4258], SEQ ID NO: 8 [pAG4588], SEQ ID NO: 9 [pAG4597], SEQ ID NO: 10 [pAG4708], SEQ ID NO: 11 [pAG4766], SEQ ID NO: 12 [pAG4767], SEQ ID NO: 13 [pAG4770], SEQ ID NO: 14 [pAG4771], SEQ ID NO: 15 [pAG4257], SEQ ID NO: 16 [pAG4692], SEQ ID NO: 17 [pAG4693], SEQ ID NO: 18 [pAG4705] and SEQ ID NO: 19 [pAG4706] is provided. The synthetic polynucleotide may include any one of the synthetic nucleic acids described herein that encode glucanase and that are capable of degrading a polysaccharide.

[0075] In an embodiment, a vector is provided. The vector may include any one of the synthetic polynucleotides or nucleic acids described herein.

[0076] In an embodiment, synthetic nucleic acids are provided having a sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. In an embodiment, isolated nucleic acids having a sequence that hybridizes to a nucleic acid having the sequence of any nucleic acid listed herein or the complement thereof are provided. In an embodiment, the hybridization conditions are low stringency conditions. In an embodiment, the hybridization conditions are moderate stringency conditions. In an embodiment, the hybridization conditions are high stringency conditions. The hybridization may be along the length of the synthetic nucleic acid. Examples of hybridization protocols and methods for optimization of hybridization protocols are described in the following publications: Molecular Cloning, T. Maniatis, E. F. Fritsch, and J. Sambrook, Cold Spring Harbor Laboratory, 1982; and, Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl, Volume 1, John Wiley & Sons, 2000 (standard protocol) and Amersham Gene Images AlkPhos Direct Labeling and Detection System (GE Healthcare UK, Ltd), which are incorporated by reference in their entirety as if fully set forth.

[0077] In an AlkPhos Direct Labeling and Detection System, moderate conditions may be as follows: membranes loaded with DNA samples are prehybridized for at least 15 minutes at 55.degree. C. in the hybridization buffer (12% (w/v) urea, 0.5M NaCl, 4% (w/v) blocking reagent). The labeled probe is added to the same solution and hybridization is carried overnight at 55.degree. C. The membranes are washed for 10 minutes at 55.degree. C. in the primary wash solution (2M urea, 0.1% (W/v) SDS, 50 mM of 0.5M Na phosphate pH 7.0, 150 mM NaCl, 1 mM of 1.0 M Mg Cl.sub.2 and 0.2% (w/v) of blocking reagent). The washing procedure is repeated. The membranes are placed in a clean container and washed for 5 minutes in a secondary buffer (1M Tris base, and 2M NaCl). The washing in the secondary solution is performed two more time. Chemoluminescence was detected using CDP-STAR.RTM. substrate for alkaline phosphatase. Low stringency refers to hybridization conditions at low temperatures, for example, between 37.degree. C. and 60.degree. C. High stringency refers to hybridization conditions at high temperatures, for example, over 68.degree. C.

[0078] In the standard protocol, moderate conditions may be as follows: filters loaded with DNA samples are pretreated for 2-4 hours at 68.degree. C. in a solution containing 6.times.citrate buffered saline (SSC; Amresco, Inc., Solon, Ohio), 0.5% sodium dodecyl sulfate (SDS; Amresco, Inc., Solon, Ohio), 5.times.Denhardt's solution (Amresco, Inc., Solon, Ohio), and denatured salmon sperm (Invitrogen Life Technologies, Inc. Carlsbad, Calif.). Hybridization is carried in the same solution with the following modifications: 0.01 M EDTA (Amresco, Inc., Solon, Ohio), 100 .mu.g/ml salmon sperm DNA, and 5-20.times.10.sup.6 cpm .sup.32P-labeled or fluorescently labeled probes. Filters are incubated in hybridization mixture for 16-20 hours and then washed for 15 minutes in a solution containing 2.times.SSC and 0.1% SDS. The wash solution is replaced for a second wash with a solution containing 0.1.times.SSC and 0.5% SDS and incubated an additional 2 hours at 20.degree. C. to 29.degree. C. below Tm (melting temperature in .degree. C.). Tm=81.5+16.61 Log.sub.10[Na.sup.+]/(1.0+0.7[Na.sup.+]))+0.41(%[G+C])-(500/n)-P-F. [Na+]=Molar concentration of sodium ions. %[G+C]=percent of G+C bases in DNA sequence. N=length of DNA sequence in bases. P=a temperature correction for % mismatched base pairs (1.degree. C. per 1% mismatch). F=correction for formamide concentration (=0.63.degree. C. per 1% formamide). Filters are exposed for development in an imager or by autoradiography. Low stringency conditions refers to hybridization conditions at low temperatures, for example, between 37.degree. C. and 60.degree. C., and the second wash with higher [Na.sup.+] (up to 0.825M) and at a temperature 40.degree. C. to 48.degree. C. below Tm. High stringency refers to hybridization conditions at high temperatures, for example, over 68.degree. C., and the second wash with [Na+]=0.0165 to 0.0330M at a temperature 5.degree. C. to 10.degree. C. below Tm. In an embodiment, synthetic nucleic acids having a sequence that has at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity along its length to a contiguous portion of a nucleic acid having any one of the sequences set forth herein or the complements thereof are provided. The contiguous portion may be the entire length of a sequence set forth herein or the complement thereof.

[0079] In an embodiment a synthetic nucleic acid may encode the fragment of a glucanase that have 316, 317, or 322 amino acids. The synthetic nucleic acids may encode the fragments that include 20, 30, 40, 50, 100, 150, 200, or 300 contiguous amino acids and retain at least 40% activity of the glucanase. The functionality of a glucanase, variants, or fragments thereof, may be determined using any methods. The functionality of a glucanase may be measured by any one of the assays described in Example 3.

[0080] Determining percent identity of two amino acid sequences or two nucleic acid sequences may include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. If all positions in two sequences are occupied by identical amino acid residues or nucleotides then the sequences are said to be 100% identical. Percent identity can be measured by the Smith Waterman algorithm (Smith T F, Waterman M S 1981 "Identification of Common Molecular Subsequences," Journal of Molecular Biology 147: 195-197, which is incorporated by reference in its entirety as if fully set forth).

[0081] In an embodiment, synthetic nucleic acids, polynucleotides, or oligonucleotides are provided having a portion of the sequence as set forth in any one of the nucleic acids listed herein or the complement thereof. These isolated nucleic acids, polynucleotides, or oligonucleotides are not limited to but may have a length in the range from 10 to full length, 10 to 800, 10 to 10 to 600, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, or 20 to 30 nucleotides or 10, 15, 20 or 25 nucleotides. A synthetic nucleic acid, polynucleotide, or oligonucleotide having a length within one of the above ranges may have any specific length within the range recited, endpoints inclusive. In an embodiment, a hybridization probe or primer is 85 to 100%, 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or 100% complementary to a nucleic acid with the same length as the probe or primer and having a sequence chosen from a length of nucleotides corresponding to the probe or primer length within a portion of a sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, a hybridization probe or primer hybridizes along its length to a corresponding length of a nucleic acid having the sequence as set forth in any one of the nucleic acids listed herein. In an embodiment, the hybridization conditions are low stringency. In an embodiment, the hybridization conditions are moderate stringency. In an embodiment, the hybridization conditions are high stringency.

[0082] In an embodiment, a transgenic plant comprising a synthetic nucleic acid encoding any one or more of the glucanases described herein is provided. The one or more glucanases expressed in the transgenic plant herein may have activity at a pH ranging from 2.0 to 10.00. The pH may be 2.0, 3.0, 4.0, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 9.0, 9.5, or 10, or a pH within a range between any two of the foregoing pH values (endpoints inclusive). The one or more glucanases expressed in a transgenic plant herein may have activity when exposed to a temperature in the range of 25.degree. C. to 130.degree. C., endpoints inclusive. The temperature may be 25.degree. C., 30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C., 55.degree. C., 65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C., 85.degree. C., 90.degree. C., 95.degree. C., 100.degree. C., 105.degree. C., 110.degree. C., 115.degree. C., 120.degree. C., 125.degree. C., 130.degree. C., 25.degree. C., to 30.degree. C., 25.degree. C. to 35.degree. C., 25.degree. C. to 40.degree. C., 25.degree. C. to 45.degree. C., 25.degree. C. to 50.degree. C., 25.degree. C. to 55.degree. C., 25.degree. C. to 60.degree. C., 25.degree. C. to 65.degree. C., 25.degree. C. to 70.degree. C., 25.degree. C. to 75.degree. C., 25.degree. C. to 80.degree. C., 25.degree. C. to 85.degree. C., 25.degree. C. to 90.degree. C., 25.degree. C. to 95.degree. C., 25.degree. C. to 100.degree. C., 25.degree. C. to 105.degree. C., 25.degree. C. to 110.degree. C., 25.degree. C. to 115.degree. C., 25.degree. C. to 120.degree. C., 25.degree. C. to 125.degree. C., or less than 130.degree. C. The glucanase expressed in the transgenic plant may have the improved activity compared to the glucanase having an identical amino acid sequence but expressed in a bacterial cell. The glucanase may have improved thermal stability compared to the activity of the glucanase expressed in the bacterial cell.

[0083] The one or more glucanase may be produced in any transgenic plant. The transgenic plant may be but is not limited to wheat, maize, soybean, barley, and sorghum.

[0084] In an embodiment, a method of making a transgenic plant that includes a glucanase is provided. The method may include contacting a plant cell with any one of the synthetic nucleic acids herein. The synthetic nucleic acids may be part of any one of the vectors described herein. The vector may include a synthetic nucleic acid encoding a glucanase. The glucanase may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514]. The method may also include regenerating a transgenic plant from the transgenic plant cell. The method may include selecting the transgenic plant expressing a glucanase.

[0085] The transgenic plant herein is also referred to as an "event." An event is characterized by presence of the transgene comprising a synthetic nucleic acid encoding a glucanase. The term "event" also refers to the genomic region of the transformed parent comprising the inserted synthetic nucleic acid sequence and the parent genomic sequences flanking the insertion. The term "event" also refers to progeny produced by crossing of the transgenic plant and a non-transgenic plant of the same genetic background. The term "line" also refers to progeny produced by crossing of the transgenic plant and a non-transgenic plant with any genetic background. After repeated crosses, the transgene and the flanking sequences of the originally transformed parent may be present in a progeny plant in the same location in the genome or on the same chromosome as in the transformed parent.

[0086] The transgenic plant may be homozygous for the transgene comprising a synthetic nucleic acid encoding a glucanase.

[0087] The transgenic plant may be hemizygous for the transgene comprising a synthetic nucleic acid encoding a glucanase. To produce homozygous plants expressing a glucanase, a hemizygous transgenic plant may be self-crossed. Progeny may be obtained from such crosses. The progeny may include homozygous, hemizygous and wild type plants. A hemizygous plant may be phenotypically indistinguishable from the wild type plants. The method may include analyzing the progeny for the presence of the transgene and selecting a progeny plant that includes the transgene. A method of identifying the homozygous event by PCR is described herein in Example 8.

[0088] In an embodiment, the method may further include crossing a hemizygous transgenic plant to another transgenic plant hemizygous for the same transgene. The method may include selecting a first progeny plant that is homozygous for the transgene. The method may further include crossing the transgenic plant to a wild type plant of the same, or different, genetic background. Progeny may be obtained from such crosses. The progeny may include hemizygous and wild type plants. The method may include selecting a first progeny plant that is hemizygous for the transgene. The method may further include selfing the first hemizygous progeny plant and selecting a second progeny plant that is homozygous for the transgene comprising a synthetic nucleic acid sequence encoding a glucanase.

[0089] The glucanase may have activity and improved thermal stability when exposed to high temperatures as here described.

[0090] It has been unexpectedly discovered that expression and accumulation of an enzyme in a plant provides the enzyme with additional thermal stability relative to the same enzyme that is produced microbially.

[0091] In an embodiment, the method of making a transgenic plant includes transformation. For transformation, the nucleic acid may be introduced into a vector. Suitable vectors may be cloning vectors, transformation vectors, expression vectors, or virus-based vectors. The expression cassette portion of a vector may further include a regulatory element operably linked to a nucleic acid encoding a glucanase. In this context, operably linked means that the regulatory element imparts its function on the nucleic acid. For example, a regulatory element may be a promoter, and the operably linked promoter would control expression of the nucleic acid.

[0092] The expression of a nucleic acid encoding a glucanase from the expression cassette may be under the control of a promoter which provides for transcription of the nucleic acid in a plant. The promoter may be a constitutive promoter or, tissue specific, or an inducible promoter. A constitutive promoter may provide transcription of the nucleic acid throughout most cells and tissues of the plant and during many stages of development but not necessarily all stages. An inducible promoter may initiate transcription of the nucleic acid sequence only when exposed to a particular chemical or environmental stimulus. A tissue specific promoter may be capable of initiating transcription in a particular plant tissue. Plant tissue may be, but is not limited to, a stem, leaves, trichomes, anthers, cob, seed, endosperm, or embryo. The constitutive promoter may be, but is not limited to the maize ubiquitin promoter (ZmUbil), Cauliflower Mosaic Virus (CAMV) 35S promoter, the Cestrum Yellow Leaf Curling Virus promoter (CMP), the actin promoter, or the Rubisco small subunit promoter. The tissue specific promoter may be the maize globulin promoter (ZmGlb1), the rice glutelin promoter (prGTL), the maize zein promoter (ZmZ27), or the maize oleosin promoter (ZmOle). The promoter may provide transcription of a synthetic polynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of SEQ ID NO: 7 [pAG4258], SEQ ID NO: 8 [pAG4588], SEQ ID NO: 9 [pAG4597], SEQ ID NO: 10 [pAG4708], SEQ ID NO: 11 [pAG4766], SEQ ID NO: 12 [pAG4767], SEQ ID NO: 13 [pAG4770], SEQ ID NO: 14 [pAG4771], SEQ ID NO: 15 [pAG4257], SEQ ID NO: 16 [pAG4692], SEQ ID NO: 17 [pAG4693], SEQ ID NO: 18 [pAG4705] and SEQ ID NO: 19 [pAG4706] and expression of glucanase that is capable of degrading a polysaccharide.

[0093] In an embodiment, the transformation in the method of making a transgenic plant may be stable transformation, wherein the nucleic acid encoding the glucanase integrates into the genome of the transformed plant. The transformation may be Agrobacterium-mediated transformation using a vector suitable for stable transformation described herein. The method of making a transgenic plant may include any other methods for transforming plants, for example, particle bombardment, or protoplast transformation via direct DNA uptake. The transgenic plant may include any synthetic nucleic acid, amino acid sequence, or vector herein.

[0094] In an embodiment, the method of making a transgenic plant may include transient transformation to transiently express the recombinant protein. The term "transient expression" refers to the expression of an exogenous nucleic acid molecule delivered into a cell: e.g., a plant cell, and not integrated in the plant's cell chromosome. Expression from extra-chromosomal exogenous nucleic acid molecules can be detected after a period of time following a DNA-delivery. Virus-based vectors may be used to carry and express exogenous nucleic acid molecules. Virus-based vectors may replicate and spread systemically within the plant. Use of virus based vectors may lead to very high levels of glucanase accumulation in transgenic plants.

[0095] Methods of making a transgenic plant, methods of increasing utilization of non-starch polysaccharides in an animal, methods for enhancing production of fermentable sugars from grains, methods for increasing metabolizable energy of a diet, methods preparing and animal feedstock and methods for producing genetically engineered plants homozygous for a synthetic nucleic acid that encodes a glucanase may comprise a method of detection herein as part of making transgenic plants and/or identifying plants, plant biomass or animal feed that comprise a synthetic nucleic acid herein.

[0096] An embodiment comprises a kit for identifying maize event 4588.259, 4588.757 or 4588.652 in a sample. The kit may comprise a first primer and a second primer.

[0097] The first primer and the second primer may be capable of amplifying a target sequence specific to an event. The target sequence may include a nucleic acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NOS: 51-55. The target sequence may be a sequence included in a junction between a genomic sequence of a transformed plant and a sequence of the T-DNA insertion. The target sequence may be included in a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from SEQ ID NOS: 22-31.

[0098] The kit may comprise the first primer comprising a nucleic acid sequence selected from SEQ ID NOS: a nucleic acid sequence selected from SEQ ID NOS: 38, 41, and 47. The kit may comprise the second primer comprising a nucleic acid sequence selected from SEQ ID NOS: 39, 42, 43, 45, and 46. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 38 and the second primer comprising the nucleic sequence of SEQ ID NO: 39. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 41 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 42. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 41 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 43. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 47 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 45. The kit may comprise the first primer comprising the nucleic acid sequence of SEQ ID NO: 47 and the second primer comprising the nucleic acid sequence of SEQ ID NO: 46. The first primer and the second primer may be capable of amplifying the target sequence to produce an amplified product. The amplified product or the target sequence may be capable of hybridizing to the sequence of the nucleic acid comprising a sequence of SEQ ID NO: 40, or SEQ ID NO: 44 under conditions of high stringency. The target sequence may be used as a probe for diagnosing any one of the events described herein.

[0099] A sample may include any sample in which nucleic acids from plant matter are present. A sample may be a protein sample. A protein sample may include any sample in which proteins from plant matter are present. The sample or protein sample may include any plant matter. The plant matter may derive from a plant or part thereof. The plant material may derive from an animal feed or food.

[0100] In an embodiment, a method of identifying maize event 4588.259, 4588.757 or 4588.652 in a sample is provided. The method may include contacting a sample with a first primer and a second primer. The method may include amplifying a synthetic polynucleotide comprising a target sequence specific to the maize event. The target sequence may be any target sequence described herein. The first primer and the second primer may be capable of amplifying the target sequence to produce an amplified product. The amplified product may be used to determine whether a plant resulted from a sexual crossing or selfing contains one or more of the target sequences and diagnose specific events. The length of the amplified product from the sample of the maize event may differ from the length of the amplified product from the sample of wild type plant of the same genetic background. The amplified product from the event sample may be further used as probe that hybridizes to a synthetic polynucleotide comprising a specific region encoding a glucanase under conditions of high stringency. The method may include further detecting hybridization of the at least one probe to the specific region of the target sequence.

[0101] In an embodiment, an animal feedstock comprising any one or more of the transgenic plants described herein or parts of the transgenic plants is provided. The term "animal feedstock" refers to any food, feed, feed composition, diet, preparation, additive, supplement, or mixture suitable and intended for intake by animals for their nourishment, maintenance, or growth. The glucanases included in the transgenic plants and in the animal feedstock may be active in the gastrointestinal or rumen environment of animals. The animal may be a monogastric animal. The animal may be a ruminant animal. The monogastric animal may be but is not limited to a chicken, a turkey, a duck, a swine, a fish, a cat, or a dog. The ruminant animal may be but is not limited to cattle, a cow, a steer, a sheep, or a goat. The glucanases may be active after preparation of the animal feed. The temperatures which feeds are exposed to during ensiling may be within range of 20.degree. C. to 70.degree. C. The temperatures which feeds are exposed to during pelleting may be within range of 70.degree. C. to 130.degree. C. The glucanases may have improved thermal stability and may retain activity after being exposed to high temperatures during feed pelleting. The glucanase with improved thermal stability may comprise, consist essentially of, or consist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a reference sequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514].

[0102] In an embodiment, a glucanase may be isolated from the transgenic plant prior to being included into the animal feedstock. The glucanase may be any one of the glucanases described herein.

[0103] In an embodiment, the animal feedstock may further include a feed supplement. The feed supplement may be any plant material. The plant material may be a non-transgenic plant or a part thereof. The plant material may include an engineered plant or a mutant plant. The feed supplement may be a mineral. The mineral may be a trace mineral. The mineral may be a macro mineral. The feed supplement may be at least one vitamin. The at least one vitamin may be a fat-soluble vitamin. The feed supplement may include one or more exogenous enzymes. The one or more exogenous enzymes may include a hydrolytic enzyme. The hydrolytic enzyme. The hydrolytic enzyme may be an enzyme classified under EC3.4 as hydrolase. The hydrolytic enzymes may be but are not limited to xylanases, mannanases, carbohydrases, proteases, peptidases, phytases, cellulases, lipases, phospholipases, pectinases, galactosidases, laccases, amylases, hemicellulases, or cellobiohydrolases. The hydrolytic enzymes may be expressed in the engineered plants or parts thereof included in the feed supplement. The feed supplement may include purified hydrolytic enzymes. The feed supplements may be but are not limited to growth improving additives, coloring agents, flavorings, stabilizers, limestone, stearine, starch, saccharides, fatty acids, or a gum. The coloring agents may be carotenoids. The carotenoids may be but are not limited to cantaxanthin, beta-carotene, astaxanthin, or lutein. The fatty acids may be polyunsaturated fatty acids. The polyunsaturated fatty acids may include but are not limited to arachidonic acid, docosohexaenoic acid (DHA), eicosapentaenoic acid (EPA) or gamma-linoleic acid. The feed supplement may be a non-transgenic plant or a part thereof. The non-transgenic plant or part thereof may include at least one component selected from the group consisting of: barley, wheat, rye, oat, corn, rice, triticale beet, sugar beet, spinach, cabbage, quinoa, corn meal, corn pellets, corn oil, distillers grains, forage, wheat meal, wheat pellets, wheat grain, barley grain, barley pellets, soybean meal, soybean oilcake, lupin meal, rapeseed meal, sorghum grain, sorghum pellets, rapeseed, sunflower seed, and cotton seed.

[0104] The feed supplement may include at least one component selected from the group consisting of: soluble solids, fat, vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, monensin sodium COBAN.RTM. PREMIX, vitamin premix, inorganic feed phosphates, monocalcium phosphate, dicalcium phosphate, tricalcium phosphate, monodicalcium phosphate, rock phosphate, selenium premix, choline chloride, sodium chloride, and mineral premix.

[0105] The feed supplement may include fish meal, fish oil, bone meal, feather meal and animal fat. The feed supplement may include yeast or yeast extract.

[0106] In an embodiment, a method of producing an animal feedstock is provided. The method may comprise including a transgenic plant that includes any one or more glucanase described herein in the animal feedstock. The animal feedstock may comprise, consist essentially of or consist of the transgenic plant. The method may further include combining the transgenic plant with a feed supplement. The feed supplement may be a non-transgenic plant or a part thereof. The transgenic plant may be produced by any one of the methods described herein. The feed supplement may be a mineral. The supplement may include one or more exogenous enzymes. The exogenous enzymes may be but are not limited to xylanases, mannanases, carbohydrases, proteases, peptidases, phytases, cellulases, lipases, phospholipases, pectinases, galactosidases, laccases, amylases, hemicellulases, or cellobiohydrolases.

[0107] In an embodiment, a method of meat production is provided. The method may include feeding an animal feedstock or one produced by any of the methods described herein to the animal. The method may include preparing an animal feedstock that includes a transgenic plant expressing a glucanase.

[0108] In an embodiment, a method of feeding an animal is provided. The method may include feeding an animal feedstock or one produced by any of the methods described herein to the animal. The method may include preparing an animal feedstock that includes a transgenic plant expressing a glucanase.

[0109] In an embodiment, a method of increasing utilization of non-starch polysaccharides in an animal is provided. The method may include feeding an animal with an animal feedstock that includes any one or more of the transgenic plants described herein. The method may include preparing the animal feedstock.

[0110] In an embodiment, a method of decreasing gastrointestinal viscosity in an animal is provided. The method may include feeding an animal with an animal feedstock that includes any one or more of the transgenic plants described herein. The method may include preparing the animal feedstock.

[0111] Addition of exogenous enzymes collectively known as carbohydrases may ameliorate the effects of non-starch polysaccharides (NSPs) in the diet of an animal. An animal feedstock that includes any one or more of glucanases described herein may increase utilization of NSPs by the animal that ingested the feedstock, or may decrease the anti-nutritional effects of the NSP on the animal that ingested the feedstock, and improve growth of the animal. Preparing the animal feedstock may include combining one or more transgenic plant herein with any other feed supplement. The glucanase may be isolated, purified and added to the animal feedstock as a pure glucanase. The glucanase may be added to the animal feedstock in admixture with other feed supplements. The transgenic plant including the glucanase or the purified glucanase may be combined with other feed supplements to form premixes.

[0112] An animal feedstock may be produced as mash feed. The animal feedstock may be produced as pellets. The milled feed stuffs may be mixed with the premix that includes any one of the transgenic plants that include a glucanase. The milled feed stuffs may include the plant material and the feed supplements described herein. The feed supplements may include one or more exogenous enzymes described herein. Enzymes may be added as liquid or solid formulations. For mash feed, a solid or liquid enzyme formulation may be added before or during the mixing step. For pelleted feed, the enzyme preparation may be added before or after the pelleting step. The glucanase may be included in a premix. The premix may also include vitamins and trace minerals. Macro minerals may be added separately to animal feedstock.

[0113] In an embodiment, a method of increasing metabolizable energy of a diet is provided. Metabolizable energy (ME) refers to the net energy of a diet or feed that is available to an animal after the utilization of some energy in the processes of digestion and absorption and the loss of some of the material as being undigested or indigestible. Metabolizable energy may be apparent metabolizable energy (AME) measured as the difference between the calories of the feed consumed by an animal and excrements collected after feed consumption. Metabolizable energy may be true metabolizable energy (TME), which is similar to AME except that it also takes into account endogenous energy. Energy contents in a diet or feed ingredients may be determined using one of several methodologies (NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, D.C., which is incorporated herein by reference as if fully set forth). Gross energy (GE) is direct measurement using an adiabatic bomb calorimeter, which measures the heat of combustion of a sample within a high oxygen atmosphere. Apparent digestible energy (DE) is GE of a feed or feedstuff minus GE of feces only. Apparent metabolizable energy (AME) is GE of a feed or feedstuff minus GE of feces, urine, and gaseous products from digestion. For poultry, the gaseous release is very low, and typically neglected due to its very small value, and the urine and feces are excreted together and are not collected separately in most cases. True metabolizable energy (TME) accounts for only the GE from excreta that is from the feed or feedstuff origin, by subtracting the endogenous energy loss from non-feed origin (i.e. sloughing of intestinal tract cells). Another energy measurement used for feedstuffs in animals is net energy (NE) which adjusts for heat increment. Since heat increment is dependent on level of productivity, which fluctuates in poultry because of short lifespan, this variable is not frequently used in poultry.

[0114] The TME rooster assay may be used to account for endogenous (non-feed) losses of GE by including a fasted rooster and collecting excreta to correct the GE from the fed (feed/feedstuff) rooster. See Sibbald, 1976, Poultry Science 55: 303-308, which is incorporated herein by reference as if fully set forth. This assay has commonly been used for determining TME of individual feedstuffs rather than complete feed, and requires cecetomized roosters (ceca surgically removed) to always be on hand. The assay involves force-feeding (into the crop) a known quantity of an ingredient (in birds that were previously fasted 24-48 hr) and then collect feces for a 24-48 hour period. The equation used to calculate TME is given as TME={(GE.sub.f.times.FI)-[(GE.sub.e.times.EO).sup.+-(GE.sub.e.times.EO).s- up.-]}/FI, where Gross Energy (GE) is determined by bomb calorimetry in kcal/kg; FI is feed intake (kg); EO is excreta output fed birds (kg); GE.sub.e is the Gross Energy of the excreta content; GE.sub.f is the Gross Energy of the feed; "k" signifies the quantity is from the fed birds energy output; and "-" signifies that the quantity is from the fasted birds energy output. The roosters (or turkeys) used in TME assays are adult birds with a fully developed digestive tract. Research has shown that there are differences in ME determinations using roosters (layer breeds), turkeys and broilers when analyzing same feed ingredients (Cozannet et al, 2010 J. Anim, Sci., 88(7):2382-2392, which is incorporated herein by reference as if fully set forth). So determining TME or AME using rooster model may not be equivalent to what is observed in a young broiler, but is a commonly used proxy in research and industry.

[0115] For broilers, the AME assay may be used for determining complete feed and some energy supplying feedstuffs, as well as the effect from adding feed ingredients that aid in digestion. There are two common methods for determining ME: 1) doing a total excreta collection and weighing and record feed consumption during the time period (Equation 1 below) or 2) using an indigestible marker in feed (chromic oxide, titanium oxide or acid insoluble ash) and taking a subsample of feces with no weighing required (Equation 2 below). The marker method of AME determination may be used, in which no weighing of feed consumption or total fecal collection and no need to separate feed spilled from feces pan are required. With the marker method, birds are fed the marker for at least two days (but preferably five or more days). Feces are collected over several days (e.g., three days) with daily collection composited into one sample.

[0116] AME using the total collection method (Equation 1) is calculated as follows:

AME=[(GE.sub.f.times.FI)-(GE.sub.e.times.EO)]/FI,

where Gross Energy (GE) is measured in bomb calorimetry (kcal/kg); FI is feed intake (kg); EO is excreta output (kg); e refers to excreta content; and f refers to the feed content. AME using the marker method is calculated as AME=[(GE.sub.e/M.sub.e)-(GE.sub.f/M.sub.f)]/(GE.sub.e/M.sub.e), where Gross Energy is GE (kcal/kg); M is the marker (ppm or %); "e"=excreta content; "f"=feed content.

[0117] Another method that may be used to determine AME of feed when investigating feed additives that aid in digestion is Ileal digestible energy (IDE). This method uses the AME marker method (described above), but the birds are euthanized and a section of ileum excised and contents removed, dried and analyzed for GE and the marker. The IDE method may be used effectively for testing and comparing feed additives used to improve digestion/absorption of feed energy. The benefit of IDE, is no cages with collection pans are required and can collect during a floor-pen study. With the marker method, birds are fed the marker for at least two days (and preferably five or more days).

[0118] AME using the IDE marker method (Equation 2) is calculated as follows:

AME=GE.sub.f-(GE.sub.d.times.M.sub.f/M.sub.d),

where GE (kcal/kg); M represents the marker; ".sub.d" represents the digesta content; and ".sub.f" signifies the feed content.

[0119] AME and TME may be corrected for nitrogen retention (AMEn and TMEn). To adjust, the grams of N are multiplied by 8.22 kcal/g (GE of uric acid; primary excretory product of protein tissue oxidized for energy), which also is subtracted off of the GE consumed. See Hill, F. W., and D. L. Anderson, 1958, "Comparison of metabolizable energy and productive energy determinations with growing chicks." J. Nutr. 64:587-603, which is incorporated herein by reference as if fully set forth. Calculations for total collection of marker method for AMEn are shown in Equation 3 and Equation 4 below, respectively. Equation 3: AMEn, total collection:

AMEn={(GE.sub.f.times.FI)-(GE.sub.e.times.EO)-[8.22.times.(N.sub.f-N.sub- .e)]}/FI,

where GE=kcal/kg; FI=feed intake (kg); EO=excreta output (kg); N=nitrogen (g); e=excreta content; f=feed content.

[0120] Equation 4: IDEn, marker method:

AMEn=GE.sub.f-(GE.sub.d.times.M.sub.f/M.sub.d)-{8.22.times.[N.sub.f-(Nd.- times.M.sub.f/M.sub.d)]},

where GE=kcal/kg; M=marker; N=nitrogen (g/kg) "d"=digesta content; "f"=feed content.

[0121] While the TME method may be used for determining ME of individual ingredients, the AME (IDE) method may be used with broilers to measure ME in individual ingredients or total diet and testing effects improving ME by use of enzymes or other feed additives.

[0122] A diet or feed may include any feed ingredient or mixture of ingredients including water. The diet may be any food, feed, feed composition, diet, preparation, additive, supplement, or mixture included in an animal feedstock described herein. The diets are known in the art and described at least in the following publications: Nutrient Requirements of Poultry, 1994, National Research Council, National Academy Press, Washington, D.C.; Broiler Performance and Nutrition Supplement, Cobb-500.TM., L-2114-07EN, July, 2015; Broiler Performance and Nutrition Supplement, Cobb-700.TM., L-21124-13EN, Dec. 21, 2012; Broiler Performance and Nutrition Supplement, CobbAvian.TM. L-2144-04EN, April, 2012; Broiler Performance and Nutrition Supplement, CobbSasso.TM., L-2154-01, May 7, 2008; Ross 308 Broiler: Nutrition Specifications, 2014 Aviagen, 0814-AVNR-035; Ross Nutrition Supplement 2009, Aviagen; Ross 708 Broiler: Nutrition Specification, 2014 Aviagen, 0814-AVNR-036; Ross PM3 Brioler Nutrition Specification, 2014 Aviagen, 0814-AVNR-037; Arbor Acres Plus Broiler Nutrition Specifications, 2014 Aviagen, 1014-AVNAA-043; Arbor Acres Broiler Nutrition Supplement, 2009 Aviagen; and Association of American Feed Control Officials (AAFCO) 2015 Official Publication, Nutrient Requirements for Poultry, all of which are incorporated herein by reference as if fully set forth.

[0123] In an embodiment, the diet may be a diet for broilers. The diet for broilers may be composed of one or more of the following ingredients: 51.49% (w/w)-61.86% (w/w) corn, 25.45% (w/w)-35.03% (w/w) soybean meal, 5.00% (w/w) corn distillers dry grains plus soluble solids, 2.00% (w/w) vermiculite, 0.30% (w/w)-1.99% (w/w) dicalcium phosphate, 1.00% (w/w) poultry fat, 0.81% (w/w)-4.01% (w/w) limestone, 0.24% (w/w)-0.50% (w/w) salt (NaCl), 0.13% (w/w)-0.45% (w/w) DL-methionine, 0.20% (w/w) choline chloride 60, 0.20% (w/w) mineral premix, 0.05% (w/w) vitamin premix, 0.13% (w/w)-0.23% (w/w) L-lysine, 0.08% (w/w)-0.14% (w/w) L-threonine, 0.05% (w/w) coban, 0.05% (w/w) selenium premix, 0.15% (w/w) sodium bicarbonate and 0.10% (w/w) sand. Digestible lysine in the diet may be 1.00% (w/w) to 1.20% (w/w). Digestible methionine in the diet may be 0.47% (w/w) to 0.54% (w/w). Digestible methionine and cysteine in the diet may be 0.98% (w/w) to 1.10% (w/w). Digestible threonine in the diet may be 0.68% (w/w) to 0.84% (w/w). Digestible tryptophan in the diet may be 0.17% (w/w) to 0.22% (w/w). Calcium in the diet may be 0.71% (w/w) to 0.96% (w/w). Available phosphorus in the diet may be 0.17% (w/w) to 0.46% (w/w). Sodium in the diet may be 0.17% (w/w) to 0.19% (w/w). The concentration of each ingredient within any one of the ranges herein may be any value between any two of the concentration points included in the range. In an embodiment, the diet may be the diet for broilers composed of one or more of the following ingredients: 30.00% (w/w)-75.00% (w/w) corn, 5.00% (w/w)-75.00% (w/w) wheat; 5.00% (w/w)-65.00% (w/w) barley; 5.00% (w/w)-30.00% (w/w) sorghum, 5.00% (w/w)-50.00% (w/w) millet, 10.00% (w/w)-45.00% (w/w) soybean meal, 5.00% (w/w)-20.00% (w/w) Canola (Rapeseed) meal, 2.00% (w/w)-15.00% (w/w) corn gluten meal, 5.00% (w/w)-15.00% (w/w) sunflower meal, 5.00% (w/w)-30.00% (w/w) corn distillers dry grains plus soluble solids, 1.00% (w/w)-8.00% (w/w) poultry/porcine/bovine meat and bone meal, 1.00% (w/w)-8.00% (w/w) fish meal, 0.10% (w/w)-2.1% (w/w) dicalcium or monocalcium or defluorinated phosphate, 0.50% (w/w)-6.00% (w/w) soy oil or vegetable oil or animal fat or grease or combination, 0.81% (w/w)-2.00% (w/w) limestone, 0.50% (w/w)-7.00% soy hulls, 0.24% (w/w)-0.50% (w/w) salt (NaCl), 0.13% (w/w)-0.50% (w/w) DL-methionine, 0.01% (w/w)-0.20% (w/w) choline chloride 60, 0.10% (w/w)-0.20% (w/w) mineral premix, 0.05% (w/w)-0.25% (w/w) vitamin premix, 0.05% (w/w)-0.30% (w/w) L-lysine, 0.10% (w/w)-0.30% (w/w) DL-Methionine or methionine analog (MHA), 0.05% (w/w)-0.20% (w/w) L-threonine, 0.05% (w/w) coban, 0.05% (w/w) selenium premix, 0.05% (w/w)-0.15% (w/w) sodium bicarbonate and 250 FTU/kg-2000 FTU/kg Phytase. Metabolizable energy of the diet may be 1225 (kcal/lb)-1491 (kcal/lb). Crude protein (CP) in the diet may be 15% (w/w) to 25% (w/w). Digestible lysine in the diet may be 0.85% (w/w) to 1.30% (w/w). Digestible methionine in the diet may be 0.45% (w/w) to 0.70% (w/w). Digestible methionine and cystine in the diet may be 0.65% (w/w) to 1.10% (w/w). Digestible threonine in the diet may be 0.60% (w/w) to 0.84% (w/w). Digestible tryptophan in the diet may be 0.10% (w/w) to 0.25% (w/w). Calcium in the diet may be 0.68% (w/w) to 1.10% (w/w). Available phosphorus in the diet may be 0.17% (w/w) to 0.50% (w/w). Sodium in the diet may be 0.17% (w/w) to 0.19% (w/w). Phytase in the diet may be 500 FTU/kg (w/w) to 8,000 FTU/kg (w/w). The concentration of each ingredient within any one of the ranges herein may be any value between any two of the concentration points included in the range. Variations in the concentrations of these ingredients may also be used in a diet.

[0124] The method may include mixing a transgenic plant or part thereof with a feed ingredient to obtain a mixture. The feed ingredient may be one or more ingredients included in the diet described herein. The transgenic plant or part thereof may be any transgenic plant or part thereof described herein. The method may include feeding an animal with the mixture. The body weight gain (BWG) in an animal fed with the mixture comprising a glucanase may be higher than the BWG in a control animal fed with identical feed ingredients not mixed with a transgenic plant including a glucanase. In an embodiment, the BWG in an animal fed with the mixture comprising a glucanase may be similar to the BWG in a control animal fed with a high energy diet or a diet that includes more or higher concentrations of the ingredients compared to the mixture including a glucanase. In an embodiment, the feed conversion ratio (FCR) in an animal fed with the mixture comprising a glucanase may be lower than the FCR in a control animal fed with identical feed ingredients not mixed with a transgenic plant including a glucanase. The FCR is defined as the mass of the feed eaten by the animal divided by the animal's mass. In an embodiment, the FCR in an animal fed with the mixture comprising a glucanase may be similar to the FCR in a control animal fed with a high energy diet or a diet that includes more or higher concentrations of the ingredients compared to the mixture including a glucanase.

[0125] In an embodiment, a method of enhancing thermal stability of a glucanase is provided. The method may include producing a transgenic plant that includes a synthetic nucleic acid encoding the glucanase. The synthetic nucleic acid may include any one the sequences described herein. The glucanase may be thermally stable upon exposure to temperatures in the range of 70.degree. C. to 130.degree. C., endpoints inclusive.

[0126] The glucanase may be thermally stable upon exposure to temperatures in the range of 25.degree. C. to 130.degree. C., endpoints inclusive. The glucanase may be thermally stable upon exposure to temperatures in the range from 25.degree. C., 30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C., 50.degree. C., 55.degree. C., 65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C., 85.degree. C., 90.degree. C., 95.degree. C., 100.degree. C., 105.degree. C., 110.degree. C., 115.degree. C., 120.degree. C., 125.degree. C., 130.degree. C., 25.degree. C., to 30.degree. C., 25.degree. C. to 35.degree. C., 25.degree. C. to 40.degree. C., 25.degree. C. to 45.degree. C., 25.degree. C. to 50.degree. C., 25.degree. C. to 55.degree. C., 25.degree. C. to 60.degree. C., 25.degree. C. to 65.degree. C., 25.degree. C. to 70.degree. C., 25.degree. C. to 75.degree. C., 25.degree. C. to 80.degree. C., 25.degree. C. to 85.degree. C., 25.degree. C. to 90.degree. C., 25.degree. C. to 95.degree. C., 25.degree. C. to 100.degree. C., 25.degree. C. to 105.degree. C., 25.degree. C. to 110.degree. C., 25.degree. C. to 115.degree. C., 25.degree. C. to 120.degree. C., 25.degree. C. to 125.degree. C., or less than 130.degree. C. The glucanase may be thermally stable upon exposure to temperatures in the range of 70.degree. C. to 130.degree. C., endpoints inclusive. The glucanase may be thermally stable upon exposure to temperatures in the range from 70.degree. C. to 75.degree. C., 70.degree. C. to 80.degree. C., 70.degree. C. to 85.degree. C., 70.degree. C. to 90.degree. C., 70.degree. C. to 95.degree. C., 70.degree. C. to 100.degree. C., 70.degree. C. to 105.degree. C., 70.degree. C. to 110.degree. C., 70.degree. C. to 115.degree. C., 70.degree. C. to 120.degree. C., or 70.degree. C. to 130.degree. C., endpoints inclusive.

[0127] The above mentioned synthetic nucleic acids may be provided in embodiments herein alone, as part of another nucleic acid, as part of a vector or as stated above as part of a transgenic plant.

[0128] In an embodiment, the transgenic plant may be derived from one of corn, rye, switchgrass, miscanthus, sugarcane or sorghum. The transgenic plant may be made by Agrobacterium mediated transformation using a vector having a nucleic sequence as set forth above.

[0129] In an embodiment, a method for enhancing production of fermentable sugars from grains is provided. The method may include mixing grains derived from any one of the transgenic plants described herein with grains from a different plant to form mixed grains. The different plant may be a non-transgenic plant. The different plant may be an engineered plant that includes a synthetic nucleic acid encoding at least one hydrolytic enzyme. The hydrolytic enzyme may be but is not limited to xylanase, an amylase, an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase, an intein-modified amylase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, a protease, an intein-modified protease, a phytase, or an intein-modified phytase. The method may include processing the mixed grains. The processing may include one or more operations selected from the group consisting of harvesting, baling, grinding, milling, chopping, size reduction, crushing, pellitizing, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, ensiling, mixing with silage or other biomass and ensiling, fermentation, chemical conversion, and chemical catalysis. The biomass may be but is not limited to hay, straw, stover, silage, compressed and pelleted feeds, soybeans, sprouted grains, legumes, feed grains, maize, rice, barley or wheat grains. The biomass may be any biomass derived from agricultural waste. The method may include converting fermentable sugars into a biochemical product. The biochemical product may be but is not limited to ethanol, butanol, lactic acid, citric acid, and acetic acid.

[0130] In an embodiment, a method for reducing the viscosity of a grain mixture is provided. The method may include mixing grains derived from any one of the transgenic plants described herein with grains from a different plant to form mixed grains. Water may be added to the mixed grains to form the grain mixture. The viscosity of the grain mixture may be lower when it includes any one of the glucanases described herein. The viscosity may be intestinal viscosity, which is typically measured from an intestinal sample removed from a bird or pig after euthanization. In this method, the digesta sample is centrifuged and the viscosity of supernatant is analyzed using a viscometer. For example, as describe by Lee et al., ileal digesta was centrifuged for 10 min at 3,500.times. gravity and 0.5 ml of supernatant was put in a Brookfield Cone and Plate Viscometer.sup.1 with a CPE-40 spindle. See Lee, J. T., C. A. Bailey, and A. L. Cartwright. 2003. .beta.-Mannanase ameliorates viscosity-associated depression of growth in broiler chickens fed guar germ and hull fractions. Poult. Sci. 82:1925-1931, which is incorporated herein by reference as if fully set forth. Samples are analyzed for 30 sec at 40.degree. C. and 5 rpm, to determine centipoise (cP units) readings. The higher the cP, the higher the viscosity of the sample.

[0131] The different plant may be a non-transgenic plant. The different plant may be an engineered plant that includes a synthetic nucleic acid encoding at least one hydrolytic enzyme. The hydrolytic enzyme may be but is not limited to xylanase, an amylase, an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase, an intein-modified amylase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, a protease, an intein-modified protease, a phytase, or an intein-modified phytase. The method may include processing the grain mixture. The processing may include one or more operations selected from the group consisting of harvesting, grinding, milling, size reduction, crushing, heating, gelotinzing, liquefaction, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, saccharifying, fermentation, chemical conversion, and chemical catalysis.

[0132] In an embodiment, a method for enhancing ethanol production from grains is provided. The method includes performing any one of the methods for enhancing production of fermentable sugars described herein.

[0133] The following list includes particular embodiments of the present invention. But the list is not limiting and does not exclude alternate embodiments, as would be appreciated by one of ordinary skill in the art.

EMBODIMENTS

[0134] 1. A transgenic plant comprising a synthetic nucleic acid encoding a glucanase, wherein the glucanase includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and is capable of degrading one or more polysaccharides. 2. The transgenic plant of embodiment 1, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 3. The transgenic plant of any one or both of the preceding embodiments, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 4.0 to 10.0. 4. The transgenic plant of any one or more of the preceding embodiments, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25.degree. C. to 130.degree. C. 5. The transgenic plant of any one or more of the preceding embodiments, wherein the glucanase activity has improved stability upon expression in the plant compared to the activity of a glucanase having an identical amino acid sequence and expressed in a bacterial cell. 6. The transgenic plant of any one or more of the preceding embodiments, wherein a plant is selected from the group consisting of: wheat, maize, barley, rice, and sorghum. 7. A transgenic plant comprising a synthetic nucleic acid including a sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one or more polysaccharides. 8. The transgenic plant of embodiment 7, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 9. The transgenic plant of any one or more of embodiments 7-8, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 4.0 to 10.0. 10. The transgenic plant of any one or more of embodiments 7-9, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25.degree. C. to 130.degree. C. 11. The transgenic plant of any one or more of embodiments 7-10, wherein the glucanase activity has improved stability upon expression in the plant compared to the activity of a glucanase having an identical amino acid sequence and expressed in a bacterial cell. 12. The transgenic plant of any one or more of embodiments 7-11, wherein the transgenic plant is a plant is selected from the group consisting of: wheat, maize, barley, rice, and sorghum. 13. The transgenic plant of any one or more of embodiments 7-12, which comprises the nucleic acid sequence of SEQ ID NO: 1 and produces an amplicon for diagnosing event 4588.259, 4588.757, or 4588.652. 14. A synthetic nucleic acid comprising a sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one or more polysaccharides. 15. The synthetic nucleic acid of embodiment 14, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 16. A synthetic polynucleotide comprising a sequence with at least 70% identity to a reference sequence selected from the group consisting of SEQ ID NO: 7-19, wherein the synthetic polynucleotide comprises a synthetic nucleic acid encoding a glucanase that is capable of degrading one or more polysaccharides. 17. The synthetic polynucleotide of embodiment 16, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 18. A vector comprising a synthetic polynucleotide, or a fragment of a synthetic polynucleotide, of embodiment 17. 19. A method of making a transgenic plant that includes a glucanase comprising:

[0135] contacting a plant cell with a synthetic nucleic acid encoding an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one or more polysaccharides; [0136] regenerating a transgenic plant from the transgenic plant cell; and

[0137] selecting the transgenic plant expressing a glucanase, wherein the glucanase is active and thermally stable upon exposure to a temperature in the range from 25.degree. C. to 130.degree. C.

20. The method of embodiment 19, wherein the one or more polysaccharide is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 21. The method of any one or both of embodiments 19-20, wherein the synthetic nucleic acid is part of a vector of embodiment 13. 22. An animal feedstock comprising a transgenic plant or part thereof of any one or more of embodiments 1-13, the product of any one or more of embodiments, 19-21, or a synthetic polypeptide of any one or more of embodiments 51-54. 23. The animal feedstock of embodiment 22 further comprising a feed supplement or feed additive. 24. The animal feedstock of any one or both of embodiments 22-23, wherein the feed supplement is plant material. 25. The animal feedstock of any one or more of embodiments 22-24, wherein the plant material is a non-transgenic plant. 26. The animal feedstock of any one or more of embodiments 22-24 wherein the plant material is an engineered plant. 27. The animal feedstock of any one or more of embodiments 22-26, wherein the feed supplement includes one or more exogenous enzymes. 28. The animal feedstock of embodiment 27, wherein the one or more exogenous enzyme includes a hydrolytic enzyme selected from the group consisting of: xylanase, endoglucanase, cellulase, exoglucanase, feruloyl esterase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified cellulase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, mannanase, amylase, an intein-modified amylase, phytase, an intein-modified phytase, protease, and an intein-modified protease. 29. The animal feedstock of any one or more embodiments 22-28, wherein the plant material includes at least one component selected from the group consisting of: forage, biomass, corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, barley grain, barley pellets, soybean meal, soybean oilcake, silage, sorghum grain and sorghum pellets. 30. The animal feedstock of any one or more of embodiments 23-29, wherein the feed supplement includes at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN.RTM., vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, and mineral premix. 31. A method of producing an animal feedstock comprising mixing 1) a transgenic plant or part thereof of any one or more of embodiments 1-13, 2) the product of any one or more of embodiments 19-41, or 3) a synthetic polypeptide of any one or more of embodiments 51-54 with plant material. 32. The method of embodiment 31 further comprising pelletizing the mixture. 33. The method of embodiment 32 further comprising adding a feed supplement to the mixture. 34. The method of embodiment 33, wherein the feed supplement includes at least one exogenous enzyme. 35. The method of embodiment 34, wherein the at least one exogenous enzyme includes a hydrolytic enzyme selected from the group consisting of xylanase, endoglucanase, cellulase, exoglucanase, feruloyl esterase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified cellulase, an intein-modified feruloyl esterase, amylase, an intein-modified amylase, mannanase, phytase, and protease. 36. A method of increasing utilization of non-starch polysaccharides in an animal comprising feeding an animal with an animal feedstock 1) including a transgenic plant of any one or more of embodiments 1-13, 2) of any or more of embodiments 22-30, 3) produced by the method of any one or more of embodiments 31-35, or 4) including a synthetic polypeptide of any one or more of embodiments 51-54. 37. The method of embodiment 36 further comprising preparing the animal feedstock. 38. The method of any or both of embodiments 36-37, wherein the animal is a monogastric animal or a ruminant animal. 39. A method of enhancing thermal stability of a glucanase comprising producing a transgenic plant that includes a synthetic nucleic acid comprising, consisting essentially of, or consisting of an amino acid a sequence having 70% identity to a reference sequence of selected fromthe group consisting of: SEQ ID NOS: 4-6, wherein the sequence encodes a glucanase capable of degrading one or more polysaccharides. 40. The method of embodiment 39, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 41. The method of any or both of embodiments 39-40, wherein expression of the nucleic acid produces the glucanase and the glucanse is thermally stable upon exposure to a temperature in the range of 25.degree. C. to 130.degree. C.

[0138] 42. A method for enhancing production of fermentable sugars from grains comprising:

[0139] mixing grains derived from a transgenic plant of any one of any one or more of embodiments 1-13 with grains derived from a different plant to form mixed grains; and

[0140] processing the mixed grains.

43. The method of embodiment 42, wherein the different plant is an engineered plant that includes a synthetic nucleic acid encoding at least one hydrolytic enzyme. 44. The method of any or both of embodiments 42-43, wherein the at least one hydrolytic enzyme is selected from the group consisting of: xylanase, an endoglucanase, an exoglucanase, cellulase, a feruloyl esterase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified cellulase, an intein-modified feruloyl esterase, amylase, phytase and protease. 45. The method of any one or more of embodiments 42-43, wherein the processing includes at least one operations selected from the group consisting of harvesting, baling, grinding, milling, chopping, size reduction, crushing, pellitizing, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, ensiling, fermentation, chemical conversion, and chemical catalysis. 46. The method of embodiment 45 further comprising producing a biochemical product. 47. The method of embodiment 46, wherein the biochemichal product is selected from the group consisting of ethanol, butanol, lactic acid, citric acid, and acetic acid. 48. A method for enhancing ethanol production from grains comprising performing a method of any one or more of embodiments 42-47. 49. A method for enhancing ethanol production from a transgenic plant comprising:

[0141] mixing a transgenic plant or part thereof of any one or more of embodiments 1-13 with a different plant or part thereof to form mixed plant material;

[0142] converting the mixed plant material into fermentable sugars; and

[0143] processing the fermentable sugars into ethanol.

50. The method of embodiment 49, wherein the plant material includes fiber, grain, or a combination thereof. 51. A synthetic polypeptide that includes an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 4-6, and capable of degrading one or more polysaccharides. 52. The synthetic polypeptide of embodiment 51, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 53. A synthetic polypeptide that includes an amino acid sequence comprising a contiguous amino acid sequence having at least 90% identity to 50 to 100, 50 to 150, 50 to 200, 50 to 250, 50 to 300, 50 to 322, or 50 to all contiguous amino acid residues of a glucanase having the sequence of any of SEQ ID NOS: 4-6, wherein the glucanase is capable of degrading one or more polysaccharides. 54. The synthetic polypeptide of embodiment 51, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 55. A method of increasing metabolizable energy of a diet comprising mixing a transgenic plant or part thereof with a feed ingredient, wherein the transgenic plant or part thereof comprises a synthetic nucleic acid encoding a glucanase comprising an amino acid sequence with at least 70% identity to a reference sequence selected from the group consisting of SEQ ID NOS: 4-6, and and capable of degrading one or more polysaccharides. 56. The synthetic polypeptide of embodiment 55, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 57. The method of any one or both of embodiments 55-56, wherein the synthetic nucleic acid comprises a sequence with at least 70% identity to a reference sequence selected from the group consisting of: SEQ ID NOS: 1-3. 58. The method of any one or more of embodiments 55-57, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 5.0 to 10.0. 59. The method of any one or more of embodiments 55-58, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25.degree. C. to 130.degree. C. 60. The method of any one or more of embodiments 55-59, wherein the feed ingredient includes at least one component selected from the group consisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, wheat middlings, barley grain, barley pellets, soybean meal, soy hulls, dried distillers grain, soybean oilcake, sorghum grain and sorghum pellets. 61. The method of any one or more of embodiments 55-60, wherein the feed ingredient includes at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN.RTM., vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, mineral premix, and one or more exogenous enzymes. 62. A method for producing an animal feedstock comprising mixing a transgenic plant or part thereof of any one or more of embodiments 1-13 with plant material. The method may also comprise the method for producing a plant that includes a glucanase of any one or more of embodiments 63-68. 63. A method for producing a plant that includes a glucanase comprising crossing a plant with a transgenic plant comprising event 4588.259, 4588.757 or 4588.652, and selecting a first progeny plant comprising event 4588.259, 4588.757 or 4588.652 and capable of degrading one or more polysaccharides. 64. The method of embodiment 63, wherein the one or more polysaccharides is selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan. 65. The method of any one or more of embodiments 63-64 further comprising selfing the first progeny plant and selecting a second progeny plant comprising event 4588.259, 4588.757 or 4588.652 and capable of degrading one or more polysaccharides. 66. The method of embodiment 65, wherein the second progeny plant is homozygous for event 4588.259, 4588.757 or 4588.652. 67. The method of embodiment 65, wherein the second progeny plant is heterozygous for event 4588.259, 4588.757 or 4588.652. 68. The method of embodiment 67 further comprising selfing the second progeny plant and selecting a third progeny plant homozygous event 4588.259, 4588.757 or 4588.652 and capable of degrading one or more polysaccharides. 69. A kit for identifying maize event 4588.259, 4588.757 or 4588.652 in a sample comprising a first primer and a second primer, wherein the first primer and the second primer are capable of amplifying a target sequence specific to maize event 4588.259, 4588.757 or 4588.652. 70. The kit of any one or more of embodiment 69 wherein, the first primer comprises a nucleic acid sequence selected from SEQ ID NOS: 38, 41, and 47. 71. The kit of any one or more of embodiments 69-70, wherein the second primer comprises a nucleic acid sequence selected from SEQ ID NOS: 39, 42, 43, 45, and 46. 72. The kit of any one or more of embodiments 69-71, wherein the target sequence comprises a sequence selected from the group consisting of SEQ ID NOS: 51-55. 73. The kit of any one or more of embodiments 69-72, wherein the target sequence is capable of hybridizing to the sequence of the nucleic acid comprising a sequence of SEQ ID NOS: 40 or 44 under conditions of high stringency. 74. The kit of any one or more of embodiments 69-73, wherein the sample comprises plant matter derived from a transgenic plant of any one or more of embodiments 1-13. 75. A method of identifying maize event 4588.259, 4588.757 or 4588.652 in a sample comprising:

[0144] contacting a sample with a first primer and a second primer of the kit of any one or more of embodiments 69-74;

[0145] amplifying a nucleic acid in the sample to obtain an amplified product; and

[0146] detecting an amplified product specific to a target sequence in maize event 4588.259, 4588.757 or 4588.65.

76. The method of embodiment 75, wherein the target sequence comprises a sequence selected from SEQ ID NOS: 51-55. The method of identifying may be added to any one or more of embodiments 63-68. 77. The method of embodiment 75, wherein the target sequence is at least one sequence selected from the group consisting of SEQ ID NOS: 22-31. 78. The method of embodiment 75, wherein the step of detecting comprises hybridizing the amplified product to the nucleic acid comprising a sequence of SEQ ID NOS: 40 under conditions of high stringency, and selecting the amplified product specific to maize event 4588.259. 79. The method of embodiment 75, wherein the step of detecting comprises hybridizing the amplified product to the nucleic acid comprising a sequence of SEQ ID NOS: 44 under conditions of high stringency, and selecting the amplified product specific to maize event 4588.652. 80. A method for reducing the viscosity of a grain mixture comprising combining grains from a transgenic plant of any one or more of embodiments 1-13, a different plant, and liquid to form a grain mixture. 81. The method of embodiment 80, wherein the different plant is a non-transgenic plant. 82. The method of embodiment 80, wherein the different plant is a genetically engineered plant. 83. The method of embodiment 80 and 82, wherein the genetically engineered plant comprises a synthetic nucleic acid encoding at least one hydrolytic enzyme. 84. The method of embodiment 83, wherein the at least one hydrolytic enzyme is selected from the group consisting of: xylanase, an amylase, an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase, an intein-modified amylase, an intein-modified xylanase, an intein-modified endoglucanase, an intein-modified exoglucanase, an intein-modified feruloyl esterase, a protease, an intein-modified protease, a phytase, or an intein-modified phytase. 85. The method of any one or more of embodiments 80-84 further comprising processing the grain mixture. 86. The method of embodiment 85, wherein the step of processing includes one or more operations selected from the group consisting of harvesting, grinding, milling, size reduction, crushing, heating, gelatinizing, liquefaction, extracting a component from the mixed grains, purifying a component or portion of the mixed grains, extracting or purifying starch, hydrolyzing polysaccharides into oligosaccharides or monosaccharides, saccharifying, fermentation, chemical conversion, and chemical catalysis.

[0147] Further embodiments herein may be formed by supplementing an embodiment with one or more element from any one or more other embodiment herein, and/or substituting one or more element from one embodiment with one or more element from one or more other embodiment herein.

EXAMPLES

[0148] The following non-limiting examples are provided to illustrate particular embodiments. The embodiments throughout may be supplemented with one or more detail from one or more example below, and/or one or more element from an embodiment may be substituted with one or more detail from one or more example below.

Example 1. Feed Glucanase Expression Vectors

[0149] A codon optimized nucleotide sequence for expression of the AGR2314 feed glucanase in maize was synthesized. For generating initial plant transformation constructs, single AGR2314 expression cassettes were assembled in vectors pAG4000 (pAG4258) or pAG4500 (pAG4588, pAG4597, and pAG4708). The vector pAG4000 has been created by replacing the rice ubiquitin 3 promoter with the first intron by the maize ubiquitin 1 promoter containing its own first intron for driving expression of the selectable marker gene encoding E. coli phosphomannose isomerase (PMI). The vector pAG4500 represents further improvement of pAG4000 and contains three modifications such as 1) insertion after the first maize ubiquitin intron of a 9 bp sequence (ATCCAGATC) representing the first three codons of the ubiquitin monomer with ATG converted into ATC; 2) insertion of the maize Kozak element (TAAACC) after the 9 bp sequence ubiquitin monomer; 3) replacement of the old multiple cloning site (MCS) by a new MCS that was synthesized by PCR and that was designed to contain multiple sites for several rare cutting enzymes (NotI, PacI, FseI, SwaI, AscI, AsiSI) to facilitate cloning of up to 4-5 expression cassettes on one T-DNA.

[0150] Sequence of the new MCS in pAG4500 (PmeI-KpnI fragment):

TABLE-US-00005 (SEQ ID NO: 20) GTTTAAACTGAAGGCGGGAAACGACAACCTGATCATGAGCGGAGAATTAA GGGAGTCACGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACG TTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTCAGCCTAAGCGG CCGCATTGGACTTAATTAAGTGAGGCCGGCCAAGCGTCGATTTAAATGTA CCACATGGCGCGCCAACTATCATGCGATCGCTTCATGTCTAACTCGAGTT ACTGGTACGTACCAAATCCATGGAATCAAGGTACC.

[0151] FIGS. 1-4 illustrate the expression vectors pAG4258, pAG4588, pAG4597, and pAG4708, respectively, carrying a single feed glucanase expression unit. The vector pAG4258 (FIG. 1; SEQ ID NO 7) has been cloned by assembling an expression cassette that was composed of the maize Glb1 promoter fused to the maize codon optimized AGR2314 sequence in KpnI-AvrII sites of pAG4000. The vectors pAG4588 (FIG. 2; SEQ ID NO 8) and pAG4597 (FIG. 3; SEQ ID NO 9) were developed by assembling their corresponding AGR2314 expression cassettes in KpnI-EcoRI sites of pAG4500, while the vector pAG4708 (FIG. 4; SEQ ID NO 10) was produced by cloning AGR2314 expression cassette into XmaI-AvrII sites of pAG4500. FIGS. 5-6 illustrate the expression vectors pAG4766 and pAG4767, respectively, carrying two feed glucanase expression units. FIGS. 7-8 illustrate the expression vectors pAG4770 and pAG4771, respectively, carrying three feed glucanase expression units. The unique rare cutting restriction sites that are available within the MCS of the pAG4500 were subsequently used in order to develop additional expression constructs containing either double AGR2314 expression units, such as pAG4766 (FIG. 5; SEQ ID NO 11) and pAG4767 (FIG. 6; SEQ ID NO 12), or triple AGR2314 expression units, such as pAG4770 (FIG. 7; SEQ ID NO 13) and pAG4771 (FIG. 8; SEQ ID NO 14), on the same T-DNA. The constructed vectors for expression of AGR2314 glucanase in plants are listed in Table 1. E. coli strains carrying the expression vectors were used for conjugation with Agrobacterium and subsequent transformation of maize.

TABLE-US-00006 TABLE 1 Description of Sequences SEQ ID Sequence NO Construct Description Type 1 AGR2314 maize--optimized protein DNA coding sequence (including C-terminal ER-retention signal "SEKDEL" 2 AGR2414 coding sequence DNA 3 AGR2514 coding sequence DNA 4 AGR2314 Mature protein sequence Amino acid (including C-terminal ER-retention signal "SEKDEL") 5 AGR2414 protein Amino acid 6 AGR2514 protein Amino acid 7 pAG4258 Glb1:mZ27:AGR2314:SEKDEL:NOS DNA 8 pAG4588 Glu1:mZ27:AGR2314:SEKDEL:T35S DNA 9 pAG4597 mZein:mZ27:AGR2314:SEKDEL:T35S DNA 10 pAG4708 Ole:mZ27:AGR2314:SEKDEL:NOS DNA 11 pAG4766 Glu1:mZ27:AGR2314:SEKDEL:NOS, DNA Glb1:mZ27:AGR2314:SEKDEL:NOS 12 pAG4767 mZein:mZ27:AGR2314:SEKDEL, DNA Glb1:mZ27:AGR2314:SEKDEL:NOS 13 pAG4770 mZein:mZ27:AGR2314:SEKDEL:NOS, DNA Glu1:mZ27:AGR2314:SEKDEL:NOS, Glb1:mZ27:AGR2314:SEKDEL:NOS 14 pAG4771 Glu1:mZ27:AGR2314:SEKDEL:NOS, DNA mZein:mZ27:AGR2314:SEKDEL:NOS, Glb1:mZ27:AGR2314:SEKDEL:NOS 15 pAG4257 mZein:mZ27:AGR2514:SEKDEL:NOS DNA 16 pAG4692 Glu1:mZ27:AGR2414:SEKDEL:T35S DNA 17 pAG4693 mZein:mZ27:AGR2414:SEKDEL:T35S DNA 18 pAG4705 Glu1:mZ27:AGR2514:SEKDEL:T35S DNA 19 pAG4706 Ole:mZ27:AGR2514:SEKDEL:NOS DNA

[0152] Expression cassettes for related beta glucanases, AGR 2414 and AGR 2514, were prepared using similar strategies, and sequences are provided for these expression cassettes as they are found in the expression vectors pAQ4257 pAQ4692, pAQ4693, pAQ4705, pAQ4706, pAQ4766, pAQ4257, pAQ4692, pAQ4693, pAQ4705, and pAQ4706.

Example 2. Feed Glucanase Protein Extraction Procedure

[0153] Flour was prepared from about 20 transgenic seeds by milling in an Udy cyclone mill or knife mill with 0.5 mm or 1 mm screen. About 0.5 ml of protein extraction buffer (100 mM sodium phosphate, pH 6.5, 0.01% Tween 20) was added to 20 mg flour in a 2 ml tube. In some cases, 2 g, 10 g, or 20 g ground samples was mixed with 10 ml, 50 ml or 100 ml of the extraction buffer in 15 ml tubes or 250 ml bottles. Larger masses and volumes can be used by scaling these amounts appropriately. After vortexing, the tubes were placed on a rotating platform in a 60.degree. C. incubator and rotated for 1 hour for protein extraction. After centrifugation at 16,000.times.g for 10 min in a tabletop centrifuge, the supernatant was diluted 20-fold for enzyme assay by adding 20 .mu.l supernatant to 380 .mu.l protein extraction buffer. In some cases, other dilution factors were used, as necessary.

Example 3. Feed Glucanase Activity Measurement

[0154] Colorimetric Assay. Fifty microliters of the diluted (20-fold to 360-fold) protein extract was mixed with 450 .mu.l of 100 mM sodium phosphate buffer, pH 6.5, 0.01% Tween 20 and 1 tablet of .beta.-glucazyme from Megazyme (Wicklow Ireland), and then incubated at 80.degree. C. for 1 hour before adding 1 ml of 2% Tris base. After centrifugation at 3000.times.g for 10 min, 100 .mu.l of supernatant was transferred to a microplate for absorbance measurement at 590 nm (A590). The activity was recorded as A590/mg flour after multiplying the dilution factors: A590xAx(500/50)/20 mg, where A is protein extraction dilution factor; 500 is the volume (ml) of buffer used for protein extraction; 50 is the volume of protein extraction (ml) used in the activity test.

[0155] Unit Activity Measurement. The assay involves the quantitation of reducing sugars that are released during a time course digestion of a model substrate (barley-.beta.-glucan) obtained from Megazyme (Wicklow, Ireland).

[0156] Hydrolysis of model substrate. Test 2 ml tubes were labeled with "+" sign, and 5 mg barley-.beta.-glucan substrate (reaction) was added to each test tube; no substrate was added to control tubes (control). Four hundred fifty microliters of 100 mM sodium phosphate buffer, pH 6.5, was added to each tube (reactions and controls), and tubes were placed into a Thermo-shaker with temperature set at 80.degree. C. and shaking speed set at 1000 rpm. Tubes were shaken at 1000 rpm at 80.degree. C. for 20 min until the substrate was completed dissolved. A tube with 2 ml of diluted grain protein extract, extracted as described above was placed in the Thermo-shaker to be pre-warmed.

[0157] Fifty microliters of the pre-warmed sample were added to the control and reaction tubes. Shaking was resumed and a timer was started. After 15 minute of shaking at 80.degree. C., 50 .mu.l of each of the reaction and control samples were removed and mixed with 10 .mu.l of 0.5N HCl in separate microplates. Shaking of the samples was resumed until all samples were removed and mixed with acid.

[0158] BCA quantification of glucose reducing equivalents. Glucose standards were prepared in protein extraction buffer at the following concentrations: 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. BCA reagent (from Thermo Scientific) was prepared by mixing reagent A with reagent B in a ratio of 50:1. To make a glucose standard curve, 75 .mu.l of buffer were dispensed into the first well of row A (A1) in a microplate and 75 .mu.l of each glucose standard were dispensed into wells A2 through A7. To detect the reducing sugars from the feed glucanase reaction and control samples, 25 .mu.l from each reaction were dispensed into rows of the microplate in the order of their incubation time with barley-.beta.-glucan (e.g., row B1-B2: 15 min-30 min), then added 25 .mu.l of corresponding control to another row of the microplate (e.g., row C1-C2: 15 min-30 min). Subsequently, 50 .mu.l of sodium phosphate buffer were dispensed in each well in these two rows (reaction and control), and 175 .mu.l BCA reagent were added to each well using a multichannel pipette. Mixing was achieved by pipetting up and down. The microplate was sealed and incubated at 80.degree. C. in a heat block. After 10 min incubation, the microplate was chilled on ice for 10 minutes and centrifuged to bring down condensate. Subsequently, the absorbance at 560 nm of each well was measured on a microplate reader.

[0159] Calculating units of feed glucanase activity from A560. The absorbance from the reagent blank was subtracted from the absorbance values for each of the glucose standards, and the resulting values were plotted according to their glucose concentrations. Linear regression was then used to calculate the "best fit" line through the data set. To determine glucose reducing equivalents in glucanase/barley-.beta.-glucan reactions, for each time point, the absorbance value from the control sample was subtracted from the reaction sample, and the resulting value was used to calculate the concentration of reducing sugars by comparison to the glucose standard curve. One unit (U) of glucanase activity is the amount of enzyme required to release 1 .mu.mol glucose reducing equivalents from 1% Barley-.beta.-glucan per minute at 80.degree. C., pH 5.3, using the BCA method of quantitation.

[0160] Unit Activity Measurement (semi-high throughput method). As described herein, the method detects the reducing sugars such as glucose released from the model substrate (barley-.beta.-glucan) by glucanase treatment at 80.degree. C. for 40 minutes or 90 minutes. When protein extract from grain or feed is appropriately diluted, the initial velocity is detected within 40 minutes (grain product) or 90 minutes (feed sample) of the reaction. The reactions were carried out in 96-well block (Costar, Cat #3960) or strip tubes (VWR, Cat #29442-610).

[0161] Substrate preparation: Barley-.beta.-glucan (low viscosity) was weighed based on the number of reactions, e.g., 10 samples, 4 dilutions for each sample needed a total of 40 reactions. Each reaction needed 5 mg substrate, therefore, at least 40.times.5=200 mg of barley-.beta.-glucan was required. The substrate was completely dissolved with the extraction buffer at 80.degree. C. water bath for 20 minutes, and vortexed at every 5 to 10 minutes.

[0162] The cluster tubes were used for 90 minutes endpoint activity unit assay of feed samples. Protein extract was diluted to 2-, 6-, 10- and 20-fold dilutions.

[0163] Purified protein diluted 100-fold was used as a positive control for assay validation. Purified glucanase protein (200,000 ppb) was stored in 50 mM MES, 150 mM sodium chloride, pH6.3 buffer plus 40% glycerol at -20.degree. C. Ten microliters of protein were mixed with 990 .mu.l of the extraction buffer, and 50 .mu.l were used for activity assay.

[0164] Barley-.beta.-glucan digestion by feed glucanase was carried out at a water bath set at 80.degree. C. In the block of cluster tubes, 450 .mu.l of the substrate were dispensed into tubes of A2 to D12 referring Table 2. These rows served as the reaction.

[0165] Four hundred fifty microliters of the extraction buffer (no substrate) were added to each control tubes from rows E2 to H12, which served as blank to correct protein content detected by BCA method for each reaction as described in Table 2 (A2 to D12).

[0166] Fifty microliters of the diluted sample extract including the negative control and positive control were added first to each blank tube, E2 to H12, and then to each reaction tube, A2 to D12, as described in Table 2.

TABLE-US-00007 TABLE 2 Example of enzyme hydrolysis of feed samples in cluster tubes 1 2 Columns 1 to 11 12 A Neg. Ctr, Sample_X, 2x dilution Pos. Ctr Reaction 2x B Neg. Ctr, Sample_X, 6x dilution Pos. Ctr 6x C Neg. Ctr, Sample_X, 10x dilution Pos. Ctr 10x D Neg. Ctr, Sample_X, 20x dilution Pos. Ctr 20x E Neg. Ctr, Sample_X, 2x dilution Pos. Ctr Blank 2x F Neg. Ctr, Sample_X, 6x dilution Pos. Ctr 6x G Neg. Ctr, Sample_X, 10x dilution Pos. Ctr 10x H Neg. Ctr, Sample_X, 20x dilution Pos. Ctr 20x

[0167] The tubes were covered with Corning.TM. Storage Mat III, the Corning Storage Mat Applicator was used to seal the tubes. The plate was shaken at a low speed. The block was placed in the water bath at 80.degree. C. for the 90 minutes incubation period. The reaction was terminated by adding 100 .mu.l of 0.5 N HCl to each well and cooling the block on ice.

[0168] BCA quantification of glucose reducing equivalents. Glucose standards were prepared in 100 mM sodium phosphate buffer, pH6, at the following concentrations: 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. BCA reagent (from Thermo Scientific) was prepared by mixing reagent A with reagent B in a ratio of 50:1. To make a glucose standard curve in column 1 on a microplate, 75 .mu.l of buffer was dispensed into the first well of row A (A1) and 75 .mu.l of each glucose standard were dispensed into wells A2 through A7. To detect the reducing sugars from the feed glucanase reaction and control samples, 25 .mu.l from each reaction were dispensed into rows of the microplate according to the order displayed on Table 2. Subsequently, 50 .mu.l of the extraction buffer was dispensed in each sample well (reaction and blank) referring to Table 2 from A2 to H12 to make a total volume 75 .mu.l. One hundred seventy five microliters of the BCA reagent were added to each well and mixed. The microplate was sealed and incubated at 80.degree. C. on a heat block. After 10 min incubation, the microplate was chilled on ice for 10 min and centrifuged to bring down condensate. Subsequently, the absorbance at 560 nm of each well was measured on a microplate reader.

[0169] Calculating units of feed glucanase activity from A560. The absorbance value for the reagent blank was subtracted from the absorbance values for each of the glucose standards, and the resulting values were plotted according to their glucose concentrations. Linear regression was then used to calculate the "best fit" line for the data set. To determine glucose reducing equivalents in the glucanase/barley-.beta.-glucan reactions, the absorbance value from the control sample was subtracted from absorbance value of the corresponding reaction sample, and the resulting value was used to calculate the concentration of reducing sugars by comparison to the glucose standard curve. One unit (U) of glucanase activity equals 1 .mu.mol glucose reducing equivalents released from 1% barley-.beta.-glucan per minute at 80.degree. C., using the BCA method of quantitation.

[0170] Calculating units of positive controls from A560 to validate the assay. The value of absorbance for blank samples (E12, F12, G12, H12) was subtracted from the value of absorbance for each reaction sample (A12, B12, C12, D12). The regression equation for the glucose standard was used to calculate the glucose content (.mu.mol). To determine the amount of reducing units produced per minute (A), the value for the amount of glucose (.mu.mol) released from barley-.beta.-glucan in the reaction was divided by the reaction time, for example 90, if the reaction time was 90 minutes. The unit value of positive controls equals the dilution x (A)/mg of protein in the assay. The dilution factor in the assay described herein equals 24. The dilution factor of 24 was determined by comparing the ratio of the total reaction volume to the portion of the reaction that was used in the BCA assay. In the assay, the total reaction volume was 600 .mu.l including 500 .mu.l reaction and 100 .mu.l of HCl used to stop the reaction. The portion of the reaction that was used in the BCA assay was 25 .mu.l. Therefore, the dilution factor of 24 was calculated by dividing 600 .mu.l by 25 .mu.l.

[0171] The amount of protein in the assay was calculated as follows. The concentration of the positive control was 2000 ng/ml, 50 .mu.l was the aliquot of the positive control used in the test (or 50/1000 if calculated in mL). The amount of protein calculated in nanograms was 2000.times.(50/1000), or 2000.times.(50/1000)/1000000 if calculated in milligrams.

Example 4. Glucanase Activity in Seed from Transgenic Maize

[0172] Silks on untransformed (wild type) maize plants were pollinated with pollen from individual transgenic maize plants that carried the pAG4588 construct. Mature, dried seeds were harvested from the resulting ears and assayed for activity via the colorimetric assay. FIG. 9 illustrates the range of activities recovered from 42 independent ears. In this figure, the numbers along the abscissa correspond to individual event identifiers. The highest activity was observed in the event 259. In the T0 transgenic maize plants 757 that also carried the pAG4588 construct, the activity was about 25 A590/mg. The average activity of the homozygous seeds derived from the first generation of the selfed plants was approximately 116 f 15 A590/mg. The activity of heterozygous seeds from this population was about 59 f 18 A590/mg.

[0173] Silks on untransformed (wild type) maize plants were pollinated with pollen from individual transgenic maize plants that carried the pAG4597 construct. Mature, dried seed were harvested from the resulting ears and assayed for activity via the colorimetric assay. FIG. 10 illustrates the range of activities recovered from 15 independent ears. In this figure, the numbers along the abscissa correspond to individual event identifiers. The highest activity was observed in the event 460.

Example 5. Maize Genomic Sequences Flanking T-DNA Integration Sites in Transgenic Events 4588.259, 4588.757 and 4588.652

[0174] Event 4588.259: The event 4588.259 carries two independent T-DNA integration sites that are located on the maize chromosomes 4 and 8. The chromosomal locations of the T-DNA integration sites were determined through BLASTN searches, in which the maize genomic DNA sequences that are contained in OB-2880, OB-2832 and OB-3252 sequences isolated from T-DNA insertion sites at the right and left T-DNA borders, were used as the queries for screening publicly available maize B73 genome sequence databases, such as the Maize Genetics and Genome Database, htt)://www.maizegdb.org/(Accessed May 8, 2016) See also Andorf, C M, Cannon, E K, Portwood, J L, Gardiner, J M, Harper, L C, Schaeffer, M L, Braun, B L, Campbell, D A, Vinnakota, A G, Sribalusu, V V, Huerta, M, Cho, K T, Wimalanathan, K, Richter, J D, Mauch, E D, Rao, B S, Birkett, S M, Richter, J D, Sen, T Z, Lawrence, C. J. (2015) MaizeGDB 2015: New tools, data, and interface for the maize model organism database. Nucleic Acids Research doi: 10.1093/nar/gkv1007; Lawrence, C J, Seigffried, T E, and Brendel, V. (2005) The Maize Genetics and Genomics Database. The community resource for access to diverse maize data. Plant Physiology 138:55-58; Lawrence, C J, Dong, Q, Polacco, M L, Seigfried, T E, and Brendel, V. (2004) Maize GDB, the community database for maize genetics and genomics. Nucleic Acids Research 32:D393-D397, all of which incorporated herein by reference as if fully set forth. Because both loci segregate independently, plants carrying both loci and each individual locus were evaluated.

[0175] In the flanks OB-2880, OB-2832 and OB-3252, which are provided below, the maize genomic DNA is shown in the uppercase letters, while the pAG4588 vector sequences are indicated in the lowercase letters and are underlined.

[0176] Integration Site on the Maize Chromosome 4:

[0177] The T-DNA integration site on the maize chromosome 4 is characterized by the 795 bp right T-DNA border flanking sequence OB-2880, which contains 677 bp of maize genomic. The isolated 677 bp maize genomic DNA flank has 99.3% sequence identity to the sequence derived from the antisense DNA strand of the maize chromosome 4 (nucleotide coordinates 56612593-56612026).

TABLE-US-00008 >OB-2880 (SEQ ID NO: 22) CTTAGATTAGAGAATGAAAATTTGATTGCTAAGGCCCAAGATTTTGATGT TTGCAAAGATACAATTACCGATCTTAGAGATAAGAATGATATACTTCGTG CTAAGATTGTTGAACTTACACCACAACCTTCTATGCCTTCTGTGACATTA ACATTACGTCACAAACAATAGTATTTTTGTCATACCTTACATGTTGGTGA CGTGATTGTGACGAAAATCACATCGTCACAGAAGGTGCGTGTTAAATGGT GTACTATGACGAATAACAAAAAAACGTCATAATAGTTTATGACGCAAACT ACAAACGTCACTAATCTATGACACTCGAATTCGTCACTAATTATGTCTAA ATACGTCACAATTCATGTAGTCGTGCCTTGCCACGTGGCTGATTACGTGG CGAGATGACATGGCAGTTGACGTGGCAGGTGATGTGGCGAAAATGTTGTG ACGAGTTCATTCGTCACAGATGTTATGACGTGGCATGCCACATGGCAGAT GATGTGGCAAAATTATGTGACAAAAATATTTGTCATAAATATCAATGAGG TGGCAATATATGTGTGACGAAATTTTTCATCACAAAGTACGATGACGTTG CAATATATTTATGACGAATTGTTCATCATAAGGCGTGATGAATTCATAGC GTCATGGAATATTATGAAATCACATGCtcaaacactgatagtttaaactg aaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacg ttatgacccccgccgatgacgcgggacaagccgttttacgtttgg

[0178] Integration site on the maize chromosome 8:

[0179] The T-DNA insertion site on the chromosome 8 is characterized by the sequences OB-2832 and OB-3252 that represent, accordingly, left and right flanks for the T-DNA integrated into this locus.

[0180] The 1211 bp OB-2832 sequence contains 864 bp of maize genomic DNA. The isolated 864 bp maize genomic DNA flank has 99.65% sequence identity to the sequence derived from the maize chromosome 8 with nucleotide coordinates 100613054-100613915.

TABLE-US-00009 >OB-2832 (SEQ ID NO: 23) tcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccg gtcttgcgatgattatcatataatttctgttgaattacgttaagcatgta ataattaacatgtaatgcatgacgttatttatgagatgggtttttatgat tagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagc gcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagat cgggaattggcgagctcgaattaattcagtacattaaaaacgtccgcaat gtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatACT AAAAAAACTCAAGGATCTGTCTCCAGAAAGGCCTTGCAGGGTTTGGCCAC GCCCACGGACATTCCATCTCAGAGCCATGATTAGAACGAAAAACACATGA GAGCCGTCGTTGCTAGGAGTCGGTTTCATATGTTCGCTAAAACAAGAGAT TTGTTTTTTTTCTCTCTCGTACATACACGAGTCAGCCCTTTTAATCTCAG GTTGACGTGCAATGTCGCTCGTCTAAGCAGAACATTTTGAGAACAAATGT GTTGTACATGAGAGTTTTGTGTACATGGTACGTACATTAAAACATCATCA TTTATCTTAGATCTAACATCTCTACTTGCTTGTTATATATTTTTTTTGTA AAATAACATCTTTCACCACTTTATATGGTGTTGTTTGCAAAATATACAGA GCAATTAGAGACGTTAGATTTGAGATGGACGGTGATAATTTAATACATGC ATAATGTACAAGAAAATCCTAACTGCACTAGATATGTTGTCAAACATTTT ACCTTTGTTACAAAAAGAAATGAATAGATGTTGAACGGTTGTCTTTCAAG CCTGTTCGCTGCGGCTTTAATTCACCAACTGCAATGAACAACCTGAAAGG TGATCGTTGCCGAACACATGCTGTTTGGCAAAGCTAGTAGTACCTTTTTT GTCTGTCACCTGGAATGATGAGAAAGGAGACAAGAGGAGAGGGCTGGCCA TTGTTTATATATATACGTATTTCCATTGCTTTGTGGCATGCAACAGTTCA AGGGTCCAAACTGGCAGGTTTTCAGCCCCGACAAATATAATAAAAAAACT ACAAAAAAAAAAGGTCCGTTTACATTCCTTTTTTGACAACGCTAGTCCGT GCGGAGCGAGC

[0181] The 696 bp OB-3252 sequence contains 95 bp of maize genomic DNA. The OB-3252 does not contain left T-DNA border sequence. The isolated 95 bp flank has 100% sequence identity to the sequence derived from the antisense DNA strand of the maize chromosome 8 with nucleotide coordinates 100613034-100612940.

TABLE-US-00010 >OB-3252 (SEQ ID NO: 24) Ggtgaaacaaggtgcagaactggacttcccgattccagtggatgattttg ccttctcgctgcatgaccttagtgataaagaaaccaccattagccagcag agtgccgccattttgttctgcgtcgaaggcgatgcaacgttgtggaaagg ttctcagcagttacagcttaaaccgggtgaatcagcgtttattgccgcca acgaatcaccggtgactgtcaaaggccacggccgtttagcgcgtgtttac aacaagctgtaagagcttactgaaaaaattaacatctcttgctaagctgg gagctctagatccccgaatttccccgatcgttcaaacatttggcaataaa gtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataa tttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgac gttatttatgagatgggtttttatgattagagtcccgcaattatacattt aatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcg cgcgcggtgtcatctatgttactagatcgggaattggcgagctcgaatta aTTCAAGTGTCTTCGTACAAACTGGGGGATGGGGCAGACCGCCAGGTTCA AACCGTTTGACTAGATGCGGCTGGCAGGCTACTTTGCAGTGCATGC

[0182] The maize genomic DNA flanks in sequences OB-2832 and OB-3252 are separated by 20 nucleotides on the maize chromosome 8, which indicates that during T-DNA integration 20 bp of the original maize genomic DNA sequence were replaced by the inserted T-DNA sequences.

[0183] There is also an OB-2861 sequence and an OB-2868 sequence within the 259 event. The 970 bp OB-2861 sequence consists of the re-arranged pAG4588 sequences including a partial 223 bp Nos terminator sequence (uppercase letters, nucleotides 3290-3512 in pAG4588); the 73 bp sequence near the left T-DNA border with the first 3 bp of the processed left T-DNA border sequence (italicized lowercase letters, nucleotides 3513-3585); the 299 bp sequence near the right T-DNA border with 5 bp of the processed right T-DNA border sequence and polylinker sequence with multiple cloning sites (lowercase letters, nucleotides 9647-9945); the 359 bp 5' sequence of the rice glutelin promoter prGTL-03 (uppercase letters, nucleotides 9946-10304). The underlined are 18 bp of a duplicated sequence that has been created during T-DNA integration process. The OB-2861 sequence is as follows:

TABLE-US-00011 (SEQ ID NO: 25) GAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATT ACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGA TGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGA AAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCAT CTATGTTACTAGATCGGGAATTGgcgagctcgaattaattcagtacatta aaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgttatca agttgtctaagcgtcaaacactgatagtttaaactgaaggcgggaaacga caacctgatcatgagcggagaattaagggagtcacgttatgacccccgcc gatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacg ttgaaggagccactcagcctaagcggccgcattggacttaattaagtgag gccggccaagcgtcgatttaaatgtaccacatggcgcgccaactatcatg cgatcgcttcatgtctaactcgagttactggtacgtaccaaatccatgga atcaaggtaccTCCATGCTGTCCTACTACTTGCTTCATCCCCTTCTACAT TTTGTTCTGGTTTTTGGCCTGCATTTCGGATCATGATGTATGTGATTTCC AATCTGCTGCAATATaAATGGAGACTCTGTGCTAACCATCAACAACATGA AATGCTTATGAGGCCTTTGCTGAGCAGCCAATCTTGCCTGTGTTTATGTC TTCACAGGCCGAATTCCTCTGTTTTGTTTTTCACCCTCAATATTTGGAAA CATTTATCTAGGTTGTTTGTGTCCAGGCCTATAAATCATACATGATGTTG TCGTATTGGATGTGAATGTGGTGGCGTGTTCAGTGCCTTGGaTTTGAGTT TGATGAGAGTTGCTTCTGGG

[0184] The 1127 bp OB-2868 sequence consists of re-arranged pAG4588 sequences including the 595 bp 3' sequence of the PMI marker gene (uppercase letters, nucleotides 2594-3188 in pAG4588); the 48 bp sequence between PMI and Nos terminator (lowercase letters, nucleotides 3189-3236); the 276 bp Nos terminator sequence (uppercase letters, nucleotides 3237-3512); the 73 bp sequence near the left T-DNA border with the first 3 bp of the processed left T-DNA border sequence (italicized lowercase letters, nucleotides 3513-3585); the 119 bp sequence near the right T-DNA border with 5 bp of the processed right T-DNA border and a partial polylinker sequence (lowercase letters, nucleotides 9647-9765). The underlined are 18 bp of a duplicated sequence that has been created during T-DNA integration process. The OB-2868 sequence is as follows:

TABLE-US-00012 (SEQ ID NO: 26) AAAATCCCGCGCGCTGGCGATTTTAAAATCGGCCCTCGATAGCCAGCAGG GTGAACCGTGGCAAACGATTCGTTTAATTTCTGAATTTTACCCGGAAGAC AGCGGTCTGTTCTCCCCGCTATTGCTGAATGTGGTGAAATTGAACCCTGG CGAAGCGATGTTCCTGTTCGCTGAAACACCGCACGCTTACCTGCAAGGCG TGGCGCTGGAAGTGATGGCAAACTCCGATAACGTGCTGCGTGCGGGTCTG ACGCCTAAATACATTGATATTCCGGAACTGGTTGCCAATGTGAAATTCGA AGCCAAACCGGCTAACCAGTTGTTGACCCAGCCGGTGAAACAAGGTGCAG AACTGGACTTCCCGATTCCAGTGGATGATTTTGCCTTCTCGCTGCATGAC CTTAGTGATAAAGAAACCACCATTAGCCAGCAGAGTGCCGCCATTTTGTT CTGCGTCGAAGGCGATGCAACGTTGTGGAAAGGTTCTCAGCAGTTACAGC TCAAACCGGGTGAATCAGCGTTTATTGCCGCCAACGAATCACCGGTGACT GTCAAAGGCCACGGCCGTTTAGCGCGTGTTTACAACAAGCTGTAAgagct tactgaaaaaattaacatctcttgctaagctgggagctctagaTCCCCGA ATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAAT CCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGT TAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGG TTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAAC AAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTAT GTTACTAGATCGGGAATTGgcgagctcgaattaattcagtacattaaaaa cgtccgcaatgtgttattaagttgtctaagcgtcaatttgttatcaagtt gtctaagcgtcaaacactgatagtttaaactgaaggcgggaaacgacaac ctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatg acgcgggacaagccgttttacgtttgg

[0185] Event 4588.757: The event 4588.757 carries one T-DNA integration site that is located on the maize chromosome 8.

[0186] The chromosomal location of the T-DNA integration site was determined through BLASTN searches, in which the maize genomic DNA sequences that are contained in OB-3170 and OB-3237 sequences isolated accordingly from T-DNA insertion sites at the right or left T-DNA borders, were used as the queries for screening publicly available maize B73 genome sequence databases, such as http://www.maizegdb.org/. See also Andorf, C M et al. (2015) Nucleic Acids Research doi: 0.10.1093/nar/gkv1007; Lawrence, C J et al, (2005) Plant Physiolgy 138:55-58; Lawrence, C J et al., (2004) Nucleic Acids Research 32:D393-D397, all of which incorporated herein by reference as if fully set forth.

[0187] In the flanks OB-3170 and OB-3237, which are provided below, the maize genomic DNA is shown in the uppercase letters, while the pAG4588 vector is indicated in the lowercase underlined letters.

[0188] The 1303 bp OB-3170 right T-DNA border flanking sequence consists of the 975 bp maize genomic DNA attached to the 328 bp of the pAG4588 vector borderless sequence proximal to the right T-DNA border site. The isolated 975 bp maize genomic DNA flank has 99.38% sequence identity to the sequence derived from the maize chromosome 8 with nucleotide coordinates 62661042-62662016.

[0189] The OB-3170 sequence is as follows:

TABLE-US-00013 (SEQ ID NO: 27) TTGGGGTTCCTTATCCTGTTGTCGGAGTTGTGCCATTATCCTTTCCATGG TTGACCTGAGCTTTAGCCTGTACACTGTAGACTCTACTAGAGGTTTACCT GAGGCTGAATTCCCGCTGCTAAGATGTGATGTTCCCGGCCATAAGCAAAG ATGCAGGTTGTCTTTGCTTTGTAAAGATGAAGGTTGTCTTTGTTTTGTAA TCGAAAAAAAAACCCTCCGACTTCGATAGCAATCCATTTCTTGAAACGAT ATAGCTATAAGCTGCAGCCACACCTTGCGTTGATGATGCCAAAGCTTTCT TTCGAGTGCGATGCATGCACTGGCCTGTTGAGATCTTATCAATATGGCAA ACAGTAACCTAACGTATATGACTACATGGTCTTCATGCTTTTGAGAGGTG CCTCATAGGAAACAGTCAGGCCAATGATTTTAGGGAATACAATATATTTT TGCTGTTTTTTTTTTGCAAATTGTCCATATTATTACAAAAAAAACTAAAC ATGCCCAAAGGCAATAGCTTTCTAAATAAAAATGAATAACGGTCCACTTA TATATGTTGGCCAGTAATCAATTCTGAGGCCTGACAAACCATGCATATAT TAACAGTAGGTTAATGGCCGTGCGTGAAAAAATTTCAATACAACAAGAGA TTGAAAAAAAAGAGTGTCTTACCAATATGTTATTTTATAAGTACCAAATG TGTAGGAAACTTGCATTCATTTTTTCCCTGAGAATGGAAAAAAACAAGAC ATACTCATTTTCAAGTTGAATTGTCATAGCAACACACATGTTGTATCTGC CGGTTCATGCAATTGTGCCAACCAAAATATCTAAATGAGATATTCAAGAC TCAACAGAATTAAAGTATGGAATAGGGTGTATATACACTCAACCATTATT AAATGGTATAATCATCTATCTATATCACTATAAAATCTACCAGTTTAAAC TTCACAAAACTCATCTAGCTAATGGaggcgggaaacgacaacctgatcat gagcggagaattaagggagtcacgttatgacccccgccgatgacgcggga caagccgttttacgtttggaactgacagaaccgcaacgttgaaggagcca ctcagcctaagcggccgcattggacttaattaagtgaggccggccaagcg tcgatttaaatgtaccacatggcgcgccaactatcatgcgatcgcttcat gtctaactcgagttactggtacgtaccaaatccatggaatcaaggtacct ccatgctgtcctactacttgcttcatccccttctacattttgttctggtt ttg

[0190] The 960 bp OB-3237 left T-DNA border flanking sequence consists of the 620 bp of the maize genomic DNA attached to the 340 bp pAG4588 vector sequence (nucleotides 3260-3599 in pAG4588), which includes 253 bp Nos terminator sequence and 70 bp sequence upstream of the 17 bp processed left T-DNA border sequence. The isolated 620 bp maize genomic DNA flank has 100% sequence identity to the sequence derived from the maize chromosome 8 with nucleotide coordinates 62662037-62662642.

[0191] The OB-3237 sequence is as follows:

TABLE-US-00014 (SEQ ID NO: 28) Aaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttg cgatgattatcatataatttctgttgaattacgttaagcatgtaataatt aacatgtaatgcatgacgttatttatgagatgggtttttatgattagagt cccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaa actaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaa ttggcgagctcgaattaattcagtacattaaaaacgtccgcaatgtgtta ttaagttgtctaagcgtcaatttgtttacaccacaatataAAATCTACCT GTTCGCTGATAAGCCGTTAGGTTGACTATGTGACTGTTGGGCGGCAAAAT GACCACGCGGACGGTCTAGCCCCAAAGCCGGACGGTCCGCGGTCCAGACA GTCTGCACTGGTGGTGTCGGCGTTTCGACCCCGGGGGGTCCCTGGACCGA CGAGTAAATTGTCGCTGCGTGTCCCAGCCCAGATGGGTCCGCGCGAGACG GAACGCGAAGATGGGAAAACAGCAAAGGGGAACCCGCGGCCTTCGTGTTG TCCTGCGCCCAGGTCGGGTGCGCTTGCAGTAGGGGGTTACAACCGTTCGC GTGGGAGAGACAGAGAGAGAGCGAGAGCCTTATGCGTCGGCCCGTTCTCC CGCGCGGCCAACCCTCTCGTACGAGAGCCCTGGACCTTCCTTTTATAGAC GTAAGGAGAGGGCCCAGGTGTACAATGGGGGGTGTAGCAGAGTGCTAACG TGTCTAGCAGAGAGGAGCCGGAGCCCTAAGTACATGTCGTCGTGGCTGTC GGAGAGGTTTTGGCGCCCTGTTCATGTGATGTCGTGGCCGTCGGAGGAGC GCTTGAGCCCCGTGGAAGTACAGCTGTCGGGGCTGTCGGATCCTTGCTGA CGTCTCCTTG

[0192] The maize genomic DNA flanks in sequences OB-3170 and OB-3237 are separated by 21 nucleotides on the maize chromosome 8, which indicates that during T-DNA integration 21 bp of the original maize genomic DNA sequence were replaced by the inserted T-DNA sequences.

[0193] Event 4588.652: FIG. 11 illustrates a diagram showing positions of the characterized flanking sequences in 4588.652.

[0194] Sequences isolated at the T-DNA insertion site in 4588.652. T-DNA in the event 4588.652 has integrated into chromosome 7 of the maize genome in BxA genotype, which was used for maize transformation with the pAG4588 construct. The T-DNA insertion occurred between nucleotides 141683320-141683357 of the publicly available reference B73 maize genome. The T-DNA integration displaced 38 bp of the native maize genomic sequence at this site. This 38 bp DNA is underlined and the sequences of pAG4588 are underlined and shown in bold characters in the sequences of the T-DNA insertions shown below. The diagram illustrated in FIG. 11 depicts locations of the sequences in the locus in 4588.652. The right and the left border T-DNA flanking sequences, OB-4448 and OB-4451 respectively, were isolated from multiple 4588.652 progeny using a PCR-based genome walking approach. The entire genomic regions between the right and the left border flanks were isolated and sequence characterized from WT genotypes BxA, 19545 (E), 15009 (G) as well as the nulls BC2ES2_512x and BC1GS2_518x. The following wild type maize genomic DNA sequences were used for reference: the WT_BxA (OB-4541; SEQ ID NO: 32), the WT_E sequence (OB-4545, OB-4546; SEQ ID NO: 33), the WT_G sequence (OB-4547, OB-4548; SEQ ID NO: 34), the Null_BC2ES2_512x sequence (OB-4578 to OB-4580; SEQ ID NO: 35), the Null_BC1GS2_518x sequence (OB-4582 to OB-4584; SEQ ID NO: 36), and the WT_B73Chr7_141681606-141685147 reference sequence (SEQ ID NO: 37). Furthermore, the right and the left border flanks were additionally isolated and entirely sequenced from the more advanced 4588_652 transgenic progeny BC2ES2_472x. The entire 4044 bp BxA genomic sequence containing the right and the left border flanking sequences have high BLASTN identity hits to two nucleotide positions 141681606-141682538 and 141682550-141685147 on the chromosome 7 in the maize B73 genome.

[0195] Analysis of Nucleotide Sequences in the Left T-DNA Border Flank

[0196] The left T-DNA border flank OB-4451 has 98.66% BLASTN sequence identity to nucleotides 141683358-141685147 on the maize chromosome 7 in B73 genome. Multiple sequence alignment of the left border specific sequences from the wild type genotypes B73, BxA, 19545 (E), 15009 (G), nulls BC2ES2_512x and BC1GS2_518x as well as 4588_652 transgenic progenies 116_F1G and BC2ES2_472x and the reference public sequence of B73 genome revealed that these 1.8 kb sequences are nearly 100% identical between all genotypes.

[0197] Analysis of Nucleotide Sequences in the Right T-DNA Border Flank

[0198] The 2218 bp right border flank OB-4448 has high BLASTN sequence identity to two nucleotide positions 141681606-141682538 and 141682550-141683319 on the maize chromosome 7 in B73 genome. Multiple sequence alignment of the right border specific sequences from 4588_652 transgenic progenies 116_F1G and BC2ES2_472x with the WT sequence of BxA revealed that these three 2.2 kb sequences are nearly 100% identical.

[0199] A 521 bp "unique" sequence that is specific to the right T-DNA border flank has originated from genotype BxA, which was used for transformation with the pAG4588 construct. No BLASTN sequence identity hits to this sequence were identified at the T-DNA integration site within the reference B73 genome. On the other hand, the 521 bp sequence has multiple BLASTN identity hits on different chromosomes in maize B73 genome indicating that this sequence is highly repetitive. The 521 bp sequence is shown in italicized lowercase letters.

[0200] Sequences characterized at 4588_652 T-DNA integration site. The OB-4448 sequence (extended right border flank in 4588_652 isolated from F1G of 4588_652 is as follows:

TABLE-US-00015 (SEQ ID NO: 29) CACCCTCGCTGTTGGTAAACGTGCGCCTTGGGTATGTCCTCACCTGCATG ATACGACATGTTGAAAAAGGTACATGGCTGGGCGGATTTAAACAGTAGAA TGAAAAGGTGCCACAAGAAAACTCGTCAAAGAATTGACTACGCGTCAATG TTCCATAGTTAAAAAGACTTGAACTCTGGATCAGGGACTTTCAAACAAGG ATAGCTGCCTGGTCACCAGTCATTAACTGTAATGTAATGGCCATAGATGA TGCATGAGTACAATAATAAAAAAACACCATCCAGCCAAATATATACTCCC TGTCACAAATGAAAATTCGTTTTAGATAATTAGTGGATTCATACAATATT TGTTGTATGTGTTTTATGTGTCTAGATTCATCATCCTCTATTTGAATATA GACAGAAAAATCATAACTAAAACGAATACTATTTGGGAACGGAGGGAGTA CTACTTTGGCAGAATGCCCCCAGGAAAGTACCAGTTTCAGGGGTAGTTTG GAAGGCTAAACCTAGGGAGGGAAAACCCCCCACATGTAACTAAATATCTT ATTCAAATGTTACCCCTAGGGATTACTCACCCTGGGAAATGAGAAGGGTC CCAAGGGGATTTCGGTTTCTATTATTTTTTCTGCAAACCATTTCAGAGCA ATGATATGAAACCAAGCTAACTACTTATAACATTTCTTAAGAATATCAGA CATAGGAAAGTGATGGCCTGGAACCAAAGTAAGACTGGTAGATAAATAGA TCACTAGAATAAACCCTGACAGTTCATAGCCTTCATAGAAGCAAAAGGAA ACACTACGGGAGCAATTGGTTGCTTGCACTAGCAATTCACTGCATTGGGT CTAATGCAGGATAGACTAAGCCAGCATAAGTGTGCGCAATGTGTTTGTGT TTGGTTGCCATGTTATAAGTAAGTTGCATTTGCTAATATctttctcctga ctctaatgagtccacttttgctgactggtgggcgaaagtaagtaagcaag tgcacaaatccaaaagaagaggctttaacagtatcatcatcttgggggct tggtgtttatggcttcatcgtaataaggtggtttttgatggtgtcagtcc ttcaattattggcataaaggcaatttttttggatgaagttgaattctgga ggcttgccggtgctaggcatcttgaggctttggttcctggtgctggaatt tttaggtcaagggttcttttgggtgattagtgaagagcaggtgtgtgtgg tctgctcgcactttttgttgttcgttctcctattgcgtgctgttgtttcc aggcgcatttatggaggctgcagttttgtgcgcagcagaagttggtggtt ttgtgttttgtgttttgcctattttggcattgtactttggtccattttgg actgttttcttctcttaatttaatgatgtgcagctctcctgcgcgtttaa gaaaaaaaaaAGTTGGCTGTTTTGTATTTCTTGTGATCACCCATGCTTGT TGTGGTCAGATTAAACTCTCACGTTTAATGCTACAGAAGCATCCATGAGA CAATGAAACACCGCTCAAAAGCCACGTAGTAGCATACCCTGACTTATGAA TAAAGCAACTCGATCTGATTTATTTGAGAAAACAGGAAACTGACAAGTTA TTTTTAACACAAAATTTCATTAAAAACGAATGGTAGACAATTACCAATCT GTAGGTCCCTGGCTTGCAAGTCCTCCCAATGTCTAAGAAATCAAATAGGA ACTGCAGGCAAGCCAGCAAGAAAGTATTAATCACTGGATATAAAATATAA AGAAAAAAGAAGGAAAGACGGCTACTCGGCTAGCATATGTTTTTGTTAGG GGTGAAAATGGATACTTATTCAGAAATCATTTTTGATCTTTTTTCTTTAA TTAGGAATAAATAGGATATAGAATATGCTAAGCAAATTCATATTCTTGTT CTTAGCATTGGGCTTGTAAAGATTCATAAAAGGTAAATCTCAAATTTATC ATATATCTTAAATGGTAGATATAAAATTCAGATACAAATATTTTTCAACT TTTTTGTTGTAGGGAACAAATTATATTAAAAAAAATTATGCACAATTCTA TTCTTATTTGTAATAATGTGCTTGATAACATAATAAAAGATTACCATCAA ATTTCACACACACCCACCCACCCACCCACCCCTGCACGCACGCGCGCGCA CACACACTATATGTGTGTtcaaacactgatagtttaaactgaaggcggga aacgacaacctgatcatgagcggagaattaagggagtcacgttatgaccc ccgccgatgacgcgggacaagccgttttacgtttggaactgacagaaccg caacgttgaaggagccactcagcctaagcggccgcattggacttaattaa gtgaggccggccaagcgtcgatttaaatgtaccacatggcgcgccaacta tcatgcgatcgcttcatgtctaactcgagttactggtacgtaccaaatcc atggaatcaaggtacctccatgctgtcctactacttgcttcatccccttc tacattttgttctggtttttggcctgcatttcggatcatgatgtatgtga tttccaatctgctgcaatatgaatggagactctgtgctaaccatcaacaa catgaaatgcttatgaggcctttgctgagcagccaatcttgcctgtgttt atgtcttcacaggccgaattcctctgttttgtttttcaccctcaatattt ggaaacatttatctaggttgttttgtgtccaggcctataaatcataaatg atgttgtcgtattggatgtgaatgtggtggcgtgttcagtgccttggatt tgagt

[0201] The RB_BC2ES2_472x sequence (extended RB flank isolated from the advanced progeny of 4588_652) is as follows:

TABLE-US-00016 (SEQ ID NO: 30) CACCCTCGCTGTTGGTAAACGTGCGCCTTGGGTATGTCCTCACCTGCATG ATACGACATGTTGAAAAAGGTACAAGGCTGGGCGGATTTAAACAGTAGAA TGAAAAGGTGCCACAAGAAAACTCGTCAAAGAATTGACTACGCGTCAATG TTCCATAGTTAAAAAGACTTGAACTCTGGATCAGGGACTTTCAAACAAGG ATAGCTGCCTGGTCACCAGTCATTAACTGTAATGTAATGGCCATAGATGA TGCATGAGTACAATAATAAAAAAACACCATCCAGCCAAATATATACTCCC TGTCACAAATGAAAATTCGTTTTAGATAATTAGTGGATTCATACAATATT TGTTGTATGTGTTTTATGTGTCTAGATTCATCATCCTCTATTTGAATATA GACAGAAAAATCATAACTAAAACGAATACTATTTGGGAACGGAGGGAGTA CTACTTTGGCAGAATGCCCCCAGGAAAGTACCAGTTTCAGGGGTAGTTTG GAAGGCTAAACCTAGGGAGGGAAAACCCCCCACATGTAACTAAATATCTT ATTCAAATGTTACCCCTAGGGATTACTCACCCTGGGAAATGAGAAGGGTC CCAAGGGGATTTCGGTTTCTATTATTTTTTCTGCAAACCATTTCAGAGCA ATGATATGAAACCAAGCTAACTACTTATAACATTTCTTAAGAATATCAGA CATAGGAAAGTGATGGCCTGGAACCAAAGTAAGACTGGTAGATAAATAGA TCACTAGAATAAACCCTGACAGTTCATAGCCTTCATAGAAGCAAAAGGAA ACACTACGGGAGCAATTGGTTGCTTGCACTAGCAATTCACTGCATTGGGT CTAATGCAGGATAGACTAAGCCAGCATAAGTGTGCGCAATGTGTTTGTGT TTGGTTGCCATGTTATAAGTAAGTTGCATTTGCTAATATctttctcctga ctctaatgagtccacttttgctgactggtgggcgaaagtaagtaagcaag tgcacaaatccaaaagaagaggctttaacagtatcatcatcttgggggct tggtgtttatggcttcatcgtaataaggtggtttttgatggtgtcagtcc ttcaattattggcataaaggcaatttttttggatgaagttgaattctgga ggcttgccggtgctaggcatcttgaggctttggttcctggtgctggaatt tttaggtcaagggttcttttgggtgattagtgaagagcaggtgtgtgtgg tctgctcgcactttttgttgttcgttctcctattgcgtgctgttgtttcc aggcgcatttatggaggctgcagttttgtgcgcagcagaagttggtggtt ttgtgttttgtgttttgcctattttggcattgtactttggtccattttgg actgttttcttctcttaatttaatgatgtgcagctctcctgcgcgtttaa gaaaaaaaaaAGTTGGCTGTTTTGTATTTCTTGTGATCACCCATGCTTGT TGTGGTCAGATTAAACTCTCACGTTTAATGCTACAGAAGCATCCATGAGA CAATGAAACACCGCTCAAAAGCCACGTAGTAGCATACCCTGACTTATGAA TAAAGCAACTCGATCTGATTTATTTGAGAAAACAGGAAACTGACAAGTTA TTTTTAACACAAAATTTCATTAAAAACGAATGGTAGACAATTACCAATCT GTAGGTCCCTGGCTTGCAAGTCCTCCCAATGTCTAAGAAATCAAATAGGA ACTGCAGGCAAGCCAGCAAGAAAGTATTAATCACTGGATATAAAATATAA AGAAAAAAGAAGGAAAGACGGCTACTCGGCTAGCATATGTTTTTGTTAGG GGTGAAAATGGATACTTATTCAGAAATCATTTTTGATCTTTTTTCTTTAA TTAGGAATAAATAGGATATAGAATATGCTAAGCAAATTCATATTCTTGTT CTTAGCATTGGGCTTGTAAAGATTCATAAAAGGTAAATCTCGAATTTATC ATATATCTTAAATGGTAGATATAAAATTCAGATACAAATATTTTTCAACT TTTTTGTTGTAGGGAACAAATTATATTAAAAAAAATTATGCACAATTCTA TTCTTATTTGTAATAATGTGCTTGATAACATAATAAAAGATTACCATCAA ATTTCACACACACCCACCCACCCACCCACCCCTGCACGCACGCGCGCGCA CACACACTATATGTGTGTtcaaacactgatagtttaaactgaaggcggga aacgacaacctgatcatgagcggagaattaagggagtcacgttatgaccc ccgccgatgacgcgggacaagccgttttacgtttgg

[0202] The OB-4451 sequence (extended left border flank in 4588_652 is as follows:

TABLE-US-00017 (SEQ ID NO: 31) gggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgt gttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcag acacgttctgattgctaacttgccagtgtttctctttggggaatcctggg atggctctagccgttccgcagacgggatcgatttcatgattttttttgtt tcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccg tgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgt gatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgt TACCCACTTTCATCCCTAGTTTTTGTTCTGGATTCAAGCATCTCAAAATT GTTTACCTGAAGTTTATCAGTTTTGAGAAAGCGGCGCCCCTGTCGACTAC CATCAGGCATTCGGACTACAACTGTCACAGCACCCTCTGCGTCTGGAGAC GGTTCCGGTGGTAATGATGCTTGCTTCGAAGTGAGACTGGACTCTAGCTC CTATTTAATCAAAACATCAGGGACAACATGACAAATAGTAGTCAAATATC CAGGCAAGAAAAAAAAACCATAAACAATGAAAATACTGATCAAAAGTCCT GTTTGGATCTCCTAAGAAAAATGAGAATGAGATCCAAACAATTGGATTCT AGAATCCAGCTATCTATCCCAAACCCATTATTTGGCGAGATTTTCACTAT GCAGAGGCAATGATCACTATAAGAATAAGATTCAAACACCCACTTATTAT TTTTTTAATCCAGAAACCAGATTCTACATTCACTATAGAATCCAGAACTT CAATATGGGAATGAGATCCAAATAGACCCTAAGCCAAAATGAAATTGGTG AGATGAAGTGGCTAGTTGTCATAACCTCCTGTAAAGAAGACAGCGGTTTA CAGTCCCAACACCCAAATAAACATGACATTAATATAATGACTACAACTCA CAACCTAAACCTAAACCAATATACATCCAAACATAAGACAAAAGGAGAAC TGAGTTTTATATGATCACACTGATGAACTGATGCTGTAGTCTAGCATTCA AGTGTTTAAGATAGTTGACTATAAACCCTTCACCTTGCAGATTACATGTG ACAGAAAGATACCTCTTCCTCAAGTTGTTTTTTACGCCTTTCCTCCTCCT CTTGCTTCTGTTTCTCAAGAACAGCTTCTCTCGCAGCTGTTTCTTCAAGG CGACGGAGCTCAGCCTCCTGTAGGGCCTTTAACTCCTTTTCTTGATCAGC TTGTAGCGATGCAAGGTACTCATCGTCCTACAAATTTAAAATTTATAAAA GTGCTCACCCATAGTGGCAATTATGAACAATGGATAAATCTTAGACCTCA AACCTGCTGCTCTCGTAATAACCGCTGTTCAGTTAATGCTGGTGATGGAG AATGAGATATTGGGGGATAATAAGTAGAGGTTCTGTGAGAAGGCATAGAG AAAGGATATGTTGGTCCACCAAACATTGCAGCCTCAAGCATAACAGCTTC ATCATGTTCCTCAGAAGAAATGCCACCCCACTTTATTGTAAAAAAAGACG TCAGAAATTAAACAAATCCATCTAATGTCTTAGCGCACATTGGAACCACA GATTATAATACCTCAGATGGGAAATCATCTCCATTATACTGGTGATTGTT CAGAACAGGGCTAGCACCTGGGTGCACTATTTTTGGTAATTCATTGTCCT CAGAAGGGGCACGCCGAGAACGACGCCTAACTAACGGCTGTTCTTCTACA TCTTCAGCCTCCTCCTGGAAGCTCTCGTCATCTATTGGTTGCCTAGATGT TCCAGCCTTTCCTGAAGCTAGTCCTTGCCTCCAAAAGGAAATTGCATGTA TAAGGAATCAAATGATACTGTAGTAGGGTAGCCTGAGTGAAGAGGTGGGT AGTAAAGTTAACATTCACCTCTCAACTATTCCATTTGCGGTTTCCATGCC TGCTTTATCAGAAGAATGGTCCCTCAAATTCACTTCCTCTTGATGCTGGC CTCCTTGCTCGACTATCTGATGATTATTGAAAAATTAGAGATGACATCAA GATAGGTTCAAGTAAGCATGTTGGGGA

Example 6. Feed Glucanase Expression in Subsequent Generations

[0203] Several "T1" progeny from original "T0" transgenic maize plants were grown, and individual ears were either self-pollinated or pollinated with pollen from wild-type maize plants. Mature seed from the resulting ears was then assayed for feed glucanase activity via the colorimetric assay. FIG. 12 illustrates that glucanase activity was observed in T1 events. In this figure, the numbers along the abscissa correspond to the event identifiers of the original T0 plants from which the progeny were derived. The highest activity was observed for seeds of T1 plant derived from the 4597_69 event.

Example 7. Feed Glucanase Expression in Multi-Generations of Hemizygous, Homozygous and Hybrid Seeds

[0204] Progeny from original "T0" transgenic maize plants were grown and backcrossed (pollinated with pollen from the wild type maize parents or pollinated onto the wild type parents) for 4 generations (in maize inbred line E (BC4E), or in maize inbred line G (BC4G)). At each generation, some individual ears were self-pollinated. PCR method was applied to select homozygous plants as described in Example 8. Hybrid ears were made by cross-pollinating transgenic line G plants with transgenic line E plants, or vice versa.

[0205] FIG. 13 illustrates that glucanase activity in the hemizygous, homozygous and hybrid ears of event 4588.259. The homozygous and hybrid ears contained average activity of 190 units/g, which was approximately double of ears from hemizygous plants.

Example 8. PCR Assays for Identifying and Determining Zygosity of the Glucanase Events 4588.259, 4588.652, and 4588.757

[0206] Maize glucanase events 4588.259, 4588.652, and 4588.757 carry transgenes that result in seed-specific expression of glucanase enzyme. Event 4588.259 originally carried two T-DNA insertions at independently segregating loci, but subsequently a single genetic locus was selected for propagation and development. Events 4588.652 and 4588.757 carry two or more T-DNAs at a single genetic locus. Molecular identification and tracking of these transgenes can be done using standard PCR analysis (visually scoring an endpoint in a gel-based electrophoresis and staining of the PCR products) or real-time PCR. In addition to determining whether a plant is carrying a transgene, some of these PCR assays can also determine whether a plant is hemizygous (carrying one copy of the insertion) or homozygous (carrying two copies of the insertion).

[0207] FIG. 24 illustrates general real-time PCR assay design used to determine T-DNA locus presence (standard and real-time PCR) and zygosity (real-time PCR only).

[0208] In FIG. 14, the standard and real-time PCR assays include, for each T-DNA locus, one primer (Primer A) that binds to a maize genomic region that is adjacent to where the T-DNA insert is located and one primer (Primer B) that binds to a region in the T-DNA that is close to Primer A. To determine zygosity in the real-time PCR assay, a second reference gene (GWD, glucan water dikinase) is amplified (X and Y primers) along with the locus primers. Real-time PCR amplification of product from Primers A+B would indicate that the T-DNA locus is present and its fluorescence relative to the GWD reference (ref) fluorescence from amplification of product from Primers X+Y would determine whether it is hemizygous (one-copy) or homozygous (two-copy).

[0209] The standard multiplex PCR assay includes Primer A and B, as described above, but also another primer (Primer C) that binds to a maize genomic region on the other side of the T-DNA, opposite Primer A, and would be close to Primer A if the T-DNA insertion was not present, as in a wild type (WT) locus. FIG. 15 illustrates general standard PCR assay design used to determine T-DNA locus presence and zygosity. When the T-DNA insertion is present, the distance between Primer A and Primer C would be too large to amplify a product under our PCR amplification conditions and therefore absence of this amplification product is used to determine zygosity. PCR amplification of products from Primer A+B and Primer A+C indicates that the T-DNA locus is present and is hemizygous (one-copy). PCR amplification of product from Primer A+B, but not Primer A+C, indicates that the T-DNA locus is present and is homozygous (two-copy). PCR amplification of product from Primer A+C only, indicates that no T-DNA is present and the plant is WT at this locus. Primers and probes for all of these assays are listed in Tables 3 and 4.

TABLE-US-00018 TABLE 3 Standard and real-time (RT) PCR primers and probes used to determine T-DNA locus presence and ocus presence and zygosity of 4588.259, 4588.652, and 4588.757 events Primer/ PCR Primer Probe Assay Event or Probe ID (type) Primer Sequence Fluor* Quencher Standard/RT 4588.259 Primer 509 (A) GAATTGTTCATCATAAGGCGTGA (SEQ ID NO: 38) Standard/RT 4588.259 Primer 516 (B) AACGTGACTCCCTTAATTCTCC (SEQ ID NO: 39) RT 4588.259 Probe PB5 AAACTGAAGGCGGGAAACGACAAC HEX BHQ1 (SEQ ID NO: 40) Standard/RT 4588.652 Primer 750 (A) GAGATGCTTGAATCCAGAACAAA (SEQ ID NO: 41) Standard/RT 4588.652 Primer 751 (B) TTGTCTTGGTTGTGATGATGTG (SEQ ID NO: 42) Standard 4588.652 Primer 749 (C) GATTACCATCAAATTTCACACACAC (SEQ ID NO: 43) RT 4588.652 Probe PB17 TAGAACGACCGCCCAACCAGAC HEX BHQ1 (SEQ ID NO: 44) Standard 4588.757 Primer 513 (B) AAACGTCCGCAATGTGTTATT (SEQ ID NO: 45) Standard 4588.757 Primer 608 (C) TCATGCAATTGTGCCACC (SEQ ID NO: 46) Standard 4588.757 Primer 609 (A) ACATAGTCAACCTAACGGCTTAT (SEQ ID NO: 47) RT GWDref Primer 371 (X) GGTTATAAGCCCGGTTGAAGTA (SEQ ID NO: 48) RT GWDref Primer 525 (Y) CTATTCCTTGCTCGGACTGAC (SEQ ID NO: 49) RT GWDref Probe PB2 CACCTGATATGCCAGATGTTCTGTCTCA FAM BHQ1 (SEQ ID NO: 50) *Fluor = fluorophore.

TABLE-US-00019 TABLE 4 4588.259, 4588.652, and 4588.757 event-specific PCR primer combinations and PCR product sizes Primer Primer Primer PCR Product Event A B C (bp) Assay Identifies 4588.259 509 516 137 T-DNA locus: OB-2880 (SEQ ID NO: 51) 4588.652 750 751 107 T-DNA locus: OB-4451 (SEQ ID NO: 52) 4588.652 750 749 174 WT locus (SEQ ID NO: 53) 4588.757 609 513 100 T-DNA locus: OB-3237 (SEQ ID NO: 54) 4588.757 609 608 218 WT locus: B73ref (SEQ ID NO: 55)

[0210] PCR assay using primers X (371) and Y(525) identifies the ZmGWDref locus (SEQ ID NO: 56).

[0211] DNA Extraction

[0212] These PCR assays will work with any DNA extraction method that yields DNA that can be amplified with PCR. A standard DNA extraction method (10X TE+Sarkosyl) that was used in this example is as follows: leaf tissue (standard 1 cm hole punch) is sampled into a 96 deep-well block, metal beads are added, and the block is frozen at -80.degree. C. for at least 30 min. The block is then ground for 45 sec in a Kleco Pulverizer, centrifuged at 4,000 RPM for 3 min, the lid is removed, 300 .mu.l of 10XTE+Sarkosyl is added, the block is resealed, and the block is mixed at room temperature for 10-20 min. After incubation, the block is centrifuged at 4,000 RPM for 5 min, 165 .mu.l of upper aqueous phase is removed and added to a 96-well PCR block, the PCR block is sealed, and the block is incubated at 90.degree. C. for 30 min. After incubation, 20 .mu.l of extract is added to 180 .mu.l of sterile water in a 96-well plate (1:10 dilution) to create the final DNA sample for PCR.

[0213] PCR

[0214] Events 4588.259, 4588.652, and 4588.757 standard and real-time PCR primers are listed in Table 3 and standard PCR primer combinations with expected PCR product sizes are listed in Table 4.

[0215] Standard PCR is performed with 2 .mu.l of DNA extract and GoTaq (Promega) or Kapa 3G (Kapa Biosystems) PCR Mix in 30 .mu.l reaction volumes with the following components and conditions for each event:

[0216] Event 4588.259 Standard PCR:

[0217] Components (final concentration) were as follows: PCR Mix with buffer, MgCl.sub.2, nucleotides, and enzyme (1.times.); primer 509 (400 nM) and primer 516 (400 nM). Conditions were as follows: 95.degree. C., 3 min; 33 cycles (95.degree. C., 30 sec; 55.degree. C., 30 sec; 72.degree. C., 30 sec); 72.degree. C., 8 min.

[0218] Event 4588.652 Standard PCR:

[0219] Components (final concentration) were as follows: PCR Mix with buffer, MgCl.sub.2, nucleotides, and enzyme (1.times.), primer 749 (400 nM), primer 750 (400 nM), and primer 751 (400 nM). Conditions were as follows: 95.degree. C., 3 min; 33 cycles (95.degree. C., 30 sec; 55.degree. C., 30 sec; 72.degree. C., 30 sec); 72.degree. C., 8 min.

[0220] Event 4588.757 Standard PCR:

[0221] Components (final concentration) were as follows: PCR Mix with buffer, MgCl.sub.2, nucleotides, and enzyme (1.times.), primer 513 (400 nM), primer 608 (400 nM), and primer 609 (400 nM). Conditions were as follows: 95.degree. C., 3 min; 33 cycles (95.degree. C., 30 sec; 55.degree. C., 30 sec; 72.degree. C., 30 sec); 72.degree. C., 8 min.

[0222] Standard PCR was analyzed by running approximately 15 .mu.l of PCR product on a 3% agarose gel at 95V for 30 min. An example of results from a standard PCR analysis of the 4588.652 selfed segregating plants is shown in FIG. 16. Referring to this figure, ten PCR reactions from 10 independent plants were separated on a 3% agarose gel stained with ethidium bromide. Expected locus and zygosity band sizes are indicated on the right side of the image.

[0223] Locus presence and zygosity is scored by visualizing specific bands in each lane.

[0224] Real-Time PCR was performed with 2 .mu.l of DNA extract in 20 .mu.l reaction volumes with the following components and conditions for each event:

[0225] Event 4588.259 Real-Time PCR:

[0226] Components (final concentration) were as follow: PCR Mix with buffer, MgCl.sub.2, nucleotides, and enzyme (1.times.), primer 509 (400 nM, primer 516 (400 nM), primer 371 (400 nM), primer 525 (400 nM), probe PB5 (200 nM) and probe PB2 (200 nM). Conditions were as follows: 95.degree. C., 4 min; 40 cycles (95.degree. C., 5 sec; 60.degree. C., 45 sec)

[0227] Event 4588. 652 Real-Time PCR:

[0228] Components (final concentration) were as follows: PCR Mix with buffer, MgCl.sub.2, nucleotides, and enzyme (1.times.), primer 750 (400 nM), primer 751 (400 nM), primer 371 (400 nM), primer 525 (400 nM), probe PB17 (200 nM) and probe PB2 (200 nM). Conditions were as follows: 95.degree. C., 4 min; 40 cycles (95.degree. C., 5 sec; 60.degree. C., 45 sec)

[0229] Event 4588. 259 Real-Time PCR. Real-Time PCR can be analyzed by any real-time PCR machine and software capable of four-channel fluorescence detection. A Bio-Rad CFX96 real-time PCR machine and CFX Manager Software were used to run an example of the 4588.259 real-time PCR assay on a selfed segregating population of 4588.259 plants. FIG. 17 illustrates an example of real-time PCR data for 4588.259 to determine locus presence and zygosity. In this figure, "RFU" refers to relative fluorescence units; "ntc" refers no target control. Presence of the 4588.259 locus and zygosity was scored by the clustering of data points on the graph.

Example 9. Germination Rates Among Seed from Independent Transgenic Plants that Express Feed Glucanase

[0230] Silks on wild-type plants were pollinated with pollen from individual transgenic plants (WT.times.Transgenic), or silks on transgenic plants were pollinated with pollen from wild-type plants (Transgenic.times.WT). Mature, dried seed were collected from the resulting ears and planted into soil. Following 1-2 weeks of incubation, germination rates were calculated. In some cases, this test was repeated following a second generation of growth and pollination (T2). Examples of results from such germination tests are shown in Table 5.

TABLE-US-00020 TABLE 5 Germination rates among seed expressing beta glucanase Germi- WT .times. Transgenic .times. nation Vector Event Transgenic WT Generation Sow % 4588 54 x T1 50 92 4588 17 x T1 50 82 4588 11 x T1 30 80 4588 161 x T1 15 67 4588 162 x T1 11 27 4588 215 x T1 13 62 4588 219 x T1 10 80 4597 18 x T1 30 37 4597 54 x T1 10 100 4597 56 x T1 10 100 4597 69 x T1 14 14 4597 69 x T1 30 73 4588 161 x T1 20 100 4597 101 x T1 33 90 4597 104 x T1 30 70 4588 259 x T2 17 100 4588 251 x T2 17 100 4588 54 x T2 17 47 4597 101 x T2 20 100 4597 104 x T2 20 100

[0231] The germination rate in T1 and/or T2 for events 4597_54, 4597_56, 4588_161, 4588_252, 4588_259, 4597_101, and/or 4597_104 was observed to be 100%.

Example 10. Survival of Feed Glucanase Activity During Preparation of Poultry Feed Pelleting

[0232] Milled grain from transgenic plants expressing the feed glucanase was mixed with starter and grower corn-soy diets that were formulated for broiler chickens. The basal corn-soy diet for starter broilers was composed as follows: 54.89% corn 08-2012, 32.81% soybean oilcake, 5.00% distillers dry grains plus soluble solids, 2.00% vermiculite, 1.99% dicalcium phosphate, 1.00% poultry fat, 0.81% limestone fine, 0.50% plain salt (NaCl), 0.20% DL-methionine, 0.20% choline chloride 60, 0.20% mineral premix, 0.13% L-lysine, 0.12% L-threonine, 0.05% vitamin premix, 0.05% coban, and 0.05% selenium premix. The basal corn-soy diet for grower broilers was composed as follows: 58.53% corn, 26.63% soybean oilcake, 8.00% distillers dry grains plus soluble solids, 2.00% vermiculite, 1.69% dicalcium phosphate, 1.00% poultry fat, 0.76% limestone fine, 0.50% plain salt (NaCl), 0.20% mineral premix, 0.20% choline chloride 60, 0.13% DL-methionine, 0.13% L-lysine, 0.08% L-threonine, 0.05% vitamin premix, 0.05% coban, and 0.05% selenium premix.

[0233] Fine corn was ground using the hammermill screens: no. 4/4 for the starter diet and no. 6/6 for the grower and finisher diets. Coarse corn (5% of total corn) was ground with the roller mill with 0/100 gap openings.

[0234] Four diets were formulated for the pelleting trial as follows: Diet A was a basal diet, Diet D was the basal diet mixed with a control enzyme, Diet E was the basal diet mixed with the milled transgenic corn grain containing a high level of glucanase, and Diet F was the basal diet mixed with the milled transgenic corn grain containing a low level of glucanase.

[0235] Milled grain from transgenic plants expressing the feed glucanase was mixed with the basal diets at a ratio of approximately 1 lb transgenic grain per 2000 lbs basal diet mixture. For the low dose diet, transgenic grain was first mixed with non-transgenic grain at a weight ratio of 1:4 (1 gram of transgenic grain per 4 grams non-transgenic grain) to dilute the enzyme concentration prior to adding this ingredient to the basal diets.

[0236] All feed diets were pelleted at 175-180.degree. F. into 4.4 mm pellets, and the starter diets were crumbled.

[0237] FIGS. 18A and 18B illustrate glucanase activity before and after pelleting in the Grower Diet (FIG. 18A) and the Starter Diet (FIG. 18B). Referring to these figures, samples from the resulting mixture were then removed before and after pelleting of the feed. These samples were then tested via the colorimetric glucanase assay to determine whether the enzyme survived the pelleting process. The identity of the various diets shown in FIGS. 18A and 18B were as follows: A, basal control diet (no external enzyme); D, positive control diet (commercially available enzyme added); F, low-dose diet (including milled grain from plants that express the feed glucanase); E, high-dose diet (including milled grain from plants that express the feed glucanase). It was observed that glucanase activity was high in the high-dose diet for both the Starter Diet and the Grower Diet and survived pelleting.

Example 11. Thermal Stability of Grain-Expressed Feed Glucanase

[0238] Feed glucanase was prepared via microbial expression and purification, and suspended in SEC buffer (100 mM MES, 300 mM NaCl, pH6.3). Five microliters of this preparation was mixed with 20 mg milled grain from wild-type maize. In parallel, 5 .mu.l of SEC buffer was mixed with 20 mg milled grain from transgenic plants that express feed glucanase. Replicates from each of these two sets of samples were incubated at 94.degree. C. or -130.degree. C. for various periods of time, then allowed to cool to room temperature. Subsequently, residual glucanase activity was measured via the colorimetric assay. FIGS. 19 A and 19B illustrate glucanase activity after heat treatment. FIG. 19A illustrates glucanase activity after treatment at 130.degree. C. FIG. 19B illustrates glucanase activity after treatment at 94.degree. C. In these experiments, more activity survived when the feed glucanase was produced in the grain itself than when it was added to the grain exogenously. This finding demonstrates that expression and accumulation of the enzyme in grain effectively provides the enzyme with additional thermal stability relative to the same enzyme that is produced microbially. In this particular instance both the grain-expressed and the microbially-expressed enzymes have the same primary amino acid sequence. Therefore, the enhanced thermal stability that was observed in the flour from transgenic grain is a function of the expression host.

Example 12. Activity of Microbially-Produced AGR2314 at Various pH Values

[0239] AGR2314 activity was measured at several pH values between 3 and 8.5. Each assay (500 .mu.L) contained Britton-Robinson polybuffer (40 mM sodium phosphate, 40 mM sodium borate, and 40 mM sodium acetate), 0.01% (v/v) Tween 20, one Beta-glucazyme substrate tablet (Megazyme, Wicklow, Ireland), and 20 nM of AGR2314 in a 2 mL Eppendorf tube. Samples were incubated for 1 hour at 37.degree. C. or 80.degree. C. Reactions were terminated by the addition of 1 mL of 2% (w/v) tris base. Samples were centrifuged at 15,000.times.g for 10 minutes, and 100 .mu.L of each supernatant (37.degree. C. assays) or 5 .mu.L supernatant plus 100 .mu.L of water (80.degree. C. assays) was transferred to a flat-bottomed 96-well microplate. Absorbances were read at 590 nm. Assays at each pH value were performed in triplicate. Single blank assays (containing no enzyme) were performed at each pH, and these absorbance values were subtracted from the assays containing enzyme.

[0240] FIGS. 20 and 21 illustrate the optimum pH for measuring AGR2314 activity at 37.degree. C. (FIG. 20) and 80.degree. C. (FIG. 21). Referring to FIG. 20, the optimum pH was determined to be 7-7.5 at 37.degree. C. FIG. 21 illustrates that the optimum was determined to be 6 at 80.degree. C.

[0241] FIG. 22 shows an example of pH optimum of the feed glucanase that is produced in transgenic flour.

[0242] To determine the relationship between the pH of the assay conditions and the activity of the enzyme that was derived from transgenic grain, 5 ml of water containing 0.2% Tween-20 was mixed with 200 mg of flour from transgenic seed on a rotating platform for 1 hour at 60.degree. C. Following centrifugation at 1500.times.g for 20 minutes in a clinical centrifuge, the supernatant was transferred to a 15 mL Eppendorf tube. This sample was centrifuged at 1500.times.g for 10 minutes in a tabletop centrifuge. Aliquots of this protein extract were diluted 20-fold in assays to test each pH condition by mixing 50 .mu.l of extract with 950 .mu.l of Britton-Robinson polybuffer (40 mM sodium phosphate, 40 mM sodium borate, and 40 mM sodium acetate) that had been prepared at pH 2-10. The pH of each reaction mixture was checked using a pH strip. Five hundred microliters from each mixture was transferred to a 96 deep-well plate for the assay. One beta-glucazyme tablet was added to each well and mixed by gentle vortexing, the plate was sealed and incubated at 80.degree. C. for 1 hour. The reactions were stopped by adding 1 mL of 2% (w/v) Tris-base to each well. The 96-well plate was centrifuged at 3000.times.g for 10 minutes in a clinical centrifuge, then 100 .mu.L of the supernatant from each of the samples was transferred to wells in a flat-bottom microplate, and the absorbance at 590 nm was determined on a microplate spectrophotometer. As shown in FIG. 22, the seed-produced enzyme has a pH optimum between pH6 and pH7, but still retains a large fraction of its activity at a pH as high as 10.

Example 13. Activity of Microbially-Produced AGR2314 and AGR2414 on Various Substrates

[0243] All reactions used 5 nM of AGR2314, AGR2414, or 5 .mu.L of control enzyme at the indicated concentrations, in 200 mM sodium phosphate, 0.01% (v/v) Tween 20, pH 6.5. Reactions were carried out for one hour at either 37.degree. C. or 80.degree. C. and terminated as described.

[0244] Beta-glucosidase assays: the substrate was 1 mM pNP-D-glucopyranoside (Sigma Chemical Co. catalog # N:7006) and the positive control enzyme was Rhizobium etli beta-glucosidase (Prozomix, catalog No. PRO-E0110; 315.9 Unit/mL). Reaction volumes were 500 .mu.L; reactions were terminated by the addition of 500 L of 2% (w/v) tris base. After centrifugation at 3000.times.g for 10 minutes, 100 .mu.L of supernatant was transferred to a microplate and the absorbance at 405 nm was recorded.

[0245] Endocellulase assays: each assay contained one tablet of Cellazyme C substrate (Megazyme, catalog No, T-CCZ) in 500 .mu.L buffer. Reactions were terminated by the addition of 1 mL of 2% tris base. Samples were centrifuged for 10 minutes at 15,000.times.g, 100 .mu.L of supernatant was transferred to a microplate, and the absorbance at 590 nm was recorded.

[0246] Exocellulase (cellobiohydrolase) assays: the substrate was 1 mM pNP-D-cellobioside (Sigma catalog No. N5759) and the positive control enzyme was CBHI from Trichoderma longibrachiatum (Megazyme catalog No. E-CBHI; 0.5 Units/.mu.L). Reaction conditions were as described above for the beta-glucosidase assays.

[0247] Anylase assays: the substrate was Red Starch (Megazyme catalog No. S-RTAR; prepared as directed by the manufacturer) and the positive control enzyme was .alpha.-amylase from Bacillus licheniformis (Megazyme catalog No. E-BLAAM: 3000 Units/mL). 245 .mu.L buffer, 5 .mu.L of enzyme, and 125 .mu.L of Red Starch reagent were mixed and incubated as described above. Reactions were terminated by the addition of 625 .mu.L ethanol. After incubating at room temperature for 10 minutes, samples were centrifuged for 10 minutes at 3000.times.g, and 100 .mu.L of supernatant was transferred to a microplate; absorbance at 510 nm was recorded.

[0248] Endoxylanase assays: each assay contained one tablet of Xylazyme AX substrate (legazyme catalog No, No. XAX-1000) and the positive control enzyme was 100 mg/mL of Thermomyces lanuginosis xylanase (Sigma catalog #X2753) in assay buffer. Reactions were carried out as described above for the endocellulase assays.

[0249] Pectinase assays: the substrate was 25 mg/mL pectin (Sigma catalog No. P7536) in assay buffer and the positive control enzyme was pectinase from Aspergillus niger (Sigma catalog No. 17389) at 100 mg/mL in assay buffer. Five p L of enzyme was added to 35 PL of pectin solution and incubated as described above. Reactions were terminated by the addition of 60 .mu.L of DNS stop/reagent solution (Wicher et al. [2001], Appl. Microbiol. Biotechnol. 55, p. 578) followed by heating at 950.degree. C. for 15 minutes. Samples were centrifuged at 3000.times.g for 10 minutes, 20 .mu.L supernatant was mixed with 100 .mu.L water in a microplate, and the absorbance at 550 nm was recorded.

[0250] 1,3-beta-glucosidase assays: each assay contained one tablet of 1,3-beta-glucazyme HS substrate (Megazyme catalog No. ET-CUR200) and the positive control enzyme was Trichoderma sp. 1,3-.beta.-D-glucanase (Megazyme catalog No. E-LAMSE; 50 Units/mL). Reactions were carried out as described above for the endocellulase assays.

[0251] 1,4-beta-glucosidase assays: each assay contained one tablet of Beta-glucazyme substrate (Megazyme catalog No. TBGZ-1000T). Reactions were carried out as described above for the endocellulase assays, except that 5 .mu.L of supernatant was mixed with 100 PL of water in a microplate for recording of absorbance.

[0252] FIGS. 23A and 23B illustrate the glucanase activity for hydrolyzing starch, cellobiose (pNP-D-cellobioside), xylan (Xylazyme AX), HE-cellulose (Cellazyme C), barley-B-glucan (Beta-glucazyme), pectin and PNP-D-gluopyranoside at 37.degree. C. (FIG. 23A) and 80.degree. C. (FIG. 23B). Referring to these figures, it was observed that both AGR2314 and AGR2414 enzymes were highly active in hydrolyzing cellobiose and HE-cellulose at 37.degree. C. and 80.degree. C.

Example 14. Glucanase Activity on Seed Fiber

[0253] Glucose release from untreated seed fiber 20 mg seed fiber was digested at pH 5.0 with 5 .mu.M AGR2314 protein for 72 hours at 55 C. A commercial enzyme cocktail was used at full loading (FCT) as a positive control. After enzymatic hydrolysis, the soluble sugars in reaction supernatant were hydrolyzed into monomers via acid hydrolysis at 121.degree. C. FIGS. 24A and 24B illustrate release of monomeric sugars after enzymatic hydrolysis of the seed fiber. FIG. 24A shows glucose yield and FIG. 24B shows xylose yield. Pre-acid hydrolysis (light gray bars) and total (dark gray bars) sugars were separated and quantified via HPLC using a Bio-Rad Aminex HPX-87-P ion-exclusion column. AGR2314 does not release monomeric glucose or xylose from untreated seed fiber. However, this enzyme was able to solubilize untreated seed fiber into oligosaccharides which account for approximately 80% of the total sugars released by a commercially-available cellulase-enzyme cocktail.

[0254] Glucose release from dilute acid-pretreated seed fiber 20 mg seed fiber was pretreated at 80 C in 0.5% H.sub.2SO.sub.4 for 16 hours, then neutralized to pH 5.0. The pretreated seed fiber was digested at pH 5.0 with 2 .mu.M AGR2314 and a suite of glucanase, cellobiohydrolase, endoglucanase, and beta-glucosidase (all at 2 .mu.M loading) for 72 hours at 55.degree. C. A commercial enzyme cocktail (Accellerase XY, Genencor) was used at full loading (FCT) as a positive control. After enzymatic hydrolysis, the soluble sugars in reaction supernatant were hydrolyzed into monomers via acid hydrolysis at 121.degree. C. FIGS. 25A and 25B illustrate release of monomeric sugars after enzymatic hydrolysis of the seed fiber. FIG. 25A shows glucose yield and FIG. 25B shows xylose yield. Pre-acid hydrolysis (light gray bars) and total (dark gray bars) sugars were separated and quantified via HPLC using a Bio-Rad Aminex HPX-87-P ion-exclusion column.

[0255] AGR2314 did not release sugars from pretreated seed fiber at greater levels than the pretreatment itself. When combined with other cell-wall degrading enzymes, approximately 90% of total sugars were released as compared to a commercially-available cellulase-enzyme cocktail.

Example 15. The Use of Glucanase Enzymes on Broiler Live Performance

[0256] The chemical energy contained within an animal's diet, and its availability to the animal eating the diet, are critical characteristics influencing the nutritional value of any diet. Diets rich in energy, provide adequate nutrition and promote rapid growth to higher levels than diets that are deficient in energy. Therefore, determining the energy within a diet, and altering energy availability by using glucanase enzymes, provides an important set of tools to improve animal nutrition and therefore animal performance.

[0257] To demonstrate the use of glucanase enzymes in broiler production, metabolizable energy and nutrient digestibility, male broilers were fed with alternative feed ingredients (wheat, barley and low-fat DDGS) with or without supplemental glucanase. Broiler body weight gain, feed consumption and feed conversion rate were determined and feed glucanase enzyme was evaluated.

[0258] Dietary Treatments and Procedures

[0259] Day-old male broiler were obtained from a commercial hatchery and randomly allocated to 64 battery cages in groups of 10. Experimental diets were fed from 0 to 28 d of ages. Initial group weights were obtained and equalized amongst the treatments. Feed disappearance and body weight were measured weekly (7, 14, 21, and 28 d of age) to calculate live performance parameters (feed consumption, body weight gain, and feed conversion ratio). In addition, excreta were collected twice to determine apparent metabolizable energy (AME) of the diets at 14 and 29 d of age.

[0260] Dietary treatments were fed in a 4.times.2 factorial design and further delineated below. Four different diets (corn/soybean meal based, corn/wheat based, corn/barley based, and corn/LF-DDGS based diets) and two levels of glucanase (with or without) were fed. Diets were formulated to be isocaloric and all nutrients, with the exception of energy, were formulated to meet or exceed the nutrient requirements. The enzyme treatments had the enzyme added on top of the diet. In addition, titanium dioxide was added to the diets as an indigestible marker to determine AME and nutrient digestibility. Table 6 describes dietary treatments.

TABLE-US-00021 TABLE 6 Treatment delineation Trt Diet Enzyme 1 Corn-soybean meal - 2 Corn-soybean meal + 3 Corn-soybean meal-wheat - 4 Corn-soybean meal-wheat + 5 Corn-soybean meal-LF- - DDGS 6 Corn-soybean meal-LF- + DDGS 7 Corn-soybean meal-barley - 8 Corn-soybean meal-barley + Total 640 male broilers Total Males needed: 8 treatments .times. 8 reps .times. 10 birds/cage = 640 male broilers + 60 for equating = 700 male broilers

[0261] Temperature

[0262] Battery cages: 92.degree. F. from placement to 4 d, 90.degree. F. from 5 to 9 d, 84.degree. F. from 10 to 15 d, 80.degree. F. from 16 to 24 d, 78.degree. F. from 25 to 29 d,

[0263] Room Setup

[0264] Prior to bird placement, lighting and temperature in the battery cage rooms were set 48 hours in advance. Wire bottoms were added to the battery cages until day 0 to 4. Unless noted in the schedule, for collection periods, excreta pans were scrapped on a regular basis to avoid any pest and odor issues.

[0265] Lighting Program

[0266] A 23 light:1 dark with a lighting intensity of 3.0 ft.sup.2 was implemented from placement until 7 d of age. A 23 light: 1 dark lighting schedule was implemented from 8-21d with a lighting intensity of 1.0 ft candles. A 23 light: 1 dark lighting schedule was implemented from 22-28 d of age with a lighting intensity of 0.3 ft candles.

[0267] Special Instructions

[0268] Wire bottoms were placed into all battery cages prior to the start ofthe experiment or day-old chicks will fall through the floors. Wire bottoms were removed at d 7. In addition, cage doors were modified to accommodate smaller birds from 0 to 7 d of age.

[0269] Any mortality was not replaced.

[0270] All diets were fed in mash form. Mix sheets were forthcoming.

[0271] To avoid cross contamination of the enzyme, separate feed scoops were needed. Table 7 describes experimental timeline.

TABLE-US-00022 TABLE 7 Timeline of dietary treatments Age of Day birds Experimental Timeline Lighting Temperature Tue -2 23L: 1D 92.degree. F. 3.0 ft.sup.2 Thu 0 Place 720 male broilers in battery cages Equate weights Start experimental diets Tue 5 90.degree. Thu 7 Weigh all birds and feed Remove wire bottoms Fri 8 23L: 1D 1.0 ft.sup.2 Sat 9 Sun 10 84.degree. Mon 11 Clean excreta pans Start 14 d collection period Thu 14 Weigh all birds and feed Collect excreta for 14 d collection period Sat 16 80.degree. Sun 17 Wed 20 80.degree..sup.B-east Thu 21 Weigh all birds and feed Fri 22 23L: 1D 0.3 ft.sup.2 Tues 25 Clean excreta pans 78.degree. Start d 29 collection period Fri 28 Weigh all birds and feed 75.degree. Collect excreta for 29 d collection period END OF EXPERIMENT

[0272] FIG. 26 illustrates the body weight gain (BWG) during the 28-day poultry feeding trial. Referring to FIG. 26, four different diets, corn/soybean meal based, corn/barley based, corn/wheat based, and corn/LF-DDGS based, with (+) or without (-) a glucanase were tested. Still referring to FIG. 26, average of BWG with or without glucanase across the four diets was shown. It was observed, that body wait gain was on average higher in chickens fed with the diets that included a glucanase.

[0273] FIG. 27 illustrates the changes in poultry BWG per time interval during 28 day feeding trial. Referring to FIG. 27, initial group weights were obtained and equalized amongst the treatments. BWG was measured weekly (7, 14, 21, and 28 d of age) for broilers feed using the corn/LF-DDGS diet with (+) or without (-) a glucanase. Still referring to FIG. 27, it was observed across all treatments that body wait gain in chickens fed with the glucanase including diets was higher than in chickens fed with diets without glucanase.

[0274] FIG. 28 illustrates feed consumption during the 28-day poultry feeding trial using two different diets (corn/barley based and corn/LF-DDGS based) with (+) or without (-) a glucanase. Referring to FIG. 28, it was found that feed consumption was higher for the diets that included a glucanase.

[0275] FIG. 29 illustrates the feed conversion rate (FCR) during the 28-day poultry feeding trial with two different diets (corn/barley based and corn/LF-DDGS based diets) with (+) or without (-) a glucanase. The feed conversion rate refers to the feed consumption required for gaining the body weight for a tested animal. The FCR is calculated by dividing the value of feed consumption by the value of body weight gain. Referring to FIG. 29, it was observed that the FCR was lower for the diets that included a glucanase. These data indicates that diets that included a glucanase facilitated digestion and feed consumption in the tested animals.

Example 16. The Use of Glucanase Enzymes for Broiler Live Performance

[0276] To demonstrate the effect of glucanase enzymes in broiler production, 936 male broilers were feed with varies glucanase concentrations in a 17 day battery trial. Birds were weighed at day 17. Feed composition includes corn, soybean meal and fat (soybean oil). Table 8 describes experimental details of the 17 day battery trial.

TABLE-US-00023 TABLE 8 Experimental Treatments: (diets were fed through day 17)--total of 9 TRTs Trt Code Description Dose 1 Positive Control (PC) -- 2 Negative Control -- (NC, less 50-60 kcal/lb of PC) 3 NC + Industry Std Enzyme_1* 0.25 lb/ton 4 NC + Industry Std Enzyme_2** 0.2 lb/ton 5 NC + Glucanase 5 6 NC + Glucanase 50 7 NC + Glucanase 100 8 NC + Glucanase 250 9 NC + Glucanase 500 *Industry Std Enzyme_l refers to ENSPIRAT .TM. (JBS United) **Industry Std Enzyme_2 refers to HOSTAZYM X .RTM. (Huvepharma) No. of treatments 9 Broilers per replicate 8 Replicates per treatment 13 Broilers per treatment 104 Total No. of replicates 117 Total No. of broilers 936

[0277] Referring to FIG. 30, the inclusion of beta-glucanase in treatments 7 (Trt_7) and 8 (Trt_8) significantly (p-value <0.05) increased body weight gain compared to the positive control (PC), negative control (NC), and treatments 3 (Trt_3) and 4 (Trt_4), which contained a commercial NSPase inclusion. The glucanase inclusion in treatments 5 (Trt_5), 6 (Trt_6), and 9 (Trt_9) produced intermediate results.

REFERENCES

[0278] Leeson, S. and L. Caston. 2000. Commercial enzyme and their influence on broilers fed wheat or barley. J. Appl. Poult. Res. 9:242-251. [0279] Yu, B. and T. K. Chung. 2004. Effects of multiple-enzyme mixtures on growth performance of broilers fed corn-soybean meal diets. J. Appl. Poult. Res. 13:178-182.

[0280] The references cited throughout this application, are incorporated for all purposes apparent herein and in the references themselves as if each reference was fully set forth. For the sake of presentation, specific ones of these references are cited at particular locations herein. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0281] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and/or shown in the attached drawings.

Sequence CWU 1

1

561966DNAArtificial SequenceSynthetic construct, AGR2314 coding sequence 1ggcgtggacc cgttcgagag gaacaagatc ctgggcaggg gcatcaacat cggcaacgcc 60ctggaggccc cgaacgaggg cgactggggc gtggtgatca aggacgagtt cttcgacatc 120atcaaggagg ccggcttcag ccacgtgaga atcccgatca ggtggagcac ccacgcccag 180gccttcccgc cgtacaagat cgagccgagc ttcttcaaga gggtggacga ggtgatcaac 240ggcgccctga agaggggcct ggccgtggtg atcaacatcc accactacga ggagctgatg 300aacgacccgg aggagcacaa ggagaggttc ctggccctgt ggaagcagat cgccgacagg 360tacaaggact acccggagac cctgttcttc gagatcctga acgagccgca cggcaacctg 420accccggaga agtggaacga gctgctggag gaggccctga aggtgatcag gagcatcgac 480aagaagcaca ccgtgatcat cggcaccgcc gagtggggcg gcatcagcgc cctggagaag 540ctgagggtgc cgaagtggga gaagaacgcc atcgtgacca tccactacta caacccgttc 600gagttcaccc accagggcgc cgagtgggtg ccgggcagcg agaagtggct gggcaggaag 660tggggcagcc cggacgacca gaagcacctg atcgaggagt tcaacttcat cgaggagtgg 720agcaagaaga acaagaggcc gatctacatc ggcgagttcg gcgcctacag gaaggccgac 780ctggagagca ggatcaagtg gaccagcttc gtggtgaggg aggccgagaa gaggggctgg 840agctgggcct actgggagtt ctgcagcggc ttcggcgtgt acgacccgct gaggaagcag 900tggaacaagg acctgctgga ggccctgatc ggcggcgaca gcatcgagag cgagaaggac 960gagctg 9662948DNAArtificial SequenceSynthetic construct, AGR2414 coding sequence 2ggcgtggacc cgttcgagag gaacaagatc ctgggcaggg gcatcaacat cggcaacgcc 60ctggaggccc cgaacgaggg cgactggggc gtggtgatca aggacgagtt cttcgacatc 120atcaaggagg ccggcttcag ccacgtgaga atcccgatca ggtggagcac ccacgcctac 180gccttcccgc cgtacaagat catggacagg ttcttcaaga gggtggacga ggtgatcaac 240ggcgccctga agaggggcct ggccgtggtg atcaacatcc accactacga ggagctgatg 300aacgacccgg aggagcacaa ggagaggttc ctggccctgt ggaagcagat cgccgacagg 360tacaaggact acccggagac cctgttcttc gagatcctga acgagccgca cggcaacctg 420accccggaga agtggaacga gctgctggag gaggccctga aggtgatcag gagcatcgac 480aagaagcaca ccatcatcat cggcaccgcc gagtggggcg gcatcagcgc cctggagaag 540ctgagcgtgc cgaagtggga gaagaactcc atcgtgacca tccactacta caacccgttc 600gagttcaccc accagggcgc cgagtgggtg gagggcagcg agaagtggct gggcaggaag 660tggggcagcc cggacgacca gaagcacctg atcgaggagt tcaacttcat cgaggagtgg 720agcaagaaga acaagaggcc gatctacatc ggcgagttcg gcgcctacag gaaggccgac 780ctggagagca ggatcaagtg gaccagcttc gtggtgaggg agatggagaa gaggcgctgg 840agctgggcct actgggagtt ctgcagcggc ttcggcgtgt acgacaccct gaggaagacc 900tggaacaagg acctgctgga ggccctgatc ggcggcgaca gcatcgag 9483951DNAArtificial SequenceSynthetic construct, AGR2514 coding sequence 3agcggcgtgg acccgttcga gaggaacaag atcctgggca ggggcatcaa catcggcaac 60gccctggagg ccccgaacga gggcgactgg ggcgtggtga tcaaggacga gtacttcgac 120atcatcaagg aggccggctt cagccacgtg agaatcccga tcaggtggag cacccacgcc 180caggccttcc cgccgtacaa gatcgaggac aggttcttca agagggtgga cgaggtgatc 240aacggcgccc tgaagagggg cctggccgtg gtgatcaacc agcaccacta cgaggagctg 300atgaacgacc cggaggagca caaggagagg ttcctggccc tgtggaagca gatcgccgac 360aggtacaagg actacccgga gaccctgttc ttcgagatcc tgaacgagcc gcacggcaac 420ctgaccccgg agaagtggaa cgagctgctg gaggaggccc tgaaggtgat caggagcatc 480gacaagaagc acaccatcat catcggcacc gccgagtggg gcggcatcag cgccctggag 540aagctgaggg tgccgaagtg ggagaagaac gccatcgtga ccatccacta ctacaacccg 600ttcgagttca cccaccaggg cgccgagtgg gtggagggca gcgagaagtg gctgggcagg 660aagtggggca gcccggacga ccagaagcac ctgatcgagg agttcaactt catcgaggag 720tggagcaaga agaacaagag gccgatctac atcggcgagt tcggcgccta caggaaggcc 780gacctggaga gcaggatcaa gtggaccagc ttcgtggtga gggaggccga gaagaggagg 840tggagctggg cctactggga gttctgcagc ggcttcggcg tgtacgacac cctgaggaag 900acctggaaca aggacctgct ggaggccctg atcggcggcg acagcatcga g 9514322PRTArtificial SequenceSyntehtic construct, AGR2314 protein 4Gly Val Asp Pro Phe Glu Arg Asn Lys Ile Leu Gly Arg Gly Ile Asn1 5 10 15Ile Gly Asn Ala Leu Glu Ala Pro Asn Glu Gly Asp Trp Gly Val Val 20 25 30Ile Lys Asp Glu Phe Phe Asp Ile Ile Lys Glu Ala Gly Phe Ser His 35 40 45Val Arg Ile Pro Ile Arg Trp Ser Thr His Ala Gln Ala Phe Pro Pro 50 55 60Tyr Lys Ile Glu Pro Ser Phe Phe Lys Arg Val Asp Glu Val Ile Asn65 70 75 80Gly Ala Leu Lys Arg Gly Leu Ala Val Val Ile Asn Ile His His Tyr 85 90 95Glu Glu Leu Met Asn Asp Pro Glu Glu His Lys Glu Arg Phe Leu Ala 100 105 110Leu Trp Lys Gln Ile Ala Asp Arg Tyr Lys Asp Tyr Pro Glu Thr Leu 115 120 125Phe Phe Glu Ile Leu Asn Glu Pro His Gly Asn Leu Thr Pro Glu Lys 130 135 140Trp Asn Glu Leu Leu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile Asp145 150 155 160Lys Lys His Thr Val Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile Ser 165 170 175Ala Leu Glu Lys Leu Arg Val Pro Lys Trp Glu Lys Asn Ala Ile Val 180 185 190Thr Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala Glu 195 200 205Trp Val Pro Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser Pro 210 215 220Asp Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu Trp225 230 235 240Ser Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe Gly Ala Tyr 245 250 255Arg Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr Ser Phe Val Val 260 265 270Arg Glu Ala Glu Lys Arg Gly Trp Ser Trp Ala Tyr Trp Glu Phe Cys 275 280 285Ser Gly Phe Gly Val Tyr Asp Pro Leu Arg Lys Gln Trp Asn Lys Asp 290 295 300Leu Leu Glu Ala Leu Ile Gly Gly Asp Ser Ile Glu Ser Glu Lys Asp305 310 315 320Glu Leu5316PRTArtificial SequenceSynthetic construct, AGR2414 protein 5Gly Val Asp Pro Phe Glu Arg Asn Lys Ile Leu Gly Arg Gly Ile Asn1 5 10 15Ile Gly Asn Ala Leu Glu Ala Pro Asn Glu Gly Asp Trp Gly Val Val 20 25 30Ile Lys Asp Glu Phe Phe Asp Ile Ile Lys Glu Ala Gly Phe Ser His 35 40 45Val Arg Ile Pro Ile Arg Trp Ser Thr His Ala Tyr Ala Phe Pro Pro 50 55 60Tyr Lys Ile Met Asp Arg Phe Phe Lys Arg Val Asp Glu Val Ile Asn65 70 75 80Gly Ala Leu Lys Arg Gly Leu Ala Val Val Ile Asn Ile His His Tyr 85 90 95Glu Glu Leu Met Asn Asp Pro Glu Glu His Lys Glu Arg Phe Leu Ala 100 105 110Leu Trp Lys Gln Ile Ala Asp Arg Tyr Lys Asp Tyr Pro Glu Thr Leu 115 120 125Phe Phe Glu Ile Leu Asn Glu Pro His Gly Asn Leu Thr Pro Glu Lys 130 135 140Trp Asn Glu Leu Leu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile Asp145 150 155 160Lys Lys His Thr Ile Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile Ser 165 170 175Ala Leu Glu Lys Leu Ser Val Pro Lys Trp Glu Lys Asn Ser Ile Val 180 185 190Thr Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala Glu 195 200 205Trp Val Glu Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser Pro 210 215 220Asp Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu Trp225 230 235 240Ser Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe Gly Ala Tyr 245 250 255Arg Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr Ser Phe Val Val 260 265 270Arg Glu Met Glu Lys Arg Arg Trp Ser Trp Ala Tyr Trp Glu Phe Cys 275 280 285Ser Gly Phe Gly Val Tyr Asp Thr Leu Arg Lys Thr Trp Asn Lys Asp 290 295 300Leu Leu Glu Ala Leu Ile Gly Gly Asp Ser Ile Glu305 310 3156317PRTArtificial SequenceSynthetic construct, AGR2514 protein 6Ser Gly Val Asp Pro Phe Glu Arg Asn Lys Ile Leu Gly Arg Gly Ile1 5 10 15Asn Ile Gly Asn Ala Leu Glu Ala Pro Asn Glu Gly Asp Trp Gly Val 20 25 30Val Ile Lys Asp Glu Tyr Phe Asp Ile Ile Lys Glu Ala Gly Phe Ser 35 40 45His Val Arg Ile Pro Ile Arg Trp Ser Thr His Ala Gln Ala Phe Pro 50 55 60Pro Tyr Lys Ile Glu Asp Arg Phe Phe Lys Arg Val Asp Glu Val Ile65 70 75 80Asn Gly Ala Leu Lys Arg Gly Leu Ala Val Val Ile Asn Gln His His 85 90 95Tyr Glu Glu Leu Met Asn Asp Pro Glu Glu His Lys Glu Arg Phe Leu 100 105 110Ala Leu Trp Lys Gln Ile Ala Asp Arg Tyr Lys Asp Tyr Pro Glu Thr 115 120 125Leu Phe Phe Glu Ile Leu Asn Glu Pro His Gly Asn Leu Thr Pro Glu 130 135 140Lys Trp Asn Glu Leu Leu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile145 150 155 160Asp Lys Lys His Thr Ile Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile 165 170 175Ser Ala Leu Glu Lys Leu Arg Val Pro Lys Trp Glu Lys Asn Ala Ile 180 185 190Val Thr Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala 195 200 205Glu Trp Val Glu Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser 210 215 220Pro Asp Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu225 230 235 240Trp Ser Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe Gly Ala 245 250 255Tyr Arg Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr Ser Phe Val 260 265 270Val Arg Glu Ala Glu Lys Arg Arg Trp Ser Trp Ala Tyr Trp Glu Phe 275 280 285Cys Ser Gly Phe Gly Val Tyr Asp Thr Leu Arg Lys Thr Trp Asn Lys 290 295 300Asp Leu Leu Glu Ala Leu Ile Gly Gly Asp Ser Ile Glu305 310 31573004DNAArtificial SequenceSyntehtic construct, pAG4258 7cggtatgaat ttggaaacaa attcagtact tttaaaaaaa tttgttgtag ggagcaaata 60atacataaaa taatttatgc attattttat tttttatttg taataatatg cttgaaacga 120taattcagta tgcatgttgt gccagtgtac tacacgggcg gggggagggg attgagtggg 180ccagcgcggt gcgtagggta gatgggctga aattgataac tcaagtccga ctaggttctc 240tttttatttc ccttcctttt ctattttcct ttcttttaat tttcatgctt tcaaactaaa 300ttcaaattcg agttttgaat ttcagcttct aaattgtaca ctaaaattat atgataaggt 360aacccctact attactttta atttttttat tctaccccat attgtttact taggggagaa 420taattgactt aatcacattc ttccttaggt ttcaattctc aatctttcaa atccacattt 480ttagatttct attttgaatt taaataccag tttggattta gagttcaatt tcaaaataca 540caaccaaaat accagcatga atgcaaatat attttatgtt tatgtattta cttttctttt 600atactttgct caaaatagtt attttcatgt atgaaactca ataagcaagg aactcacgtt 660attatataac ctaataggaa taatttaggt aacataattt atcatcctct tgatttaaaa 720gagatatgcc tccagaataa gacacatact aaaaataact ctaatattga ataactaaag 780tcgtacaaat ctctactatt attcctataa aataataaag aactagctac aacttcttta 840aggcattatt cagggtttac agcttgagag gcatgaaccc atcctgtata ctcctggact 900tggaagacaa aatgtcaacc aaagtgaaag gttttcttat ggttgctgct aagagataga 960ttgaacacta gatctctcct aagacgtcag ggcatgcgtt tagactccta cacatgcgaa 1020aactgcatct tacagttgga agaaactata tctcaccact tcctgcggtg taactttgcc 1080caaagatgtt ggctcactgt tggaatcact ccgccccgaa ctttggatct aacgcttgca 1140gtgctacata ttagagcaag actaacaatg ccgtggagaa tggaaggtat tataaccatg 1200tcatggtgca tatggaaatg tcgaaataac tggatattcg aaaacatacc gccaacggtg 1260gcggcctgca aggaaatgtt caagactgaa atgaactaca tctgctacca agttaagctc 1320gagacaggag ctaaaagtag aaactggata caacactttg taacatagtg acactcccct 1380tttcctttct tttaccttag aactatacat acaatccaca ttcaataaaa atttgtaggt 1440acgccataca cactaccgga atccggctct ttgccgagtg tgaggcgctt tgtcgagtgc 1500tttttgtcca gcactcggca aaaaagtctt tgccatgtgc cgcactcggc aaagtcctgc 1560tctcggtaac gaccgcgttt accgagagca ggactctcga cacagaaata cactcgacaa 1620agaaatcttt gccgagagcc aaacactcgg cgaacggcag cgctcggcaa agggtcgtca 1680gccgccgtct aaagctgacg gtcgttatct ttgtcgagtg ccccctcgtc cgacactcag 1740tagagcaagc ttgccgagtg ccatccttgg acactcgata aagtatattt tatttttttt 1800tattttgcca accaaacttt ttgtggtatg ttcctacact atgtagatct acatgtacca 1860ttttggcaca attacaaaaa tgttttctat aactattaga tttagttcgt ttatttgaat 1920ttcttcggaa aattcacata tgaactgcaa gtcactcgaa acatgaaaaa ccgtgcatgc 1980aaaataaatg atatgcatgt tatctagcac aagttacgac cgaattcaga agcagaccag 2040aatcttcaag caccatgctc actaaacatg accgtgaact tgttatccag ttgtttaaaa 2100attgtataaa acacaaataa agtcagaaat taatgaaact tgtccacatg tcatgatatc 2160atatatagag gttgtgataa aaatttgata atgtttcggt aaagttgtga cgtactatgt 2220gtagaaacct aagtgaccta cacataaaat catagagttt caatgtagtt cactcgacaa 2280agactttgtc aagtgtccga taaaaagtat tcagcaaaga agccgttgtc gatttactgt 2340tcgtcgagat ctctttgccg agtgtcacac taggcaaagt ctttacggag tgtttttcag 2400gctttgacac tcggcaaagc gctcgattcc agtagtgaca gtaatttgca tcaaaaatag 2460ccgagagatt taaaatgagt caactaatag accaactaat tattagctat tagtcgttag 2520cttctttaat ctaagctaaa accaactaat agcttatttg ttgaattaca attagctcaa 2580cggaattctc tgttttttct ataaaaaaaa gggaaactgc ccctcattta cagcaaactg 2640tccgctgcct gtcgtccaga tacaatgaac gtacctagta ggaactcttt tacacgctcg 2700gtcgctcgcc gcggatcgga gtcccaggaa cacgacacca ctgtggaaca cgacaaagtc 2760tgctcagagg cggccacacc ctggcgtgca ccgagccgga gcccggataa gcacggtaag 2820gagagtacgg cgggacgtgg cgacccgtgt gtctgctgcc acgcagcctt cctccacgta 2880gccgcgcggc cgcgccacgt accagggccc ggcgctggta taaatgcgcg ccacctccgc 2940tttagttctg catacagcca acccaacaca cacccgagca tatcacagtg acagacacta 3000cacg 300483292DNAArtificial SequenceSynthetic construct, pAG4588 8tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt ttttggcctg 60catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg agactctgtg 120ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa tcttgcctgt 180gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat atttggaaac 240atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt cgtattggat 300gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt gcttctgggt 360caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc cctgtttacg 420ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat gccatggaaa 480gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc catttgtgtt 540gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag attatataac 600ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat atagtgaatt 660tttaagagca tgttttccta aagaagtata tattttctat gtacaaaggc cattgaagta 720attgtagata caggataatg tagacttttt ggacttacac tgctaccttt aagtaacaat 780catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct tgatttccat 840gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat gcataggaaa 900gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac ttaattaatc 960atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac cagcactcaa 1020gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac ctcttgtaaa 1080gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac aactgacaag 1140gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt agttagcagt 1200agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc aacccaaata 1260aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca aaactaaaag 1320taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg tgtgggcatt 1380atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat gagcctcaaa 1440agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca ttctccactg 1500acataatgca aaataagata tcatcgatga catagcaact catgcatcat atcatgcctc 1560tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg ttagaacata 1620aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca cagactcaag 1680caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat attgcaaaaa 1740gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt cgtgtcaaaa 1800agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa gaacaacaca 1860atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct ttcgtgtacc 1920acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta tcttaacatt 1980tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg tttctcacaa 2040aaagcattca gttcattagt cctacaacaa cggatccacc atgagggtgt tgctcgttgc 2100cctcgctctc ctggctctcg ctgcgagcgc caccagcggc gtggacccgt tcgagaggaa 2160caagatcctg ggcaggggca tcaacatcgg caacgccctg gaggccccga acgagggcga 2220ctggggcgtg gtgatcaagg acgagttctt cgacatcatc aaggaggccg gcttcagcca 2280cgtgagaatc ccgatcaggt ggagcaccca cgcccaggcc ttcccgccgt acaagatcga 2340gccgagcttc ttcaagaggg tggacgaggt gatcaacggc gccctgaaga ggggcctggc 2400cgtggtgatc aacatccacc actacgagga gctgatgaac gacccggagg agcacaagga 2460gaggttcctg gccctgtgga agcagatcgc cgacaggtac aaggactacc cggagaccct 2520gttcttcgag atcctgaacg agccgcacgg caacctgacc ccggagaagt ggaacgagct 2580gctggaggag gccctgaagg tgatcaggag catcgacaag aagcacaccg tgatcatcgg 2640caccgccgag tggggcggca tcagcgccct ggagaagctg agggtgccga agtgggagaa 2700gaacgccatc gtgaccatcc actactacaa cccgttcgag ttcacccacc agggcgccga 2760gtgggtgccg ggcagcgaga agtggctggg caggaagtgg ggcagcccgg acgaccagaa 2820gcacctgatc

gaggagttca acttcatcga ggagtggagc aagaagaaca agaggccgat 2880ctacatcggc gagttcggcg cctacaggaa ggccgacctg gagagcagga tcaagtggac 2940cagcttcgtg gtgagggagg ccgagaagag gggctggagc tgggcctact gggagttctg 3000cagcggcttc ggcgtgtacg acccgctgag gaagcagtgg aacaaggacc tgctggaggc 3060cctgatcggc ggcgacagca tcgagagcga gaaggacgag ctgtgaccta ggctgcacaa 3120agtggagtag tcagtcatcg atcaggaacc agacaccaga cttttattca tacagtgaag 3180tgaagtgaag tgcagtgcag tgagttgctg gtttttgtac aacttagtat gtatttgtat 3240ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc ag 329292584DNAArtificial SequenceSynthetic construct, pAG4597 9aaagtaatca tattatttta tgtgtgaatc ttctttactt tttcatttga ttatgattat 60gaaggtatga ccttcataac cttcgtccga aatccattat atccaaagga aaataatgct 120tcgaaggacg aaggattttg atatttaaca ttttatgttg ccttgttctt aattcatagc 180atttgagaac aagtccccaa caccaatctt tatctttact atattaaagc accagttcaa 240cgatcgtctc gtgtcaatat tattaaaaaa ctcctacatt tctttataat caacccgcac 300tcttataatc tcttctctta ctactataat aagagagttt atgtacaaaa taaggtgaaa 360ttatgtataa gtgttctgga ccttggttgt tggctcatat tcacacaacc taatcaatag 420aaaacatatg ttttattaaa acaaaattta tcatatatat atatatatat atatatatat 480atatatatat atataatata aaccgtagca atgcacaggc atatgactag tggcaactta 540ataccatgtg tgtattaaga tgaataagag gtatccaaat aaataacttg ttcgcttacg 600tctggatcga aaggggttgg aaacgattaa atctcttcct agtcaaaatt aaatagaagg 660agatttaatc gatttctccc aatccccttc gatccaggtg caaccgaata agtccttaaa 720tgttgaggaa cacgaaacaa ccatgcattg gcatgtaaag ctccaagaat tcgttgtatc 780cttaacaact cacagaacat caaccaaaat tgcacgtcaa gggtattggg taagaaacaa 840tcaaacaaat cctctctgtg tgcaaagaaa cacggtgagt catgccgaga tcatactcat 900ctgatataca tgcttacagc tcacaagaca ttacaaacaa ctcatattgc attacaaaga 960tcgtttcatg aaaaataaaa taggccggaa caggacaaaa atccttgacg tgtaaagtaa 1020atttacaaca aaaaaaaagc catatgtcaa gctaaatcta attcgtttta cgtagatcaa 1080caacctgtag aaggcaacaa aactgagcca cgcagaagta cagaatgatt ccagatgaac 1140catcgacgtg ctacgtaaag agagtgacga gtcatataca tttggcaaga aaccatgaag 1200ctgcctacag ccgtctcggt ggcataagaa cacaagaaat tgtgttaatt aatcaaagct 1260ataaataacg ctcgcatgcc tgtgcacttc tccatcacca ccactgggtc ttcagaccat 1320tagctttatc tactccagag cgcagaagaa cccgatcgac accggatcca ccatgagggt 1380gttgctcgtt gccctcgctc tcctggctct cgctgcgagc gccaccagcg gcgtggaccc 1440gttcgagagg aacaagatcc tgggcagggg catcaacatc ggcaacgccc tggaggcccc 1500gaacgagggc gactggggcg tggtgatcaa ggacgagttc ttcgacatca tcaaggaggc 1560cggcttcagc cacgtgagaa tcccgatcag gtggagcacc cacgcccagg ccttcccgcc 1620gtacaagatc gagccgagct tcttcaagag ggtggacgag gtgatcaacg gcgccctgaa 1680gaggggcctg gccgtggtga tcaacatcca ccactacgag gagctgatga acgacccgga 1740ggagcacaag gagaggttcc tggccctgtg gaagcagatc gccgacaggt acaaggacta 1800cccggagacc ctgttcttcg agatcctgaa cgagccgcac ggcaacctga ccccggagaa 1860gtggaacgag ctgctggagg aggccctgaa ggtgatcagg agcatcgaca agaagcacac 1920cgtgatcatc ggcaccgccg agtggggcgg catcagcgcc ctggagaagc tgagggtgcc 1980gaagtgggag aagaacgcca tcgtgaccat ccactactac aacccgttcg agttcaccca 2040ccagggcgcc gagtgggtgc cgggcagcga gaagtggctg ggcaggaagt ggggcagccc 2100ggacgaccag aagcacctga tcgaggagtt caacttcatc gaggagtgga gcaagaagaa 2160caagaggccg atctacatcg gcgagttcgg cgcctacagg aaggccgacc tggagagcag 2220gatcaagtgg accagcttcg tggtgaggga ggccgagaag aggggctgga gctgggccta 2280ctgggagttc tgcagcggct tcggcgtgta cgacccgctg aggaagcagt ggaacaagga 2340cctgctggag gccctgatcg gcggcgacag catcgagagc gagaaggacg agctgtgacc 2400taggctgcac aaagtggagt agtcagtcat cgatcaggaa ccagacacca gacttttatt 2460catacagtga agtgaagtga agtgcagtgc agtgagttgc tggtttttgt acaacttagt 2520atgtatttgt atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat 2580ccag 2584103317DNAArtificial SequenceSynthetic construct, pAG4708 10ataaaatttt agtgaagcta aagcggtgaa agattataga atttgatgtg ccagattaat 60aaatcgatta actcctaaag ttcaagccga gactacagac acatgagcta cataaatgag 120ccaaggactc gagcaaagac aaatcgacac agacattata attcaagtca ttctagaaga 180ttcatgagaa gagtatcatt tatttaaatc aatgacttga tcaaataaga cctaggagct 240actattgata atatatatca tgggtatcta gatcaagcat tatgaagaag agcctaagta 300gaaggcccca tgggctcgac cacaaaccca aggactcgac aataaagtct aggagggatc 360ccatagctaa aaggactcta gaagtgtatg tatggtaaag attttatcga gacaagaaat 420acgataaaga tcttaacaga atcggagtca tacttgtaaa aatagagttg gactcgtgta 480caacttggtc ttcgacttag ttcggtcatg aattcagtaa ccgactagat atgtaccatg 540gaacccctag ggcatgaggc tatgagccat aggatcatca gatccaaaca tacaccaaca 600aatccatcac acaccgaaga tccatattaa caagggatta gctactttac aatttcagag 660taacaaatag agccaaactc atagcacagg ggaacttcat atcacaaatg gaggcattga 720attgatataa aaagctaaag ttctaaaaag tttgaagtgc tgaaacttca aagccgctaa 780ctagtgaagc accgaagcct tccggggaga gaagacatac acgacacgtt agggacgtaa 840aatgacgaaa ttatacaact acctctatat gtaacactta tgtaatagaa aagacagaat 900ccatatgaag atgtataatg gatcaaccat ataaatagat aaacaatata tctgctatgg 960ggattggcat tcttgtatcc ctacgcctgt atatcccctg tttagagaac ctccgaaggt 1020atatgatgct gaagattatt gttgtcttgt ctttcatcat atatcgagtc tttccctagg 1080atattattat tcgcaatgtg cattacatgg ttaatcgatt gagagaacat gcatctcacc 1140tttagctgat aaacgataat ccatgtttta cacttcgtag ctactcatga gtttcgatat 1200acaaatttgt tttctggact acgtaccatt ccatcctctt aggagaggag aggaagtgtc 1260ctcgatttaa ttatgttgtc attttgtagt tcttcacaaa atctcaacag gtaccaaaca 1320cattgtttcc acaagacata ttttagtcac aacaaatcta tattattatt aatcactaaa 1380actatactga ggctcagatg cttttactag ctcttgctag tatgtgatgt aggtctcttt 1440cgacatcatt ccatcaaaat catatgatta gcccatacca aacatttcta taccattcag 1500agaccagaat agtcttttct aatagaaaaa aggaaaatag agtgggccga cgacgacaca 1560aattactgcg tggaccagaa aatagtgaga cacggaagac aaaagaagta aaagaggcaa 1620ggactacggc ccacatgaga ttcggccccg ccacctccgg caaccagcgg ccgatccaac 1680ggcagtgcat cctcaacggc gcgcgcgcgc gcgcgcgcgc acaacctcgt atatatcgcc 1740gcgcggaagc ggcgcgaccg aggaagcctt gtcctcgaca ccccctacac aggtgtcgcg 1800ctgcccccga cacgagtccc gcatgcgtcc cacgcggccg cgccagatcc cgcctccgcg 1860cgttgccacg ccctctataa acacccagct ctctccctcg ccctcatcta tcgcactcgt 1920agtcgtagct caagcatcag cggcaggagc tctgggcagc gtgcgcacgt ggggtaccta 1980gctcgctctg ctagcctacc ggatccacca tgagggtgtt gctcgttgcc ctcgctctcc 2040tggctctcgc tgcgagcgcc accagcggcg tggacccgtt cgagaggaac aagatcctgg 2100gcaggggcat caacatcggc aacgccctgg aggccccgaa cgagggcgac tggggcgtgg 2160tgatcaagga cgagttcttc gacatcatca aggaggccgg cttcagccac gtgagaatcc 2220cgatcaggtg gagcacccac gcccaggcct tcccgccgta caagatcgag ccgagcttct 2280tcaagagggt ggacgaggtg atcaacggcg ccctgaagag gggcctggcc gtggtgatca 2340acatccacca ctacgaggag ctgatgaacg acccggagga gcacaaggag aggttcctgg 2400ccctgtggaa gcagatcgcc gacaggtaca aggactaccc ggagaccctg ttcttcgaga 2460tcctgaacga gccgcacggc aacctgaccc cggagaagtg gaacgagctg ctggaggagg 2520ccctgaaggt gatcaggagc atcgacaaga agcacaccgt gatcatcggc accgccgagt 2580ggggcggcat cagcgccctg gagaagctga gggtgccgaa gtgggagaag aacgccatcg 2640tgaccatcca ctactacaac ccgttcgagt tcacccacca gggcgccgag tgggtgccgg 2700gcagcgagaa gtggctgggc aggaagtggg gcagcccgga cgaccagaag cacctgatcg 2760aggagttcaa cttcatcgag gagtggagca agaagaacaa gaggccgatc tacatcggcg 2820agttcggcgc ctacaggaag gccgacctgg agagcaggat caagtggacc agcttcgtgg 2880tgagggaggc cgagaagagg ggctggagct gggcctactg ggagttctgc agcggcttcg 2940gcgtgtacga cccgctgagg aagcagtgga acaaggacct gctggaggcc ctgatcggcg 3000gcgacagcat cgagagcgag aaggacgagc tgtgacctag gtccccgaat ttccccgatc 3060gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 3120ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 3180cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 3240tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 3300tactagatcg ggaattg 3317113388DNAArtificial SequenceSynthetic construct,pAG4766 11tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt ttttggcctg 60catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg agactctgtg 120ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa tcttgcctgt 180gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat atttggaaac 240atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt cgtattggat 300gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt gcttctgggt 360caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc cctgtttacg 420ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat gccatggaaa 480gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc catttgtgtt 540gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag attatataac 600ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat atagtgaatt 660tttaagagca tgttttccta aagaagtata tattttctat gtacaaaggc cattgaagta 720attgtagata caggataatg tagacttttt ggacttacac tgctaccttt aagtaacaat 780catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct tgatttccat 840gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat gcataggaaa 900gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac ttaattaatc 960atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac cagcactcaa 1020gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac ctcttgtaaa 1080gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac aactgacaag 1140gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt agttagcagt 1200agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc aacccaaata 1260aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca aaactaaaag 1320taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg tgtgggcatt 1380atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat gagcctcaaa 1440agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca ttctccactg 1500acataatgca aaataagata tcatcgatga catagcaact catgcatcat atcatgcctc 1560tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg ttagaacata 1620aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca cagactcaag 1680caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat attgcaaaaa 1740gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt cgtgtcaaaa 1800agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa gaacaacaca 1860atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct ttcgtgtacc 1920acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta tcttaacatt 1980tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg tttctcacaa 2040aaagcattca gttcattagt cctacaacaa cggatccacc atgagggtgt tgctcgttgc 2100cctcgctctc ctggctctcg ctgcgagcgc caccagcggc gtggacccgt tcgagaggaa 2160caagatcctg ggcaggggca tcaacatcgg caacgccctg gaggccccga acgagggcga 2220ctggggcgtg gtgatcaagg acgagttctt cgacatcatc aaggaggccg gcttcagcca 2280cgtgagaatc ccgatcaggt ggagcaccca cgcccaggcc ttcccgccgt acaagatcga 2340gccgagcttc ttcaagaggg tggacgaggt gatcaacggc gccctgaaga ggggcctggc 2400cgtggtgatc aacatccacc actacgagga gctgatgaac gacccggagg agcacaagga 2460gaggttcctg gccctgtgga agcagatcgc cgacaggtac aaggactacc cggagaccct 2520gttcttcgag atcctgaacg agccgcacgg caacctgacc ccggagaagt ggaacgagct 2580gctggaggag gccctgaagg tgatcaggag catcgacaag aagcacaccg tgatcatcgg 2640caccgccgag tggggcggca tcagcgccct ggagaagctg agggtgccga agtgggagaa 2700gaacgccatc gtgaccatcc actactacaa cccgttcgag ttcacccacc agggcgccga 2760gtgggtgccg ggcagcgaga agtggctggg caggaagtgg ggcagcccgg acgaccagaa 2820gcacctgatc gaggagttca acttcatcga ggagtggagc aagaagaaca agaggccgat 2880ctacatcggc gagttcggcg cctacaggaa ggccgacctg gagagcagga tcaagtggac 2940cagcttcgtg gtgagggagg ccgagaagag gggctggagc tgggcctact gggagttctg 3000cagcggcttc ggcgtgtacg acccgctgag gaagcagtgg aacaaggacc tgctggaggc 3060cctgatcggc ggcgacagca tcgagagcga gaaggacgag ctgtgaccta ggtccccgaa 3120tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 3180tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 3240gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 3300ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 3360gtcatctatg ttactagatc gggaattg 3388124321DNAArtificial SequenceSynthetic construct, pAG4767 12cggtatgaat ttggaaacaa attcagtact tttaaaaaaa tttgttgtag ggagcaaata 60atacataaaa taatttatgc attattttat tttttatttg taataatatg cttgaaacga 120taattcagta tgcatgttgt gccagtgtac tacacgggcg gggggagggg attgagtggg 180ccagcgcggt gcgtagggta gatgggctga aattgataac tcaagtccga ctaggttctc 240tttttatttc ccttcctttt ctattttcct ttcttttaat tttcatgctt tcaaactaaa 300ttcaaattcg agttttgaat ttcagcttct aaattgtaca ctaaaattat atgataaggt 360aacccctact attactttta atttttttat tctaccccat attgtttact taggggagaa 420taattgactt aatcacattc ttccttaggt ttcaattctc aatctttcaa atccacattt 480ttagatttct attttgaatt taaataccag tttggattta gagttcaatt tcaaaataca 540caaccaaaat accagcatga atgcaaatat attttatgtt tatgtattta cttttctttt 600atactttgct caaaatagtt attttcatgt atgaaactca ataagcaagg aactcacgtt 660attatataac ctaataggaa taatttaggt aacataattt atcatcctct tgatttaaaa 720gagatatgcc tccagaataa gacacatact aaaaataact ctaatattga ataactaaag 780tcgtacaaat ctctactatt attcctataa aataataaag aactagctac aacttcttta 840aggcattatt cagggtttac agcttgagag gcatgaaccc atcctgtata ctcctggact 900tggaagacaa aatgtcaacc aaagtgaaag gttttcttat ggttgctgct aagagataga 960ttgaacacta gatctctcct aagacgtcag ggcatgcgtt tagactccta cacatgcgaa 1020aactgcatct tacagttgga agaaactata tctcaccact tcctgcggtg taactttgcc 1080caaagatgtt ggctcactgt tggaatcact ccgccccgaa ctttggatct aacgcttgca 1140gtgctacata ttagagcaag actaacaatg ccgtggagaa tggaaggtat tataaccatg 1200tcatggtgca tatggaaatg tcgaaataac tggatattcg aaaacatacc gccaacggtg 1260gcggcctgca aggaaatgtt caagactgaa atgaactaca tctgctacca agttaagctc 1320gagacaggag ctaaaagtag aaactggata caacactttg taacatagtg acactcccct 1380tttcctttct tttaccttag aactatacat acaatccaca ttcaataaaa atttgtaggt 1440acgccataca cactaccgga atccggctct ttgccgagtg tgaggcgctt tgtcgagtgc 1500tttttgtcca gcactcggca aaaaagtctt tgccatgtgc cgcactcggc aaagtcctgc 1560tctcggtaac gaccgcgttt accgagagca ggactctcga cacagaaata cactcgacaa 1620agaaatcttt gccgagagcc aaacactcgg cgaacggcag cgctcggcaa agggtcgtca 1680gccgccgtct aaagctgacg gtcgttatct ttgtcgagtg ccccctcgtc cgacactcag 1740tagagcaagc ttgccgagtg ccatccttgg acactcgata aagtatattt tatttttttt 1800tattttgcca accaaacttt ttgtggtatg ttcctacact atgtagatct acatgtacca 1860ttttggcaca attacaaaaa tgttttctat aactattaga tttagttcgt ttatttgaat 1920ttcttcggaa aattcacata tgaactgcaa gtcactcgaa acatgaaaaa ccgtgcatgc 1980aaaataaatg atatgcatgt tatctagcac aagttacgac cgaattcaga agcagaccag 2040aatcttcaag caccatgctc actaaacatg accgtgaact tgttatccag ttgtttaaaa 2100attgtataaa acacaaataa agtcagaaat taatgaaact tgtccacatg tcatgatatc 2160atatatagag gttgtgataa aaatttgata atgtttcggt aaagttgtga cgtactatgt 2220gtagaaacct aagtgaccta cacataaaat catagagttt caatgtagtt cactcgacaa 2280agactttgtc aagtgtccga taaaaagtat tcagcaaaga agccgttgtc gatttactgt 2340tcgtcgagat ctctttgccg agtgtcacac taggcaaagt ctttacggag tgtttttcag 2400gctttgacac tcggcaaagc gctcgattcc agtagtgaca gtaatttgca tcaaaaatag 2460ccgagagatt taaaatgagt caactaatag accaactaat tattagctat tagtcgttag 2520cttctttaat ctaagctaaa accaactaat agcttatttg ttgaattaca attagctcaa 2580cggaattctc tgttttttct ataaaaaaaa gggaaactgc ccctcattta cagcaaactg 2640tccgctgcct gtcgtccaga tacaatgaac gtacctagta ggaactcttt tacacgctcg 2700gtcgctcgcc gcggatcgga gtcccaggaa cacgacacca ctgtggaaca cgacaaagtc 2760tgctcagagg cggccacacc ctggcgtgca ccgagccgga gcccggataa gcacggtaag 2820gagagtacgg cgggacgtgg cgacccgtgt gtctgctgcc acgcagcctt cctccacgta 2880gccgcgcggc cgcgccacgt accagggccc ggcgctggta taaatgcgcg ccacctccgc 2940tttagttctg catacagcca acccaacaca cacccgagca tatcacagtg acagacacta 3000cacgggatcc accatgaggg tgttgctcgt tgccctcgct ctcctggctc tcgctgcgag 3060cgccaccagc ggcgtggacc cgttcgagag gaacaagatc ctgggcaggg gcatcaacat 3120cggcaacgcc ctggaggccc cgaacgaggg cgactggggc gtggtgatca aggacgagtt 3180cttcgacatc atcaaggagg ccggcttcag ccacgtgaga atcccgatca ggtggagcac 3240ccacgcccag gccttcccgc cgtacaagat cgagccgagc ttcttcaaga gggtggacga 3300ggtgatcaac ggcgccctga agaggggcct ggccgtggtg atcaacatcc accactacga 3360ggagctgatg aacgacccgg aggagcacaa ggagaggttc ctggccctgt ggaagcagat 3420cgccgacagg tacaaggact acccggagac cctgttcttc gagatcctga acgagccgca 3480cggcaacctg accccggaga agtggaacga gctgctggag gaggccctga aggtgatcag 3540gagcatcgac aagaagcaca ccgtgatcat cggcaccgcc gagtggggcg gcatcagcgc 3600cctggagaag ctgagggtgc cgaagtggga gaagaacgcc atcgtgacca tccactacta 3660caacccgttc gagttcaccc accagggcgc cgagtgggtg ccgggcagcg agaagtggct 3720gggcaggaag tggggcagcc cggacgacca gaagcacctg atcgaggagt tcaacttcat 3780cgaggagtgg agcaagaaga acaagaggcc gatctacatc ggcgagttcg gcgcctacag 3840gaaggccgac ctggagagca ggatcaagtg gaccagcttc gtggtgaggg aggccgagaa 3900gaggggctgg agctgggcct actgggagtt ctgcagcggc ttcggcgtgt acgacccgct 3960gaggaagcag tggaacaagg acctgctgga ggccctgatc ggcggcgaca gcatcgagag 4020cgagaaggac gagctgtgac ctaggtcccc gaatttcccc gatcgttcaa acatttggca 4080ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct 4140gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg 4200ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata 4260gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt 4320g 43211310425DNAArtificial SequenceSynthetic construct, pAG4770 13aaagtaatca tattatttta tgtgtgaatc ttctttactt tttcatttga ttatgattat 60gaaggtatga ccttcataac cttcgtccga aatccattat atccaaagga aaataatgct 120tcgaaggacg aaggattttg atatttaaca ttttatgttg ccttgttctt aattcatagc 180atttgagaac aagtccccaa caccaatctt tatctttact atattaaagc accagttcaa 240cgatcgtctc gtgtcaatat tattaaaaaa ctcctacatt tctttataat caacccgcac 300tcttataatc tcttctctta ctactataat aagagagttt atgtacaaaa taaggtgaaa 360ttatgtataa gtgttctgga ccttggttgt tggctcatat tcacacaacc taatcaatag 420aaaacatatg ttttattaaa acaaaattta tcatatatat atatatatat atatatatat

480atatatatat atataatata aaccgtagca atgcacaggc atatgactag tggcaactta 540ataccatgtg tgtattaaga tgaataagag gtatccaaat aaataacttg ttcgcttacg 600tctggatcga aaggggttgg aaacgattaa atctcttcct agtcaaaatt aaatagaagg 660agatttaatc gatttctccc aatccccttc gatccaggtg caaccgaata agtccttaaa 720tgttgaggaa cacgaaacaa ccatgcattg gcatgtaaag ctccaagaat tcgttgtatc 780cttaacaact cacagaacat caaccaaaat tgcacgtcaa gggtattggg taagaaacaa 840tcaaacaaat cctctctgtg tgcaaagaaa cacggtgagt catgccgaga tcatactcat 900ctgatataca tgcttacagc tcacaagaca ttacaaacaa ctcatattgc attacaaaga 960tcgtttcatg aaaaataaaa taggccggaa caggacaaaa atccttgacg tgtaaagtaa 1020atttacaaca aaaaaaaagc catatgtcaa gctaaatcta attcgtttta cgtagatcaa 1080caacctgtag aaggcaacaa aactgagcca cgcagaagta cagaatgatt ccagatgaac 1140catcgacgtg ctacgtaaag agagtgacga gtcatataca tttggcaaga aaccatgaag 1200ctgcctacag ccgtctcggt ggcataagaa cacaagaaat tgtgttaatt aatcaaagct 1260ataaataacg ctcgcatgcc tgtgcacttc tccatcacca ccactgggtc ttcagaccat 1320tagctttatc tactccagag cgcagaagaa cccgatcgac accggatcca ccatgagggt 1380gttgctcgtt gccctcgctc tcctggctct cgctgcgagc gccaccagcg gcgtggaccc 1440gttcgagagg aacaagatcc tgggcagggg catcaacatc ggcaacgccc tggaggcccc 1500gaacgagggc gactggggcg tggtgatcaa ggacgagttc ttcgacatca tcaaggaggc 1560cggcttcagc cacgtgagaa tcccgatcag gtggagcacc cacgcccagg ccttcccgcc 1620gtacaagatc gagccgagct tcttcaagag ggtggacgag gtgatcaacg gcgccctgaa 1680gaggggcctg gccgtggtga tcaacatcca ccactacgag gagctgatga acgacccgga 1740ggagcacaag gagaggttcc tggccctgtg gaagcagatc gccgacaggt acaaggacta 1800cccggagacc ctgttcttcg agatcctgaa cgagccgcac ggcaacctga ccccggagaa 1860gtggaacgag ctgctggagg aggccctgaa ggtgatcagg agcatcgaca agaagcacac 1920cgtgatcatc ggcaccgccg agtggggcgg catcagcgcc ctggagaagc tgagggtgcc 1980gaagtgggag aagaacgcca tcgtgaccat ccactactac aacccgttcg agttcaccca 2040ccagggcgcc gagtgggtgc cgggcagcga gaagtggctg ggcaggaagt ggggcagccc 2100ggacgaccag aagcacctga tcgaggagtt caacttcatc gaggagtgga gcaagaagaa 2160caagaggccg atctacatcg gcgagttcgg cgcctacagg aaggccgacc tggagagcag 2220gatcaagtgg accagcttcg tggtgaggga ggccgagaag aggggctgga gctgggccta 2280ctgggagttc tgcagcggct tcggcgtgta cgacccgctg aggaagcagt ggaacaagga 2340cctgctggag gccctgatcg gcggcgacag catcgagagc gagaaggacg agctgtgacc 2400taggtccccg aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa 2460tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 2520aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc 2580gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt 2640atcgcgcgcg gtgtcatcta tgttactaga tcgggaattg tacgtaccaa atccatggaa 2700tcaaggtacc tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt 2760ttttggcctg catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg 2820agactctgtg ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa 2880tcttgcctgt gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat 2940atttggaaac atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt 3000cgtattggat gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt 3060gcttctgggt caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc 3120cctgtttacg ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat 3180gccatggaaa gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc 3240catttgtgtt gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag 3300attatataac ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat 3360atagtgaatt tttaagagca tgttttccta aagaagtata tattttctat gtacaaaggc 3420cattgaagta attgtagata caggataatg tagacttttt ggacttacac tgctaccttt 3480aagtaacaat catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct 3540tgatttccat gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat 3600gcataggaaa gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac 3660ttaattaatc atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac 3720cagcactcaa gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac 3780ctcttgtaaa gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac 3840aactgacaag gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt 3900agttagcagt agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc 3960aacccaaata aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca 4020aaactaaaag taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg 4080tgtgggcatt atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat 4140gagcctcaaa agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca 4200ttctccactg acataatgca aaataagata tcatcgatga catagcaact catgcatcat 4260atcatgcctc tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg 4320ttagaacata aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca 4380cagactcaag caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat 4440attgcaaaaa gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt 4500cgtgtcaaaa agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa 4560gaacaacaca atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct 4620ttcgtgtacc acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta 4680tcttaacatt tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg 4740tttctcacaa aaagcattca gttcattagt cctacaacaa cggatccacc atgagggtgt 4800tgctcgttgc cctcgctctc ctggctctcg ctgcgagcgc caccagcggc gtggacccgt 4860tcgagaggaa caagatcctg ggcaggggca tcaacatcgg caacgccctg gaggccccga 4920acgagggcga ctggggcgtg gtgatcaagg acgagttctt cgacatcatc aaggaggccg 4980gcttcagcca cgtgagaatc ccgatcaggt ggagcaccca cgcccaggcc ttcccgccgt 5040acaagatcga gccgagcttc ttcaagaggg tggacgaggt gatcaacggc gccctgaaga 5100ggggcctggc cgtggtgatc aacatccacc actacgagga gctgatgaac gacccggagg 5160agcacaagga gaggttcctg gccctgtgga agcagatcgc cgacaggtac aaggactacc 5220cggagaccct gttcttcgag atcctgaacg agccgcacgg caacctgacc ccggagaagt 5280ggaacgagct gctggaggag gccctgaagg tgatcaggag catcgacaag aagcacaccg 5340tgatcatcgg caccgccgag tggggcggca tcagcgccct ggagaagctg agggtgccga 5400agtgggagaa gaacgccatc gtgaccatcc actactacaa cccgttcgag ttcacccacc 5460agggcgccga gtgggtgccg ggcagcgaga agtggctggg caggaagtgg ggcagcccgg 5520acgaccagaa gcacctgatc gaggagttca acttcatcga ggagtggagc aagaagaaca 5580agaggccgat ctacatcggc gagttcggcg cctacaggaa ggccgacctg gagagcagga 5640tcaagtggac cagcttcgtg gtgagggagg ccgagaagag gggctggagc tgggcctact 5700gggagttctg cagcggcttc ggcgtgtacg acccgctgag gaagcagtgg aacaaggacc 5760tgctggaggc cctgatcggc ggcgacagca tcgagagcga gaaggacgag ctgtgaccta 5820ggtccccgaa tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc 5880ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 5940taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc 6000aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat 6060cgcgcgcggt gtcatctatg ttactagatc gggaattggg tacccggtat gaatttggaa 6120acaaattcag tacttttaaa aaaatttgtt gtagggagca aataatacat aaaataattt 6180atgcattatt ttatttttta tttgtaataa tatgcttgaa acgataattc agtatgcatg 6240ttgtgccagt gtactacacg ggcgggggga ggggattgag tgggccagcg cggtgcgtag 6300ggtagatggg ctgaaattga taactcaagt ccgactaggt tctcttttta tttcccttcc 6360ttttctattt tcctttcttt taattttcat gctttcaaac taaattcaaa ttcgagtttt 6420gaatttcagc ttctaaattg tacactaaaa ttatatgata aggtaacccc tactattact 6480tttaattttt ttattctacc ccatattgtt tacttagggg agaataattg acttaatcac 6540attcttcctt aggtttcaat tctcaatctt tcaaatccac atttttagat ttctattttg 6600aatttaaata ccagtttgga tttagagttc aatttcaaaa tacacaacca aaataccagc 6660atgaatgcaa atatatttta tgtttatgta tttacttttc ttttatactt tgctcaaaat 6720agttattttc atgtatgaaa ctcaataagc aaggaactca cgttattata taacctaata 6780ggaataattt aggtaacata atttatcatc ctcttgattt aaaagagata tgcctccaga 6840ataagacaca tactaaaaat aactctaata ttgaataact aaagtcgtac aaatctctac 6900tattattcct ataaaataat aaagaactag ctacaacttc tttaaggcat tattcagggt 6960ttacagcttg agaggcatga acccatcctg tatactcctg gacttggaag acaaaatgtc 7020aaccaaagtg aaaggttttc ttatggttgc tgctaagaga tagattgaac actagatctc 7080tcctaagacg tcagggcatg cgtttagact cctacacatg cgaaaactgc atcttacagt 7140tggaagaaac tatatctcac cacttcctgc ggtgtaactt tgcccaaaga tgttggctca 7200ctgttggaat cactccgccc cgaactttgg atctaacgct tgcagtgcta catattagag 7260caagactaac aatgccgtgg agaatggaag gtattataac catgtcatgg tgcatatgga 7320aatgtcgaaa taactggata ttcgaaaaca taccgccaac ggtggcggcc tgcaaggaaa 7380tgttcaagac tgaaatgaac tacatctgct accaagttaa gctcgagaca ggagctaaaa 7440gtagaaactg gatacaacac tttgtaacat agtgacactc cccttttcct ttcttttacc 7500ttagaactat acatacaatc cacattcaat aaaaatttgt aggtacgcca tacacactac 7560cggaatccgg ctctttgccg agtgtgaggc gctttgtcga gtgctttttg tccagcactc 7620ggcaaaaaag tctttgccat gtgccgcact cggcaaagtc ctgctctcgg taacgaccgc 7680gtttaccgag agcaggactc tcgacacaga aatacactcg acaaagaaat ctttgccgag 7740agccaaacac tcggcgaacg gcagcgctcg gcaaagggtc gtcagccgcc gtctaaagct 7800gacggtcgtt atctttgtcg agtgccccct cgtccgacac tcagtagagc aagcttgccg 7860agtgccatcc ttggacactc gataaagtat attttatttt tttttatttt gccaaccaaa 7920ctttttgtgg tatgttccta cactatgtag atctacatgt accattttgg cacaattaca 7980aaaatgtttt ctataactat tagatttagt tcgtttattt gaatttcttc ggaaaattca 8040catatgaact gcaagtcact cgaaacatga aaaaccgtgc atgcaaaata aatgatatgc 8100atgttatcta gcacaagtta cgaccgaatt cagaagcaga ccagaatctt caagcaccat 8160gctcactaaa catgaccgtg aacttgttat ccagttgttt aaaaattgta taaaacacaa 8220ataaagtcag aaattaatga aacttgtcca catgtcatga tatcatatat agaggttgtg 8280ataaaaattt gataatgttt cggtaaagtt gtgacgtact atgtgtagaa acctaagtga 8340cctacacata aaatcataga gtttcaatgt agttcactcg acaaagactt tgtcaagtgt 8400ccgataaaaa gtattcagca aagaagccgt tgtcgattta ctgttcgtcg agatctcttt 8460gccgagtgtc acactaggca aagtctttac ggagtgtttt tcaggctttg acactcggca 8520aagcgctcga ttccagtagt gacagtaatt tgcatcaaaa atagccgaga gatttaaaat 8580gagtcaacta atagaccaac taattattag ctattagtcg ttagcttctt taatctaagc 8640taaaaccaac taatagctta tttgttgaat tacaattagc tcaacggaat tctctgtttt 8700ttctataaaa aaaagggaaa ctgcccctca tttacagcaa actgtccgct gcctgtcgtc 8760cagatacaat gaacgtacct agtaggaact cttttacacg ctcggtcgct cgccgcggat 8820cggagtccca ggaacacgac accactgtgg aacacgacaa agtctgctca gaggcggcca 8880caccctggcg tgcaccgagc cggagcccgg ataagcacgg taaggagagt acggcgggac 8940gtggcgaccc gtgtgtctgc tgccacgcag ccttcctcca cgtagccgcg cggccgcgcc 9000acgtaccagg gcccggcgct ggtataaatg cgcgccacct ccgctttagt tctgcataca 9060gccaacccaa cacacacccg agcatatcac agtgacagac actacacggg atccaccatg 9120agggtgttgc tcgttgccct cgctctcctg gctctcgctg cgagcgccac cagcggcgtg 9180gacccgttcg agaggaacaa gatcctgggc aggggcatca acatcggcaa cgccctggag 9240gccccgaacg agggcgactg gggcgtggtg atcaaggacg agttcttcga catcatcaag 9300gaggccggct tcagccacgt gagaatcccg atcaggtgga gcacccacgc ccaggccttc 9360ccgccgtaca agatcgagcc gagcttcttc aagagggtgg acgaggtgat caacggcgcc 9420ctgaagaggg gcctggccgt ggtgatcaac atccaccact acgaggagct gatgaacgac 9480ccggaggagc acaaggagag gttcctggcc ctgtggaagc agatcgccga caggtacaag 9540gactacccgg agaccctgtt cttcgagatc ctgaacgagc cgcacggcaa cctgaccccg 9600gagaagtgga acgagctgct ggaggaggcc ctgaaggtga tcaggagcat cgacaagaag 9660cacaccgtga tcatcggcac cgccgagtgg ggcggcatca gcgccctgga gaagctgagg 9720gtgccgaagt gggagaagaa cgccatcgtg accatccact actacaaccc gttcgagttc 9780acccaccagg gcgccgagtg ggtgccgggc agcgagaagt ggctgggcag gaagtggggc 9840agcccggacg accagaagca cctgatcgag gagttcaact tcatcgagga gtggagcaag 9900aagaacaaga ggccgatcta catcggcgag ttcggcgcct acaggaaggc cgacctggag 9960agcaggatca agtggaccag cttcgtggtg agggaggccg agaagagggg ctggagctgg 10020gcctactggg agttctgcag cggcttcggc gtgtacgacc cgctgaggaa gcagtggaac 10080aaggacctgc tggaggccct gatcggcggc gacagcatcg agagcgagaa ggacgagctg 10140tgacctaggt ccccgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga 10200ttgaatcctg ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag 10260catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga 10320gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat 10380aaattatcgc gcgcggtgtc atctatgtta ctagatcggg aattg 104251410413DNAArtificial SequenceSynthetic construct, pAG4771 14tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt ttttggcctg 60catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg agactctgtg 120ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa tcttgcctgt 180gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat atttggaaac 240atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt cgtattggat 300gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt gcttctgggt 360caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc cctgtttacg 420ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat gccatggaaa 480gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc catttgtgtt 540gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag attatataac 600ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat atagtgaatt 660tttaagagca tgttttccta aagaagtata tattttctat gtacaaaggc cattgaagta 720attgtagata caggataatg tagacttttt ggacttacac tgctaccttt aagtaacaat 780catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct tgatttccat 840gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat gcataggaaa 900gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac ttaattaatc 960atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac cagcactcaa 1020gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac ctcttgtaaa 1080gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac aactgacaag 1140gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt agttagcagt 1200agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc aacccaaata 1260aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca aaactaaaag 1320taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg tgtgggcatt 1380atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat gagcctcaaa 1440agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca ttctccactg 1500acataatgca aaataagata tcatcgatga catagcaact catgcatcat atcatgcctc 1560tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg ttagaacata 1620aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca cagactcaag 1680caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat attgcaaaaa 1740gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt cgtgtcaaaa 1800agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa gaacaacaca 1860atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct ttcgtgtacc 1920acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta tcttaacatt 1980tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg tttctcacaa 2040aaagcattca gttcattagt cctacaacaa cggatccacc atgagggtgt tgctcgttgc 2100cctcgctctc ctggctctcg ctgcgagcgc caccagcggc gtggacccgt tcgagaggaa 2160caagatcctg ggcaggggca tcaacatcgg caacgccctg gaggccccga acgagggcga 2220ctggggcgtg gtgatcaagg acgagttctt cgacatcatc aaggaggccg gcttcagcca 2280cgtgagaatc ccgatcaggt ggagcaccca cgcccaggcc ttcccgccgt acaagatcga 2340gccgagcttc ttcaagaggg tggacgaggt gatcaacggc gccctgaaga ggggcctggc 2400cgtggtgatc aacatccacc actacgagga gctgatgaac gacccggagg agcacaagga 2460gaggttcctg gccctgtgga agcagatcgc cgacaggtac aaggactacc cggagaccct 2520gttcttcgag atcctgaacg agccgcacgg caacctgacc ccggagaagt ggaacgagct 2580gctggaggag gccctgaagg tgatcaggag catcgacaag aagcacaccg tgatcatcgg 2640caccgccgag tggggcggca tcagcgccct ggagaagctg agggtgccga agtgggagaa 2700gaacgccatc gtgaccatcc actactacaa cccgttcgag ttcacccacc agggcgccga 2760gtgggtgccg ggcagcgaga agtggctggg caggaagtgg ggcagcccgg acgaccagaa 2820gcacctgatc gaggagttca acttcatcga ggagtggagc aagaagaaca agaggccgat 2880ctacatcggc gagttcggcg cctacaggaa ggccgacctg gagagcagga tcaagtggac 2940cagcttcgtg gtgagggagg ccgagaagag gggctggagc tgggcctact gggagttctg 3000cagcggcttc ggcgtgtacg acccgctgag gaagcagtgg aacaaggacc tgctggaggc 3060cctgatcggc ggcgacagca tcgagagcga gaaggacgag ctgtgaccta ggtccccgaa 3120tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 3180tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 3240gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 3300ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 3360gtcatctatg ttactagatc gggaattgcc atggaatcaa ggtaccaaag taatcatatt 3420attttatgtg tgaatcttct ttactttttc atttgattat gattatgaag gtatgacctt 3480cataaccttc gtccgaaatc cattatatcc aaaggaaaat aatgcttcga aggacgaagg 3540attttgatat ttaacatttt atgttgcctt gttcttaatt catagcattt gagaacaagt 3600ccccaacacc aatctttatc tttactatat taaagcacca gttcaacgat cgtctcgtgt 3660caatattatt aaaaaactcc tacatttctt tataatcaac ccgcactctt ataatctctt 3720ctcttactac tataataaga gagtttatgt acaaaataag gtgaaattat gtataagtgt 3780tctggacctt ggttgttggc tcatattcac acaacctaat caatagaaaa catatgtttt 3840attaaaacaa aatttatcat atatatatat atatatatat atatatatat atatatatat 3900aatataaacc gtagcaatgc acaggcatat gactagtggc aacttaatac catgtgtgta 3960ttaagatgaa taagaggtat ccaaataaat aacttgttcg cttacgtctg gatcgaaagg 4020ggttggaaac gattaaatct cttcctagtc aaaattaaat agaaggagat ttaatcgatt 4080tctcccaatc cccttcgatc caggtgcaac cgaataagtc cttaaatgtt gaggaacacg 4140aaacaaccat gcattggcat gtaaagctcc aagaattcgt tgtatcctta acaactcaca 4200gaacatcaac caaaattgca cgtcaagggt attgggtaag aaacaatcaa acaaatcctc 4260tctgtgtgca aagaaacacg gtgagtcatg ccgagatcat actcatctga tatacatgct 4320tacagctcac aagacattac aaacaactca tattgcatta caaagatcgt ttcatgaaaa 4380ataaaatagg ccggaacagg acaaaaatcc ttgacgtgta aagtaaattt acaacaaaaa 4440aaaagccata tgtcaagcta aatctaattc gttttacgta gatcaacaac ctgtagaagg 4500caacaaaact gagccacgca gaagtacaga atgattccag atgaaccatc gacgtgctac 4560gtaaagagag tgacgagtca tatacatttg gcaagaaacc atgaagctgc ctacagccgt 4620ctcggtggca taagaacaca agaaattgtg ttaattaatc aaagctataa ataacgctcg 4680catgcctgtg cacttctcca tcaccaccac tgggtcttca gaccattagc tttatctact 4740ccagagcgca gaagaacccg atcgacaccg gatccaccat gagggtgttg ctcgttgccc 4800tcgctctcct ggctctcgct gcgagcgcca ccagcggcgt ggacccgttc gagaggaaca 4860agatcctggg caggggcatc aacatcggca acgccctgga ggccccgaac gagggcgact 4920ggggcgtggt gatcaaggac gagttcttcg acatcatcaa ggaggccggc ttcagccacg 4980tgagaatccc gatcaggtgg agcacccacg cccaggcctt cccgccgtac aagatcgagc

5040cgagcttctt caagagggtg gacgaggtga tcaacggcgc cctgaagagg ggcctggccg 5100tggtgatcaa catccaccac tacgaggagc tgatgaacga cccggaggag cacaaggaga 5160ggttcctggc cctgtggaag cagatcgccg acaggtacaa ggactacccg gagaccctgt 5220tcttcgagat cctgaacgag ccgcacggca acctgacccc ggagaagtgg aacgagctgc 5280tggaggaggc cctgaaggtg atcaggagca tcgacaagaa gcacaccgtg atcatcggca 5340ccgccgagtg gggcggcatc agcgccctgg agaagctgag ggtgccgaag tgggagaaga 5400acgccatcgt gaccatccac tactacaacc cgttcgagtt cacccaccag ggcgccgagt 5460gggtgccggg cagcgagaag tggctgggca ggaagtgggg cagcccggac gaccagaagc 5520acctgatcga ggagttcaac ttcatcgagg agtggagcaa gaagaacaag aggccgatct 5580acatcggcga gttcggcgcc tacaggaagg ccgacctgga gagcaggatc aagtggacca 5640gcttcgtggt gagggaggcc gagaagaggg gctggagctg ggcctactgg gagttctgca 5700gcggcttcgg cgtgtacgac ccgctgagga agcagtggaa caaggacctg ctggaggccc 5760tgatcggcgg cgacagcatc gagagcgaga aggacgagct gtgacctagg tccccgaatt 5820tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 5880ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 5940aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 6000aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 6060catctatgtt actagatcgg gaattgggta cccggtatga atttggaaac aaattcagta 6120cttttaaaaa aatttgttgt agggagcaaa taatacataa aataatttat gcattatttt 6180attttttatt tgtaataata tgcttgaaac gataattcag tatgcatgtt gtgccagtgt 6240actacacggg cggggggagg ggattgagtg ggccagcgcg gtgcgtaggg tagatgggct 6300gaaattgata actcaagtcc gactaggttc tctttttatt tcccttcctt ttctattttc 6360ctttctttta attttcatgc tttcaaacta aattcaaatt cgagttttga atttcagctt 6420ctaaattgta cactaaaatt atatgataag gtaaccccta ctattacttt taattttttt 6480attctacccc atattgttta cttaggggag aataattgac ttaatcacat tcttccttag 6540gtttcaattc tcaatctttc aaatccacat ttttagattt ctattttgaa tttaaatacc 6600agtttggatt tagagttcaa tttcaaaata cacaaccaaa ataccagcat gaatgcaaat 6660atattttatg tttatgtatt tacttttctt ttatactttg ctcaaaatag ttattttcat 6720gtatgaaact caataagcaa ggaactcacg ttattatata acctaatagg aataatttag 6780gtaacataat ttatcatcct cttgatttaa aagagatatg cctccagaat aagacacata 6840ctaaaaataa ctctaatatt gaataactaa agtcgtacaa atctctacta ttattcctat 6900aaaataataa agaactagct acaacttctt taaggcatta ttcagggttt acagcttgag 6960aggcatgaac ccatcctgta tactcctgga cttggaagac aaaatgtcaa ccaaagtgaa 7020aggttttctt atggttgctg ctaagagata gattgaacac tagatctctc ctaagacgtc 7080agggcatgcg tttagactcc tacacatgcg aaaactgcat cttacagttg gaagaaacta 7140tatctcacca cttcctgcgg tgtaactttg cccaaagatg ttggctcact gttggaatca 7200ctccgccccg aactttggat ctaacgcttg cagtgctaca tattagagca agactaacaa 7260tgccgtggag aatggaaggt attataacca tgtcatggtg catatggaaa tgtcgaaata 7320actggatatt cgaaaacata ccgccaacgg tggcggcctg caaggaaatg ttcaagactg 7380aaatgaacta catctgctac caagttaagc tcgagacagg agctaaaagt agaaactgga 7440tacaacactt tgtaacatag tgacactccc cttttccttt cttttacctt agaactatac 7500atacaatcca cattcaataa aaatttgtag gtacgccata cacactaccg gaatccggct 7560ctttgccgag tgtgaggcgc tttgtcgagt gctttttgtc cagcactcgg caaaaaagtc 7620tttgccatgt gccgcactcg gcaaagtcct gctctcggta acgaccgcgt ttaccgagag 7680caggactctc gacacagaaa tacactcgac aaagaaatct ttgccgagag ccaaacactc 7740ggcgaacggc agcgctcggc aaagggtcgt cagccgccgt ctaaagctga cggtcgttat 7800ctttgtcgag tgccccctcg tccgacactc agtagagcaa gcttgccgag tgccatcctt 7860ggacactcga taaagtatat tttatttttt tttattttgc caaccaaact ttttgtggta 7920tgttcctaca ctatgtagat ctacatgtac cattttggca caattacaaa aatgttttct 7980ataactatta gatttagttc gtttatttga atttcttcgg aaaattcaca tatgaactgc 8040aagtcactcg aaacatgaaa aaccgtgcat gcaaaataaa tgatatgcat gttatctagc 8100acaagttacg accgaattca gaagcagacc agaatcttca agcaccatgc tcactaaaca 8160tgaccgtgaa cttgttatcc agttgtttaa aaattgtata aaacacaaat aaagtcagaa 8220attaatgaaa cttgtccaca tgtcatgata tcatatatag aggttgtgat aaaaatttga 8280taatgtttcg gtaaagttgt gacgtactat gtgtagaaac ctaagtgacc tacacataaa 8340atcatagagt ttcaatgtag ttcactcgac aaagactttg tcaagtgtcc gataaaaagt 8400attcagcaaa gaagccgttg tcgatttact gttcgtcgag atctctttgc cgagtgtcac 8460actaggcaaa gtctttacgg agtgtttttc aggctttgac actcggcaaa gcgctcgatt 8520ccagtagtga cagtaatttg catcaaaaat agccgagaga tttaaaatga gtcaactaat 8580agaccaacta attattagct attagtcgtt agcttcttta atctaagcta aaaccaacta 8640atagcttatt tgttgaatta caattagctc aacggaattc tctgtttttt ctataaaaaa 8700aagggaaact gcccctcatt tacagcaaac tgtccgctgc ctgtcgtcca gatacaatga 8760acgtacctag taggaactct tttacacgct cggtcgctcg ccgcggatcg gagtcccagg 8820aacacgacac cactgtggaa cacgacaaag tctgctcaga ggcggccaca ccctggcgtg 8880caccgagccg gagcccggat aagcacggta aggagagtac ggcgggacgt ggcgacccgt 8940gtgtctgctg ccacgcagcc ttcctccacg tagccgcgcg gccgcgccac gtaccagggc 9000ccggcgctgg tataaatgcg cgccacctcc gctttagttc tgcatacagc caacccaaca 9060cacacccgag catatcacag tgacagacac tacacgggat ccaccatgag ggtgttgctc 9120gttgccctcg ctctcctggc tctcgctgcg agcgccacca gcggcgtgga cccgttcgag 9180aggaacaaga tcctgggcag gggcatcaac atcggcaacg ccctggaggc cccgaacgag 9240ggcgactggg gcgtggtgat caaggacgag ttcttcgaca tcatcaagga ggccggcttc 9300agccacgtga gaatcccgat caggtggagc acccacgccc aggccttccc gccgtacaag 9360atcgagccga gcttcttcaa gagggtggac gaggtgatca acggcgccct gaagaggggc 9420ctggccgtgg tgatcaacat ccaccactac gaggagctga tgaacgaccc ggaggagcac 9480aaggagaggt tcctggccct gtggaagcag atcgccgaca ggtacaagga ctacccggag 9540accctgttct tcgagatcct gaacgagccg cacggcaacc tgaccccgga gaagtggaac 9600gagctgctgg aggaggccct gaaggtgatc aggagcatcg acaagaagca caccgtgatc 9660atcggcaccg ccgagtgggg cggcatcagc gccctggaga agctgagggt gccgaagtgg 9720gagaagaacg ccatcgtgac catccactac tacaacccgt tcgagttcac ccaccagggc 9780gccgagtggg tgccgggcag cgagaagtgg ctgggcagga agtggggcag cccggacgac 9840cagaagcacc tgatcgagga gttcaacttc atcgaggagt ggagcaagaa gaacaagagg 9900ccgatctaca tcggcgagtt cggcgcctac aggaaggccg acctggagag caggatcaag 9960tggaccagct tcgtggtgag ggaggccgag aagaggggct ggagctgggc ctactgggag 10020ttctgcagcg gcttcggcgt gtacgacccg ctgaggaagc agtggaacaa ggacctgctg 10080gaggccctga tcggcggcga cagcatcgag agcgagaagg acgagctgtg acctaggtcc 10140ccgaatttcc ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt 10200gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt 10260aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 10320tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc 10380gcggtgtcat ctatgttact agatcgggaa ttg 10413152680DNAArtificial SequenceSynthetic construct, expression cassette from pAG4257 15aaagtaatca tattatttta tgtgtgaatc ttctttactt tttcatttga ttatgattat 60gaaggtatga ccttcataac cttcgtccga aatccattat atccaaagga aaataatgct 120tcgaaggacg aaggattttg atatttaaca ttttatgttg ccttgttctt aattcatagc 180atttgagaac aagtccccaa caccaatctt tatctttact atattaaagc accagttcaa 240cgatcgtctc gtgtcaatat tattaaaaaa ctcctacatt tctttataat caacccgcac 300tcttataatc tcttctctta ctactataat aagagagttt atgtacaaaa taaggtgaaa 360ttatgtataa gtgttctgga ccttggttgt tggctcatat tcacacaacc taatcaatag 420aaaacatatg ttttattaaa acaaaattta tcatatatat atatatatat atatatatat 480atatatatat atataatata aaccgtagca atgcacaggc atatgactag tggcaactta 540ataccatgtg tgtattaaga tgaataagag gtatccaaat aaataacttg ttcgcttacg 600tctggatcga aaggggttgg aaacgattaa atctcttcct agtcaaaatt aaatagaagg 660agatttaatc gatttctccc aatccccttc gatccaggtg caaccgaata agtccttaaa 720tgttgaggaa cacgaaacaa ccatgcattg gcatgtaaag ctccaagaat tcgttgtatc 780cttaacaact cacagaacat caaccaaaat tgcacgtcaa gggtattggg taagaaacaa 840tcaaacaaat cctctctgtg tgcaaagaaa cacggtgagt catgccgaga tcatactcat 900ctgatataca tgcttacagc tcacaagaca ttacaaacaa ctcatattgc attacaaaga 960tcgtttcatg aaaaataaaa taggccggaa caggacaaaa atccttgacg tgtaaagtaa 1020atttacaaca aaaaaaaagc catatgtcaa gctaaatcta attcgtttta cgtagatcaa 1080caacctgtag aaggcaacaa aactgagcca cgcagaagta cagaatgatt ccagatgaac 1140catcgacgtg ctacgtaaag agagtgacga gtcatataca tttggcaaga aaccatgaag 1200ctgcctacag ccgtctcggt ggcataagaa cacaagaaat tgtgttaatt aatcaaagct 1260ataaataacg ctcgcatgcc tgtgcacttc tccatcacca ccactgggtc ttcagaccat 1320tagctttatc tactccagag cgcagaagaa cccgatcgac accggatcca ccatgagggt 1380gttgctcgtt gccctcgctc tcctggctct cgctgcgagc gccaccagcg gcgtggaccc 1440gttcgagagg aacaagatcc tgggcagggg catcaacatc ggcaacgccc tggaggcccc 1500gaacgagggc gactggggcg tggtgatcaa ggacgagtac ttcgacatca tcaaggaggc 1560cggcttcagc cacgtgagaa tcccgatcag gtggagcacc cacgcccagg ccttcccgcc 1620gtacaagatc gaggacaggt tcttcaagag ggtggacgag gtgatcaacg gcgccctgaa 1680gaggggcctg gccgtggtga tcaaccagca ccactacgag gagctgatga acgacccgga 1740ggagcacaag gagaggttcc tggccctgtg gaagcagatc gccgacaggt acaaggacta 1800cccggagacc ctgttcttcg agatcctgaa cgagccgcac ggcaacctga ccccggagaa 1860gtggaacgag ctgctggagg aggccctgaa ggtgatcagg agcatcgaca agaagcacac 1920catcatcatc ggcaccgccg agtggggcgg catcagcgcc ctggagaagc tgagggtgcc 1980gaagtgggag aagaacgcca tcgtgaccat ccactactac aacccgttcg agttcaccca 2040ccagggcgcc gagtgggtgg agggcagcga gaagtggctg ggcaggaagt ggggcagccc 2100ggacgaccag aagcacctga tcgaggagtt caacttcatc gaggagtgga gcaagaagaa 2160caagaggccg atctacatcg gcgagttcgg cgcctacagg aaggccgacc tggagagcag 2220gatcaagtgg accagcttcg tggtgaggga ggccgagaag aggaggtgga gctgggccta 2280ctgggagttc tgcagcggct tcggcgtgta cgacaccctg aggaagacct ggaacaagga 2340cctgctggag gccctgatcg gcggcgacag catcgagagc gagaaggacg agctgtgacc 2400taggtccccg aatttccccg atcgttcaaa catttggcaa taaagtttct taagattgaa 2460tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 2520aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc 2580gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt 2640atcgcgcgcg gtgtcatcta tgttactaga tcgggaattg 2680163292DNAArtificial SequenceSynthetic construct, expression cassetter from pAG4692 16tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt ttttggcctg 60catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg agactctgtg 120ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa tcttgcctgt 180gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat atttggaaac 240atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt cgtattggat 300gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt gcttctgggt 360caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc cctgtttacg 420ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat gccatggaaa 480gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc catttgtgtt 540gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag attatataac 600ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat atagtgaatt 660tttaagagca tgttttccta aagaagtata tattttctat gtacaaaggc cattgaagta 720attgtagata caggataatg tagacttttt ggacttacac tgctaccttt aagtaacaat 780catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct tgatttccat 840gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat gcataggaaa 900gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac ttaattaatc 960atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac cagcactcaa 1020gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac ctcttgtaaa 1080gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac aactgacaag 1140gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt agttagcagt 1200agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc aacccaaata 1260aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca aaactaaaag 1320taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg tgtgggcatt 1380atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat gagcctcaaa 1440agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca ttctccactg 1500acataatgca aaataagata tcatcgatga catagcaact catgcatcat atcatgcctc 1560tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg ttagaacata 1620aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca cagactcaag 1680caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat attgcaaaaa 1740gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt cgtgtcaaaa 1800agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa gaacaacaca 1860atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct ttcgtgtacc 1920acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta tcttaacatt 1980tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg tttctcacaa 2040aaagcattca gttcattagt cctacaacaa cggatccacc atgagggtgt tgctcgttgc 2100cctcgctctc ctggctctcg ctgcgagcgc caccagcggc gtggacccgt tcgagaggaa 2160caagatcctg ggcaggggca tcaacatcgg caacgccctg gaggccccga acgagggcga 2220ctggggcgtg gtgatcaagg acgagttctt cgacatcatc aaggaggccg gcttcagcca 2280cgtgagaatc ccgatcaggt ggagcaccca cgcctacgcc ttcccgccgt acaagatcat 2340ggacaggttc ttcaagaggg tggacgaggt gatcaacggc gccctgaaga ggggcctggc 2400cgtggtgatc aacatccacc actacgagga gctgatgaac gacccggagg agcacaagga 2460gaggttcctg gccctgtgga agcagatcgc cgacaggtac aaggactacc cggagaccct 2520gttcttcgag atcctgaacg agccgcacgg caacctgacc ccggagaagt ggaacgagct 2580gctggaggag gccctgaagg tgatcaggag catcgacaag aagcacacca tcatcatcgg 2640caccgccgag tggggcggca tcagcgccct ggagaagctg agcgtgccga agtgggagaa 2700gaactccatc gtgaccatcc actactacaa cccgttcgag ttcacccacc agggcgccga 2760gtgggtggag ggcagcgaga agtggctggg caggaagtgg ggcagcccgg acgaccagaa 2820gcacctgatc gaggagttca acttcatcga ggagtggagc aagaagaaca agaggccgat 2880ctacatcggc gagttcggcg cctacaggaa ggccgacctg gagagcagga tcaagtggac 2940cagcttcgtg gtgagggaga tggagaagag gcgctggagc tgggcctact gggagttctg 3000cagcggcttc ggcgtgtacg acaccctgag gaagacctgg aacaaggacc tgctggaggc 3060cctgatcggc ggcgacagca tcgagagcga gaaggacgag ctgtgaccta ggctgcacaa 3120agtggagtag tcagtcatcg atcaggaacc agacaccaga cttttattca tacagtgaag 3180tgaagtgaag tgcagtgcag tgagttgctg gtttttgtac aacttagtat gtatttgtat 3240ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc ag 3292172584DNAArtificial SequenceSynthetic construct, expression cassette from pAG 4693 17aaagtaatca tattatttta tgtgtgaatc ttctttactt tttcatttga ttatgattat 60gaaggtatga ccttcataac cttcgtccga aatccattat atccaaagga aaataatgct 120tcgaaggacg aaggattttg atatttaaca ttttatgttg ccttgttctt aattcatagc 180atttgagaac aagtccccaa caccaatctt tatctttact atattaaagc accagttcaa 240cgatcgtctc gtgtcaatat tattaaaaaa ctcctacatt tctttataat caacccgcac 300tcttataatc tcttctctta ctactataat aagagagttt atgtacaaaa taaggtgaaa 360ttatgtataa gtgttctgga ccttggttgt tggctcatat tcacacaacc taatcaatag 420aaaacatatg ttttattaaa acaaaattta tcatatatat atatatatat atatatatat 480atatatatat atataatata aaccgtagca atgcacaggc atatgactag tggcaactta 540ataccatgtg tgtattaaga tgaataagag gtatccaaat aaataacttg ttcgcttacg 600tctggatcga aaggggttgg aaacgattaa atctcttcct agtcaaaatt aaatagaagg 660agatttaatc gatttctccc aatccccttc gatccaggtg caaccgaata agtccttaaa 720tgttgaggaa cacgaaacaa ccatgcattg gcatgtaaag ctccaagaat tcgttgtatc 780cttaacaact cacagaacat caaccaaaat tgcacgtcaa gggtattggg taagaaacaa 840tcaaacaaat cctctctgtg tgcaaagaaa cacggtgagt catgccgaga tcatactcat 900ctgatataca tgcttacagc tcacaagaca ttacaaacaa ctcatattgc attacaaaga 960tcgtttcatg aaaaataaaa taggccggaa caggacaaaa atccttgacg tgtaaagtaa 1020atttacaaca aaaaaaaagc catatgtcaa gctaaatcta attcgtttta cgtagatcaa 1080caacctgtag aaggcaacaa aactgagcca cgcagaagta cagaatgatt ccagatgaac 1140catcgacgtg ctacgtaaag agagtgacga gtcatataca tttggcaaga aaccatgaag 1200ctgcctacag ccgtctcggt ggcataagaa cacaagaaat tgtgttaatt aatcaaagct 1260ataaataacg ctcgcatgcc tgtgcacttc tccatcacca ccactgggtc ttcagaccat 1320tagctttatc tactccagag cgcagaagaa cccgatcgac accggatcca ccatgagggt 1380gttgctcgtt gccctcgctc tcctggctct cgctgcgagc gccaccagcg gcgtggaccc 1440gttcgagagg aacaagatcc tgggcagggg catcaacatc ggcaacgccc tggaggcccc 1500gaacgagggc gactggggcg tggtgatcaa ggacgagttc ttcgacatca tcaaggaggc 1560cggcttcagc cacgtgagaa tcccgatcag gtggagcacc cacgcctacg ccttcccgcc 1620gtacaagatc atggacaggt tcttcaagag ggtggacgag gtgatcaacg gcgccctgaa 1680gaggggcctg gccgtggtga tcaacatcca ccactacgag gagctgatga acgacccgga 1740ggagcacaag gagaggttcc tggccctgtg gaagcagatc gccgacaggt acaaggacta 1800cccggagacc ctgttcttcg agatcctgaa cgagccgcac ggcaacctga ccccggagaa 1860gtggaacgag ctgctggagg aggccctgaa ggtgatcagg agcatcgaca agaagcacac 1920catcatcatc ggcaccgccg agtggggcgg catcagcgcc ctggagaagc tgagcgtgcc 1980gaagtgggag aagaactcca tcgtgaccat ccactactac aacccgttcg agttcaccca 2040ccagggcgcc gagtgggtgg agggcagcga gaagtggctg ggcaggaagt ggggcagccc 2100ggacgaccag aagcacctga tcgaggagtt caacttcatc gaggagtgga gcaagaagaa 2160caagaggccg atctacatcg gcgagttcgg cgcctacagg aaggccgacc tggagagcag 2220gatcaagtgg accagcttcg tggtgaggga gatggagaag aggcgctgga gctgggccta 2280ctgggagttc tgcagcggct tcggcgtgta cgacaccctg aggaagacct ggaacaagga 2340cctgctggag gccctgatcg gcggcgacag catcgagagc gagaaggacg agctgtgacc 2400taggctgcac aaagtggagt agtcagtcat cgatcaggaa ccagacacca gacttttatt 2460catacagtga agtgaagtga agtgcagtgc agtgagttgc tggtttttgt acaacttagt 2520atgtatttgt atttgtaaaa tacttctatc aataaaattt ctaattccta aaaccaaaat 2580ccag 2584183292DNAArtificial SequenceSynthetic construct, expression cassetter from pAG4705 18tccatgctgt cctactactt gcttcatccc cttctacatt ttgttctggt ttttggcctg 60catttcggat catgatgtat gtgatttcca atctgctgca atatgaatgg agactctgtg 120ctaaccatca acaacatgaa atgcttatga ggcctttgct gagcagccaa tcttgcctgt 180gtttatgtct tcacaggccg aattcctctg ttttgttttt caccctcaat atttggaaac 240atttatctag gttgtttgtg tccaggccta taaatcatac atgatgttgt cgtattggat 300gtgaatgtgg tggcgtgttc agtgccttgg atttgagttt gatgagagtt gcttctgggt 360caccactcac cattatcgat gctcctcttc agcataaggt aaaagtcttc cctgtttacg 420ttattttacc cactatggtt gcttgggttg gttttttcct gattgcttat gccatggaaa 480gtcatttgat atgttgaact tgaattaact gtagaattgt atacatgttc catttgtgtt 540gtacttcctt cttttctatt agtagcctca gatgagtgtg aaaaaaacag attatataac 600ttgccctata aatcatttga aaaaaatatt gtacagtgag aaattgatat atagtgaatt 660tttaagagca tgttttccta aagaagtata tattttctat

gtacaaaggc cattgaagta 720attgtagata caggataatg tagacttttt ggacttacac tgctaccttt aagtaacaat 780catgagcaat agtgttgcaa tgatatttag gctgcattcg tttactctct tgatttccat 840gagcacgctt cccaaactgt taaactctgt gttttttgcc aaaaaaaaat gcataggaaa 900gttgctttta aaaaatcata tcaatccatt ttttaagtta tagctaatac ttaattaatc 960atgcgctaat aagtcactct gtttttcgta ctagagagat tgttttgaac cagcactcaa 1020gaacacagcc ttaacccagc caaataatgc tacaacctac cagtccacac ctcttgtaaa 1080gcatttgttg catggaaaag ctaagatgac agcaacctgt tcaggaaaac aactgacaag 1140gtcataggga gagggagctt ttggaaaggt gccgtgcagt tcaaacaatt agttagcagt 1200agggtgttgg tttttgctca cagcaataag aagttaatca tggtgtaggc aacccaaata 1260aaacaccaaa atatgcacaa ggcagtttgt tgtattctgt agtacagaca aaactaaaag 1320taatgaaaga agatgtggtg ttagaaaagg aaacaatatc atgagtaatg tgtgggcatt 1380atgggaccac gaaataaaaa gaacattttg atgagtcgtg tatcctcgat gagcctcaaa 1440agttctctca ccccggataa gaaaccctta agcaatgtgc aaagtttgca ttctccactg 1500acataatgca aaataagata tcatcgatga catagcaact catgcatcat atcatgcctc 1560tctcaaccta ttcattccta ctcatctaca taagtatctt cagctaaatg ttagaacata 1620aacccataag tcacgtttga tgagtattag gcgtgacaca tgacaaatca cagactcaag 1680caagataaag caaaatgatg tgtacataaa actccagagc tatatgtcat attgcaaaaa 1740gaggagagct tataagacaa ggcatgactc acaaaaattc atttgccttt cgtgtcaaaa 1800agaggagggc tttacattat ccatgtcata ttgcaaaaga aagagagaaa gaacaacaca 1860atgctgcgtc aattatacat atctgtatgt ccatcattat tcatccacct ttcgtgtacc 1920acacttcata tatcatgagt cacttcatgt ctggacatta acaaactcta tcttaacatt 1980tagatgcaag agcctttatc tcactataaa tgcacgatga tttctcattg tttctcacaa 2040aaagcattca gttcattagt cctacaacaa cggatccacc atgagggtgt tgctcgttgc 2100cctcgctctc ctggctctcg ctgcgagcgc caccagcggc gtggacccgt tcgagaggaa 2160caagatcctg ggcaggggca tcaacatcgg caacgccctg gaggccccga acgagggcga 2220ctggggcgtg gtgatcaagg acgagtactt cgacatcatc aaggaggccg gcttcagcca 2280cgtgagaatc ccgatcaggt ggagcaccca cgcccaggcc ttcccgccgt acaagatcga 2340ggacaggttc ttcaagaggg tggacgaggt gatcaacggc gccctgaaga ggggcctggc 2400cgtggtgatc aaccagcacc actacgagga gctgatgaac gacccggagg agcacaagga 2460gaggttcctg gccctgtgga agcagatcgc cgacaggtac aaggactacc cggagaccct 2520gttcttcgag atcctgaacg agccgcacgg caacctgacc ccggagaagt ggaacgagct 2580gctggaggag gccctgaagg tgatcaggag catcgacaag aagcacacca tcatcatcgg 2640caccgccgag tggggcggca tcagcgccct ggagaagctg agggtgccga agtgggagaa 2700gaacgccatc gtgaccatcc actactacaa cccgttcgag ttcacccacc agggcgccga 2760gtgggtggag ggcagcgaga agtggctggg caggaagtgg ggcagcccgg acgaccagaa 2820gcacctgatc gaggagttca acttcatcga ggagtggagc aagaagaaca agaggccgat 2880ctacatcggc gagttcggcg cctacaggaa ggccgacctg gagagcagga tcaagtggac 2940cagcttcgtg gtgagggagg ccgagaagag gaggtggagc tgggcctact gggagttctg 3000cagcggcttc ggcgtgtacg acaccctgag gaagacctgg aacaaggacc tgctggaggc 3060cctgatcggc ggcgacagca tcgagagcga gaaggacgag ctgtgaccta ggctgcacaa 3120agtggagtag tcagtcatcg atcaggaacc agacaccaga cttttattca tacagtgaag 3180tgaagtgaag tgcagtgcag tgagttgctg gtttttgtac aacttagtat gtatttgtat 3240ttgtaaaata cttctatcaa taaaatttct aattcctaaa accaaaatcc ag 3292193317DNAArtificial SequenceSynthetic construct, expression cassette from pAG4706 19ataaaatttt agtgaagcta aagcggtgaa agattataga atttgatgtg ccagattaat 60aaatcgatta actcctaaag ttcaagccga gactacagac acatgagcta cataaatgag 120ccaaggactc gagcaaagac aaatcgacac agacattata attcaagtca ttctagaaga 180ttcatgagaa gagtatcatt tatttaaatc aatgacttga tcaaataaga cctaggagct 240actattgata atatatatca tgggtatcta gatcaagcat tatgaagaag agcctaagta 300gaaggcccca tgggctcgac cacaaaccca aggactcgac aataaagtct aggagggatc 360ccatagctaa aaggactcta gaagtgtatg tatggtaaag attttatcga gacaagaaat 420acgataaaga tcttaacaga atcggagtca tacttgtaaa aatagagttg gactcgtgta 480caacttggtc ttcgacttag ttcggtcatg aattcagtaa ccgactagat atgtaccatg 540gaacccctag ggcatgaggc tatgagccat aggatcatca gatccaaaca tacaccaaca 600aatccatcac acaccgaaga tccatattaa caagggatta gctactttac aatttcagag 660taacaaatag agccaaactc atagcacagg ggaacttcat atcacaaatg gaggcattga 720attgatataa aaagctaaag ttctaaaaag tttgaagtgc tgaaacttca aagccgctaa 780ctagtgaagc accgaagcct tccggggaga gaagacatac acgacacgtt agggacgtaa 840aatgacgaaa ttatacaact acctctatat gtaacactta tgtaatagaa aagacagaat 900ccatatgaag atgtataatg gatcaaccat ataaatagat aaacaatata tctgctatgg 960ggattggcat tcttgtatcc ctacgcctgt atatcccctg tttagagaac ctccgaaggt 1020atatgatgct gaagattatt gttgtcttgt ctttcatcat atatcgagtc tttccctagg 1080atattattat tcgcaatgtg cattacatgg ttaatcgatt gagagaacat gcatctcacc 1140tttagctgat aaacgataat ccatgtttta cacttcgtag ctactcatga gtttcgatat 1200acaaatttgt tttctggact acgtaccatt ccatcctctt aggagaggag aggaagtgtc 1260ctcgatttaa ttatgttgtc attttgtagt tcttcacaaa atctcaacag gtaccaaaca 1320cattgtttcc acaagacata ttttagtcac aacaaatcta tattattatt aatcactaaa 1380actatactga ggctcagatg cttttactag ctcttgctag tatgtgatgt aggtctcttt 1440cgacatcatt ccatcaaaat catatgatta gcccatacca aacatttcta taccattcag 1500agaccagaat agtcttttct aatagaaaaa aggaaaatag agtgggccga cgacgacaca 1560aattactgcg tggaccagaa aatagtgaga cacggaagac aaaagaagta aaagaggcaa 1620ggactacggc ccacatgaga ttcggccccg ccacctccgg caaccagcgg ccgatccaac 1680ggcagtgcat cctcaacggc gcgcgcgcgc gcgcgcgcgc acaacctcgt atatatcgcc 1740gcgcggaagc ggcgcgaccg aggaagcctt gtcctcgaca ccccctacac aggtgtcgcg 1800ctgcccccga cacgagtccc gcatgcgtcc cacgcggccg cgccagatcc cgcctccgcg 1860cgttgccacg ccctctataa acacccagct ctctccctcg ccctcatcta tcgcactcgt 1920agtcgtagct caagcatcag cggcaggagc tctgggcagc gtgcgcacgt ggggtaccta 1980gctcgctctg ctagcctacc ggatccacca tgagggtgtt gctcgttgcc ctcgctctcc 2040tggctctcgc tgcgagcgcc accagcggcg tggacccgtt cgagaggaac aagatcctgg 2100gcaggggcat caacatcggc aacgccctgg aggccccgaa cgagggcgac tggggcgtgg 2160tgatcaagga cgagtacttc gacatcatca aggaggccgg cttcagccac gtgagaatcc 2220cgatcaggtg gagcacccac gcccaggcct tcccgccgta caagatcgag gacaggttct 2280tcaagagggt ggacgaggtg atcaacggcg ccctgaagag gggcctggcc gtggtgatca 2340accagcacca ctacgaggag ctgatgaacg acccggagga gcacaaggag aggttcctgg 2400ccctgtggaa gcagatcgcc gacaggtaca aggactaccc ggagaccctg ttcttcgaga 2460tcctgaacga gccgcacggc aacctgaccc cggagaagtg gaacgagctg ctggaggagg 2520ccctgaaggt gatcaggagc atcgacaaga agcacaccat catcatcggc accgccgagt 2580ggggcggcat cagcgccctg gagaagctga gggtgccgaa gtgggagaag aacgccatcg 2640tgaccatcca ctactacaac ccgttcgagt tcacccacca gggcgccgag tgggtggagg 2700gcagcgagaa gtggctgggc aggaagtggg gcagcccgga cgaccagaag cacctgatcg 2760aggagttcaa cttcatcgag gagtggagca agaagaacaa gaggccgatc tacatcggcg 2820agttcggcgc ctacaggaag gccgacctgg agagcaggat caagtggacc agcttcgtgg 2880tgagggaggc cgagaagagg aggtggagct gggcctactg ggagttctgc agcggcttcg 2940gcgtgtacga caccctgagg aagacctgga acaaggacct gctggaggcc ctgatcggcg 3000gcgacagcat cgagagcgag aaggacgagc tgtgacctag gtccccgaat ttccccgatc 3060gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 3120ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 3180cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 3240tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 3300tactagatcg ggaattg 331720285DNAArtificial SequenceSynthetic construct, pAG4500_multiple cloning site 20gtttaaactg aaggcgggaa acgacaacct gatcatgagc ggagaattaa gggagtcacg 60ttatgacccc cgccgatgac gcgggacaag ccgttttacg tttggaactg acagaaccgc 120aacgttgaag gagccactca gcctaagcgg ccgcattgga cttaattaag tgaggccggc 180caagcgtcga tttaaatgta ccacatggcg cgccaactat catgcgatcg cttcatgtct 240aactcgagtt actggtacgt accaaatcca tggaatcaag gtacc 28521315PRTArtificial SequenceSynthetic construct, catalytic domain of AGR2314 21Gly Val Asp Pro Phe Glu Arg Asn Lys Ile Leu Gly Arg Gly Ile Asn1 5 10 15Ile Gly Asn Ala Leu Glu Ala Pro Asn Glu Gly Asp Trp Gly Val Val 20 25 30Ile Lys Asp Glu Phe Phe Asp Ile Ile Lys Glu Ala Gly Phe Ser His 35 40 45Val Arg Ile Pro Ile Arg Trp Ser Thr His Ala Ala Phe Pro Pro Tyr 50 55 60Lys Ile Glu Pro Ser Phe Phe Lys Arg Val Asp Glu Val Ile Asn Gly65 70 75 80Ala Leu Lys Arg Gly Leu Ala Val Val Ile Asn Ile His His Tyr Glu 85 90 95Glu Leu Met Asn Asp Pro Glu Glu His Lys Glu Arg Phe Leu Ala Leu 100 105 110Trp Lys Gln Ile Ala Asp Arg Tyr Lys Asp Tyr Pro Glu Thr Leu Phe 115 120 125Phe Glu Ile Leu Asn Glu Pro His Gly Asn Leu Thr Pro Glu Lys Trp 130 135 140Asn Glu Leu Leu Glu Glu Ala Leu Lys Val Ile Arg Ser Ile Asp Lys145 150 155 160Lys His Thr Val Ile Ile Gly Thr Ala Glu Trp Gly Gly Ile Ser Ala 165 170 175Leu Glu Lys Leu Arg Val Pro Lys Trp Glu Lys Asn Ala Ile Val Thr 180 185 190Ile His Tyr Tyr Asn Pro Phe Glu Phe Thr His Gln Gly Ala Glu Trp 195 200 205Val Pro Gly Ser Glu Lys Trp Leu Gly Arg Lys Trp Gly Ser Pro Asp 210 215 220Asp Gln Lys His Leu Ile Glu Glu Phe Asn Phe Ile Glu Glu Trp Ser225 230 235 240Lys Lys Asn Lys Arg Pro Ile Tyr Ile Gly Glu Phe Gly Ala Tyr Arg 245 250 255Lys Ala Asp Leu Glu Ser Arg Ile Lys Trp Thr Ser Phe Val Val Arg 260 265 270Glu Ala Glu Lys Arg Gly Trp Ser Trp Ala Tyr Trp Glu Phe Cys Ser 275 280 285Gly Phe Gly Val Tyr Asp Pro Leu Arg Lys Gln Trp Asn Lys Asp Leu 290 295 300Leu Glu Ala Leu Ile Gly Gly Asp Ser Ile Glu305 310 31522795DNAArtificial SequenceSynthetic construct, OB-2880 22cttagattag agaatgaaaa tttgattgct aaggcccaag attttgatgt ttgcaaagat 60acaattaccg atcttagaga taagaatgat atacttcgtg ctaagattgt tgaacttaca 120ccacaacctt ctatgccttc tgtgacatta acattacgtc acaaacaata gtatttttgt 180cataccttac atgttggtga cgtgattgtg acgaaaatca catcgtcaca gaaggtgcgt 240gttaaatggt gtactatgac gaataacaaa aaaacgtcat aatagtttat gacgcaaact 300acaaacgtca ctaatctatg acactcgaat tcgtcactaa ttatgtctaa atacgtcaca 360attcatgtag tcgtgccttg ccacgtggct gattacgtgg cgagatgaca tggcagttga 420cgtggcaggt gatgtggcga aaatgttgtg acgagttcat tcgtcacaga tgttatgacg 480tggcatgcca catggcagat gatgtggcaa aattatgtga caaaaatatt tgtcataaat 540atcaatgagg tggcaatata tgtgtgacga aatttttcat cacaaagtac gatgacgttg 600caatatattt atgacgaatt gttcatcata aggcgtgatg aattcatagc gtcatggaat 660attatgaaat cacatgctca aacactgata gtttaaactg aaggcgggaa acgacaacct 720gatcatgagc ggagaattaa gggagtcacg ttatgacccc cgccgatgac gcgggacaag 780ccgttttacg tttgg 795231211DNAArtificial SequenceSynthetic construct, OB-2832 23tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 60gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 120gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 180gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 240gttactagat cgggaattgg cgagctcgaa ttaattcagt acattaaaaa cgtccgcaat 300gtgttattaa gttgtctaag cgtcaatttg tttacaccac aatatatact aaaaaaactc 360aaggatctgt ctccagaaag gccttgcagg gtttggccac gcccacggac attccatctc 420agagccatga ttagaacgaa aaacacatga gagccgtcgt tgctaggagt cggtttcata 480tgttcgctaa aacaagagat ttgttttttt tctctctcgt acatacacga gtcagccctt 540ttaatctcag gttgacgtgc aatgtcgctc gtctaagcag aacattttga gaacaaatgt 600gttgtacatg agagttttgt gtacatggta cgtacattaa aacatcatca tttatcttag 660atctaacatc tctacttgct tgttatatat tttttttgta aaataacatc tttcaccact 720ttatatggtg ttgtttgcaa aatatacaga gcaattagag acgttagatt tgagatggac 780ggtgataatt taatacatgc ataatgtaca agaaaatcct aactgcacta gatatgttgt 840caaacatttt acctttgtta caaaaagaaa tgaatagatg ttgaacggtt gtctttcaag 900cctgttcgct gcggctttaa ttcaccaact gcaatgaaca acctgaaagg tgatcgttgc 960cgaacacatg ctgtttggca aagctagtag tacctttttt gtctgtcacc tggaatgatg 1020agaaaggaga caagaggaga gggctggcca ttgtttatat atatacgtat ttccattgct 1080ttgtggcatg caacagttca agggtccaaa ctggcaggtt ttcagccccg acaaatataa 1140taaaaaaact acaaaaaaaa aaggtccgtt tacattcctt ttttgacaac gctagtccgt 1200gcggagcgag c 121124696DNAArtificial SequenceSynthetic construct, OB-3252 24ggtgaaacaa ggtgcagaac tggacttccc gattccagtg gatgattttg ccttctcgct 60gcatgacctt agtgataaag aaaccaccat tagccagcag agtgccgcca ttttgttctg 120cgtcgaaggc gatgcaacgt tgtggaaagg ttctcagcag ttacagctta aaccgggtga 180atcagcgttt attgccgcca acgaatcacc ggtgactgtc aaaggccacg gccgtttagc 240gcgtgtttac aacaagctgt aagagcttac tgaaaaaatt aacatctctt gctaagctgg 300gagctctaga tccccgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag 360attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 420gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 480agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 540taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattggcga gctcgaatta 600attcaagtgt cttcgtacaa actgggggat ggggcagacc gccaggttca aaccgtttga 660ctagatgcgg ctggcaggct actttgcagt gcatgc 69625970DNAArtificial SequenceSynthetic construct, OB-2861 25gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca 60tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt 120cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa 180attatcgcgc gcggtgtcat ctatgttact agatcgggaa ttggcgagct cgaattaatt 240cagtacatta aaaacgtccg caatgtgtta ttaagttgtc taagcgtcaa tttgttatca 300agttgtctaa gcgtcaaaca ctgatagttt aaactgaagg cgggaaacga caacctgatc 360atgagcggag aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt 420tttacgtttg gaactgacag aaccgcaacg ttgaaggagc cactcagcct aagcggccgc 480attggactta attaagtgag gccggccaag cgtcgattta aatgtaccac atggcgcgcc 540aactatcatg cgatcgcttc atgtctaact cgagttactg gtacgtacca aatccatgga 600atcaaggtac ctccatgctg tcctactact tgcttcatcc ccttctacat tttgttctgg 660tttttggcct gcatttcgga tcatgatgta tgtgatttcc aatctgctgc aatataaatg 720gagactctgt gctaaccatc aacaacatga aatgcttatg aggcctttgc tgagcagcca 780atcttgcctg tgtttatgtc ttcacaggcc gaattcctct gttttgtttt tcaccctcaa 840tatttggaaa catttatcta ggttgtttgt gtccaggcct ataaatcata catgatgttg 900tcgtattgga tgtgaatgtg gtggcgtgtt cagtgccttg gatttgagtt tgatgagagt 960tgcttctggg 970261127DNAArtificial SequenceSynthetic construct, OB-2868 26aaaatcccgc gcgctggcga ttttaaaatc ggccctcgat agccagcagg gtgaaccgtg 60gcaaacgatt cgtttaattt ctgaatttta cccggaagac agcggtctgt tctccccgct 120attgctgaat gtggtgaaat tgaaccctgg cgaagcgatg ttcctgttcg ctgaaacacc 180gcacgcttac ctgcaaggcg tggcgctgga agtgatggca aactccgata acgtgctgcg 240tgcgggtctg acgcctaaat acattgatat tccggaactg gttgccaatg tgaaattcga 300agccaaaccg gctaaccagt tgttgaccca gccggtgaaa caaggtgcag aactggactt 360cccgattcca gtggatgatt ttgccttctc gctgcatgac cttagtgata aagaaaccac 420cattagccag cagagtgccg ccattttgtt ctgcgtcgaa ggcgatgcaa cgttgtggaa 480aggttctcag cagttacagc tcaaaccggg tgaatcagcg tttattgccg ccaacgaatc 540accggtgact gtcaaaggcc acggccgttt agcgcgtgtt tacaacaagc tgtaagagct 600tactgaaaaa attaacatct cttgctaagc tgggagctct agatccccga atttccccga 660tcgttcaaac atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat 720gattatcata taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat 780gacgttattt atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc 840gatagaaaac aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat 900gttactagat cgggaattgg cgagctcgaa ttaattcagt acattaaaaa cgtccgcaat 960gtgttattaa gttgtctaag cgtcaatttg ttatcaagtt gtctaagcgt caaacactga 1020tagtttaaac tgaaggcggg aaacgacaac ctgatcatga gcggagaatt aagggagtca 1080cgttatgacc cccgccgatg acgcgggaca agccgtttta cgtttgg 1127271303DNAArtificial SequenceSynthetic construct, OB-3170 27ttggggttcc ttatcctgtt gtcggagttg tgccattatc ctttccatgg ttgacctgag 60ctttagcctg tacactgtag actctactag aggtttacct gaggctgaat tcccgctgct 120aagatgtgat gttcccggcc ataagcaaag atgcaggttg tctttgcttt gtaaagatga 180aggttgtctt tgttttgtaa tcgaaaaaaa aaccctccga cttcgatagc aatccatttc 240ttgaaacgat atagctataa gctgcagcca caccttgcgt tgatgatgcc aaagctttct 300ttcgagtgcg atgcatgcac tggcctgttg agatcttatc aatatggcaa acagtaacct 360aacgtatatg actacatggt cttcatgctt ttgagaggtg cctcatagga aacagtcagg 420ccaatgattt tagggaatac aatatatttt tgctgttttt tttttgcaaa ttgtccatat 480tattacaaaa aaaactaaac atgcccaaag gcaatagctt tctaaataaa aatgaataac 540ggtccactta tatatgttgg ccagtaatca attctgaggc ctgacaaacc atgcatatat 600taacagtagg ttaatggccg tgcgtgaaaa aatttcaata caacaagaga ttgaaaaaaa 660agagtgtctt accaatatgt tattttataa gtaccaaatg tgtaggaaac ttgcattcat 720tttttccctg agaatggaaa aaaacaagac atactcattt tcaagttgaa ttgtcatagc 780aacacacatg ttgtatctgc cggttcatgc aattgtgcca accaaaatat ctaaatgaga 840tattcaagac tcaacagaat taaagtatgg aatagggtgt atatacactc aaccattatt 900aaatggtata atcatctatc tatatcacta taaaatctac cagtttaaac ttcacaaaac 960tcatctagct aatggaggcg ggaaacgaca acctgatcat gagcggagaa ttaagggagt 1020cacgttatga cccccgccga tgacgcggga caagccgttt tacgtttgga actgacagaa 1080ccgcaacgtt gaaggagcca ctcagcctaa gcggccgcat tggacttaat taagtgaggc 1140cggccaagcg tcgatttaaa tgtaccacat ggcgcgccaa ctatcatgcg atcgcttcat 1200gtctaactcg agttactggt acgtaccaaa tccatggaat caaggtacct ccatgctgtc 1260ctactacttg cttcatcccc ttctacattt tgttctggtt ttg

130328960DNAArtificial SequenceSynthetic construct, OB-3237 28aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat 60catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt 120atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga 180aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact 240agatcgggaa ttggcgagct cgaattaatt cagtacatta aaaacgtccg caatgtgtta 300ttaagttgtc taagcgtcaa tttgtttaca ccacaatata aaatctacct gttcgctgat 360aagccgttag gttgactatg tgactgttgg gcggcaaaat gaccacgcgg acggtctagc 420cccaaagccg gacggtccgc ggtccagaca gtctgcactg gtggtgtcgg cgtttcgacc 480ccggggggtc cctggaccga cgagtaaatt gtcgctgcgt gtcccagccc agatgggtcc 540gcgcgagacg gaacgcgaag atgggaaaac agcaaagggg aacccgcggc cttcgtgttg 600tcctgcgccc aggtcgggtg cgcttgcagt agggggttac aaccgttcgc gtgggagaga 660cagagagaga gcgagagcct tatgcgtcgg cccgttctcc cgcgcggcca accctctcgt 720acgagagccc tggaccttcc ttttatagac gtaaggagag ggcccaggtg tacaatgggg 780ggtgtagcag agtgctaacg tgtctagcag agaggagccg gagccctaag tacatgtcgt 840cgtggctgtc ggagaggttt tggcgccctg ttcatgtgat gtcgtggccg tcggaggagc 900gcttgagccc cgtggaagta cagctgtcgg ggctgtcgga tccttgctga cgtctccttg 960292855DNAArtificial SequenceSynthetic construct, OB-4448 29caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgacatg 60ttgaaaaagg tacatggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttgtatgt 360gttttatgtg tctagattca tcatcctcta tttgaatata gacagaaaaa tcataactaa 420aacgaatact atttgggaac ggagggagta ctactttggc agaatgcccc caggaaagta 480ccagtttcag gggtagtttg gaaggctaaa cctagggagg gaaaaccccc cacatgtaac 540taaatatctt attcaaatgt tacccctagg gattactcac cctgggaaat gagaagggtc 600ccaaggggat ttcggtttct attatttttt ctgcaaacca tttcagagca atgatatgaa 660accaagctaa ctacttataa catttcttaa gaatatcaga cataggaaag tgatggcctg 720gaaccaaagt aagactggta gataaataga tcactagaat aaaccctgac agttcatagc 780cttcatagaa gcaaaaggaa acactacggg agcaattggt tgcttgcact agcaattcac 840tgcattgggt ctaatgcagg atagactaag ccagcataag tgtgcgcaat gtgtttgtgt 900ttggttgcca tgttataagt aagttgcatt tgctaatatc tttctcctga ctctaatgag 960tccacttttg ctgactggtg ggcgaaagta agtaagcaag tgcacaaatc caaaagaaga 1020ggctttaaca gtatcatcat cttgggggct tggtgtttat ggcttcatcg taataaggtg 1080gtttttgatg gtgtcagtcc ttcaattatt ggcataaagg caattttttt ggatgaagtt 1140gaattctgga ggcttgccgg tgctaggcat cttgaggctt tggttcctgg tgctggaatt 1200tttaggtcaa gggttctttt gggtgattag tgaagagcag gtgtgtgtgg tctgctcgca 1260ctttttgttg ttcgttctcc tattgcgtgc tgttgtttcc aggcgcattt atggaggctg 1320cagttttgtg cgcagcagaa gttggtggtt ttgtgttttg tgttttgcct attttggcat 1380tgtactttgg tccattttgg actgttttct tctcttaatt taatgatgtg cagctctcct 1440gcgcgtttaa gaaaaaaaaa agttggctgt tttgtatttc ttgtgatcac ccatgcttgt 1500tgtggtcaga ttaaactctc acgtttaatg ctacagaagc atccatgaga caatgaaaca 1560ccgctcaaaa gccacgtagt agcataccct gacttatgaa taaagcaact cgatctgatt 1620tatttgagaa aacaggaaac tgacaagtta tttttaacac aaaatttcat taaaaacgaa 1680tggtagacaa ttaccaatct gtaggtccct ggcttgcaag tcctcccaat gtctaagaaa 1740tcaaatagga actgcaggca agccagcaag aaagtattaa tcactggata taaaatataa 1800agaaaaaaga aggaaagacg gctactcggc tagcatatgt ttttgttagg ggtgaaaatg 1860gatacttatt cagaaatcat ttttgatctt ttttctttaa ttaggaataa ataggatata 1920gaatatgcta agcaaattca tattcttgtt cttagcattg ggcttgtaaa gattcataaa 1980aggtaaatct caaatttatc atatatctta aatggtagat ataaaattca gatacaaata 2040tttttcaact tttttgttgt agggaacaaa ttatattaaa aaaaattatg cacaattcta 2100ttcttatttg taataatgtg cttgataaca taataaaaga ttaccatcaa atttcacaca 2160cacccaccca cccacccacc cctgcacgca cgcgcgcgca cacacactat atgtgtgttc 2220aaacactgat agtttaaact gaaggcggga aacgacaacc tgatcatgag cggagaatta 2280agggagtcac gttatgaccc ccgccgatga cgcgggacaa gccgttttac gtttggaact 2340gacagaaccg caacgttgaa ggagccactc agcctaagcg gccgcattgg acttaattaa 2400gtgaggccgg ccaagcgtcg atttaaatgt accacatggc gcgccaacta tcatgcgatc 2460gcttcatgtc taactcgagt tactggtacg taccaaatcc atggaatcaa ggtacctcca 2520tgctgtccta ctacttgctt catccccttc tacattttgt tctggttttt ggcctgcatt 2580tcggatcatg atgtatgtga tttccaatct gctgcaatat gaatggagac tctgtgctaa 2640ccatcaacaa catgaaatgc ttatgaggcc tttgctgagc agccaatctt gcctgtgttt 2700atgtcttcac aggccgaatt cctctgtttt gtttttcacc ctcaatattt ggaaacattt 2760atctaggttg ttttgtgtcc aggcctataa atcataaatg atgttgtcgt attggatgtg 2820aatgtggtgg cgtgttcagt gccttggatt tgagt 2855302336DNAArtificial SequenceSynthetic construct, RB_BC2ES2_472x 30caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgacatg 60ttgaaaaagg tacaaggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttgtatgt 360gttttatgtg tctagattca tcatcctcta tttgaatata gacagaaaaa tcataactaa 420aacgaatact atttgggaac ggagggagta ctactttggc agaatgcccc caggaaagta 480ccagtttcag gggtagtttg gaaggctaaa cctagggagg gaaaaccccc cacatgtaac 540taaatatctt attcaaatgt tacccctagg gattactcac cctgggaaat gagaagggtc 600ccaaggggat ttcggtttct attatttttt ctgcaaacca tttcagagca atgatatgaa 660accaagctaa ctacttataa catttcttaa gaatatcaga cataggaaag tgatggcctg 720gaaccaaagt aagactggta gataaataga tcactagaat aaaccctgac agttcatagc 780cttcatagaa gcaaaaggaa acactacggg agcaattggt tgcttgcact agcaattcac 840tgcattgggt ctaatgcagg atagactaag ccagcataag tgtgcgcaat gtgtttgtgt 900ttggttgcca tgttataagt aagttgcatt tgctaatatc tttctcctga ctctaatgag 960tccacttttg ctgactggtg ggcgaaagta agtaagcaag tgcacaaatc caaaagaaga 1020ggctttaaca gtatcatcat cttgggggct tggtgtttat ggcttcatcg taataaggtg 1080gtttttgatg gtgtcagtcc ttcaattatt ggcataaagg caattttttt ggatgaagtt 1140gaattctgga ggcttgccgg tgctaggcat cttgaggctt tggttcctgg tgctggaatt 1200tttaggtcaa gggttctttt gggtgattag tgaagagcag gtgtgtgtgg tctgctcgca 1260ctttttgttg ttcgttctcc tattgcgtgc tgttgtttcc aggcgcattt atggaggctg 1320cagttttgtg cgcagcagaa gttggtggtt ttgtgttttg tgttttgcct attttggcat 1380tgtactttgg tccattttgg actgttttct tctcttaatt taatgatgtg cagctctcct 1440gcgcgtttaa gaaaaaaaaa agttggctgt tttgtatttc ttgtgatcac ccatgcttgt 1500tgtggtcaga ttaaactctc acgtttaatg ctacagaagc atccatgaga caatgaaaca 1560ccgctcaaaa gccacgtagt agcataccct gacttatgaa taaagcaact cgatctgatt 1620tatttgagaa aacaggaaac tgacaagtta tttttaacac aaaatttcat taaaaacgaa 1680tggtagacaa ttaccaatct gtaggtccct ggcttgcaag tcctcccaat gtctaagaaa 1740tcaaatagga actgcaggca agccagcaag aaagtattaa tcactggata taaaatataa 1800agaaaaaaga aggaaagacg gctactcggc tagcatatgt ttttgttagg ggtgaaaatg 1860gatacttatt cagaaatcat ttttgatctt ttttctttaa ttaggaataa ataggatata 1920gaatatgcta agcaaattca tattcttgtt cttagcattg ggcttgtaaa gattcataaa 1980aggtaaatct cgaatttatc atatatctta aatggtagat ataaaattca gatacaaata 2040tttttcaact tttttgttgt agggaacaaa ttatattaaa aaaaattatg cacaattcta 2100ttcttatttg taataatgtg cttgataaca taataaaaga ttaccatcaa atttcacaca 2160cacccaccca cccacccacc cctgcacgca cgcgcgcgca cacacactat atgtgtgttc 2220aaacactgat agtttaaact gaaggcggga aacgacaacc tgatcatgag cggagaatta 2280agggagtcac gttatgaccc ccgccgatga cgcgggacaa gccgttttac gtttgg 2336312127DNAArtificial SequenceSynthetic construct, OB-4451 31gggcccggta gttctacttc tgttcatgtt tgtgttagat ccgtgtttgt gttagatccg 60tgctgctagc gttcgtacac ggatgcgacc tgtacgtcag acacgttctg attgctaact 120tgccagtgtt tctctttggg gaatcctggg atggctctag ccgttccgca gacgggatcg 180atttcatgat tttttttgtt tcgttgcata gggtttggtt tgcccttttc ctttatttca 240atatatgccg tgcacttgtt tgtcgggtca tcttttcatg cttttttttg tcttggttgt 300gatgatgtgg tctggttggg cggtcgttct agatcggagt agaattctgt tacccacttt 360catccctagt ttttgttctg gattcaagca tctcaaaatt gtttacctga agtttatcag 420ttttgagaaa gcggcgcccc tgtcgactac catcaggcat tcggactaca actgtcacag 480caccctctgc gtctggagac ggttccggtg gtaatgatgc ttgcttcgaa gtgagactgg 540actctagctc ctatttaatc aaaacatcag ggacaacatg acaaatagta gtcaaatatc 600caggcaagaa aaaaaaacca taaacaatga aaatactgat caaaagtcct gtttggatct 660cctaagaaaa atgagaatga gatccaaaca attggattct agaatccagc tatctatccc 720aaacccatta tttggcgaga ttttcactat gcagaggcaa tgatcactat aagaataaga 780ttcaaacacc cacttattat ttttttaatc cagaaaccag attctacatt cactatagaa 840tccagaactt caatatggga atgagatcca aatagaccct aagccaaaat gaaattggtg 900agatgaagtg gctagttgtc ataacctcct gtaaagaaga cagcggttta cagtcccaac 960acccaaataa acatgacatt aatataatga ctacaactca caacctaaac ctaaaccaat 1020atacatccaa acataagaca aaaggagaac tgagttttat atgatcacac tgatgaactg 1080atgctgtagt ctagcattca agtgtttaag atagttgact ataaaccctt caccttgcag 1140attacatgtg acagaaagat acctcttcct caagttgttt tttacgcctt tcctcctcct 1200cttgcttctg tttctcaaga acagcttctc tcgcagctgt ttcttcaagg cgacggagct 1260cagcctcctg tagggccttt aactcctttt cttgatcagc ttgtagcgat gcaaggtact 1320catcgtccta caaatttaaa atttataaaa gtgctcaccc atagtggcaa ttatgaacaa 1380tggataaatc ttagacctca aacctgctgc tctcgtaata accgctgttc agttaatgct 1440ggtgatggag aatgagatat tgggggataa taagtagagg ttctgtgaga aggcatagag 1500aaaggatatg ttggtccacc aaacattgca gcctcaagca taacagcttc atcatgttcc 1560tcagaagaaa tgccacccca ctttattgta aaaaaagacg tcagaaatta aacaaatcca 1620tctaatgtct tagcgcacat tggaaccaca gattataata cctcagatgg gaaatcatct 1680ccattatact ggtgattgtt cagaacaggg ctagcacctg ggtgcactat ttttggtaat 1740tcattgtcct cagaaggggc acgccgagaa cgacgcctaa ctaacggctg ttcttctaca 1800tcttcagcct cctcctggaa gctctcgtca tctattggtt gcctagatgt tccagccttt 1860cctgaagcta gtccttgcct ccaaaaggaa attgcatgta taaggaatca aatgatactg 1920tagtagggta gcctgagtga agaggtgggt agtaaagtta acattcacct ctcaactatt 1980ccatttgcgg tttccatgcc tgctttatca gaagaatggt ccctcaaatt cacttcctct 2040tgatgctggc ctccttgctc gactatctga tgattattga aaaattagag atgacatcaa 2100gataggttca agtaagcatg ttgggga 2127324044DNAArtificial SequenceSynthetic construct, WT_BxA (OB-4541) 32caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgacatg 60ttgaaaaagg tacaaggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttgtatgt 360gttttatgtg tctagattca tcatcctcta tttgaatata gacagaaaaa tcataactaa 420aacgaatact atttgggaac ggagggagta ctactttggc agaatgcccc caggaaagta 480ccagtttcag gggtagtttg gaaggctaaa cctagggagg gaaaaccccc cacatgtaac 540taaatatctt attcaaatgt tacccctagg gattactcac cctgggaaat gagaagggtc 600ccaaggggat ttcggtttct attatttttt ctgcaaacca tttcagagca atgatatgaa 660accaagctaa ctacttataa catttcttaa gaatatcaga cataggaaag tgatggcctg 720gaaccaaagt aagactggta gataaataga tcactagaat aaaccctgac agttcatagc 780cttcatagaa gcaaaaggaa acactacggg agcaattggt tgcttgcact agcaattcac 840tgcattgggt ctaatgcagg atagactaag ccagcataag tgtgcgcaat gtgtttgtgt 900ttggttgcca tgttataagt aagttgcatt tgctaatatc tttctcctga ctctaatgag 960tccacttttg ctgactggtg ggcgaaagta agtaagcaag tgcacaaatc caaaagaaga 1020ggctttaaca gtatcatcat cttgggggct tggtgtttat ggcttcatcg taataaggtg 1080gttttgatgg tgtcagtcct tcaattattg gcataaaggc aatttttttg gatgaagttg 1140aattctggag gcttgccggt gctaggcatc ttgaggcttt ggttcctggt gctggaattt 1200ttaggtcaag ggttcttttg ggtgattagt gaagagcagg tgtgtgtggt ctgctcgcac 1260tttttgttgt tcgttctcct attgcgtgct gttgtttcca ggcgcattta tggaggctgc 1320agttttgtgc gcagcagaag ttggtggttt tgtgttttgt gttttgccta ttttggcatt 1380gtactttggt ccattttgga ctgttttctt ctcttaattt aatgatgtgc agctctcctg 1440cgcgtttaag aaaaaaaaaa gttggctgtt ttgtatttct tgtgatcacc catgcttgtt 1500gtggtcagat taaactctca cgtttaatgc tacagaagca tccatgagac aatgaaacac 1560cgctcaaaag ccacgtagta gcataccctg acttatgaat aaagcaactc gatctgattt 1620atttgagaaa acaggaaact gacaagttat ttttaacaca aaatttcatt aaaaacgaat 1680ggtagacaat taccaatctg taggtccctg gcttgcaagt cctcccaatg tctaagaaat 1740caaataggaa ctgcaggcaa gccagcaaga aagtattaat cactggatat aaaatataaa 1800gaaaaaagaa ggaaagacgg ctactcggct agcatatgtt tttgttaggg gtgaaaatgg 1860atacttattc agaaatcatt tttgatcttt tttctttaat taggaataaa taggatatag 1920aatatgctaa gcaaattcat attcttgttc ttagcattgg gcttgtaaag attcataaaa 1980ggtaaatctc aaatttatca tatatcttaa atggtagata taaaattcag atacaaatat 2040ttttcaactt ttttgttgta gggaacaaat tatattaaaa aaaattatgc acaattctat 2100tcttatttgt aataatgtgc ttgataacat aataaaagat taccatcaaa tttcacacac 2160acccacccac ccacccaccc ctgcacgcac gcgcgcgcac acacactata tgtgtgtata 2220tatattaaat attgaattta tcctaacaat ttggataccc actttcatcc ctagtttttg 2280ttctggattc aagcatctca aaattgttta cctgaagttt atcagttttg agaaagcggc 2340gcccctgtcg actaccatca ggcattcgga ctacaactgt cacagcaccc tctgcgtctg 2400gagacggttc cggtggtaat gatgcttgct tcgaagtgag actggactct agctcctatt 2460taatcaaaac atcagggaca acatgacaaa tagtagtcaa atatccaggc aagaaaaaaa 2520aaccataaac aatgaaaata ctgatcaaaa gtcctgtttg gatctcctaa gaaaaatgag 2580aatgagatcc aaacaattgg attctagaat ccagctatct atcccaaacc cattatttgg 2640cgagattttc actatgcaga ggcaatgatc actataagaa taagattcaa acacccactt 2700attatttttt taatccagaa accagattct acattcacta tagaatccag aacttcaata 2760tgggaatgag atccaaatag accctaagcc aaaatgaaat tggtgagatg aagtggctag 2820ttgtcataac ctcctgtaaa gaagacagcg gtttacagtc ccaacaccca aataaacatg 2880acattaatat aatgactaca actcacaacc taaacctaaa ccaatataca tccaaacata 2940agacaaaagg agaactgagt tttatatgat cacactgatg aactgatgct gtagtctagc 3000attcaagtgt ttaagatagt tgactataaa cccttcacct tgcagattac atgtgacaga 3060aagatacctc ttcctcaagt tgttttttac gcctttcctc ctcctcttgc ttctgtttct 3120caagaacagc ttctctcgca gctgtttctt caaggcgacg gagctcagcc tcctgtaggg 3180cctttaactc cttttcttga tcagcttgta gcgatgcaag gtactcatcg tcctacaaat 3240ttaaaattta taaaagtgct cacccatagt ggcaattatg aacaatggat aaatcttaga 3300cctcaaacct gctgctctcg taataaccgc tgttcagtta atgctggtga tggagaatga 3360gatattgggg gataataagt agaggttctg tgagaaggca tagagaaagg atatgttggt 3420ccttcaggaa ctccaccaaa cattgcagcc tcaagcataa cagcttcatc atgttcctca 3480gaagaaatgc caccccactt tattgtaaaa aaagacgtca gaaattaaac aaatccatct 3540aatgtcttag cgcacattgg aaccacagat tataatacct cagatgggaa atcatctcca 3600ttatactggt gattgttcag aacagggcta gcacctgggt gcactatttt tggtaattca 3660ttgtcctcag aaggggcacg ccgagaacga cgcctaacta acggctgttc ttctacatct 3720tcagcctcct cctggaagct ctcgtcatct attggttgcc tagatgttcc agcctttcct 3780gaagctagtc cttgcctcca aaaggaaatt gcatgtataa ggaatcaaat gatactgtag 3840tagggtagcc tgagtgaaga ggtgggtagt aaagttaaca ttcacctctc aactattcca 3900tttgcggttt ccatgcctgc tttatcagaa gaatggtccc tcaaattcac ttcctcttga 3960tgctggcctc cttgctcgac tatctgatga ttattgaaaa attagagatg acatcaagat 4020aggttcaagt aagcatgttg ggga 4044333539DNAArtificial SequenceSynthetic construct, WT_E (OB-4545, OB-4546) 33caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgaaatg 60ttgaaaaagg tacaaggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttttatgg 360gtgtctagat tcatcatcct ccatttgaat atagaaagaa aaatcataac taaaacgaat 420actatttggg aacggaggga gttactattt tgcccgaatg ctcccaggaa agcacgagtt 480tcagggctag tttggaaggc taaacctagg gagggaaaac cccccacatg taactaaata 540tcttattcaa atgttacccc tagggattac tcaccctggg aaatgagaag ggtcccaagg 600ggatttcggt ttctattatt ttttctgcaa accatttcag agcaatgata tgaaaccaac 660ctaactactt ataacatttc ttaagaatat cagacatagg aaagtgatgg cctggaagca 720aagtaagact gatagataaa tagatcacta gaataaaccc tgacagttca tagccttcat 780agaagcaaaa ggaaacacta cgggagcaat tggttgcttg cactagcaat tcactgcatt 840gggtctaatg caggatagac taagccagca taagtgtgca caatgtgttt gtgtttggtt 900gccatgttat aagtaagttg catttgctaa tataatgttt ggttagttgg ctgttttgta 960tttcttgtga tcacccatgc ttgttgtggt gagattaaac tctcacgttt aatgctacag 1020aagcatccat gagacaatga aacatcgctc aaaagccacg tagtagcata ccctgactta 1080tgaataaagc aactcgatct gatttatttg agaaaacagg aaactgacaa gttattttta 1140acacaaaatt tcattaaaaa cgaatggtag acaattacca atctgtaggt ccctggcttg 1200caagtcctcc caatgtctaa gaaatcaaat aggaactgca ggcaagccag caagaaagta 1260ttaatcactg gatataaaat ataaagaaaa aagaaggaaa gacggctact cggctagcat 1320atgtttttgt taggggtgaa aatggatact tattcagaaa tcattttttg atcttttttc 1380tttaattagg aataaatagg atatagaata tgctaagcaa attcatattc ttgttcttag 1440cattgggctt gtaaagattc ataaaaggta aatctcaaat ttatcatata tcttaaatgg 1500tagatataaa attcagatac aaatattttt caactttttt gctgtaggga acaaataata 1560taaaaaaatt tatgcacaat tctattctta tttgtaataa tgtgcttgat aacataataa 1620aagattacca tcaaatttca cacacacaca cacacacacc cacccccccc acacccccac 1680gcacgagcgc acacacacac tatatgtgtg tatatatatt aaatattgaa tttatcctaa 1740caatttggat acccactttc atccctagtt tttgttctgg attcaagcat ctcaaaattg 1800tttacctgaa gtttatcagt tttgagaaag cggcgcccct gtcgactacc atcaggcatt 1860cggactacaa ctgtcacagc accctctgcg tctggagacg gttccggtgg taatgatgct 1920tgcttcgaag tgagactgga ctctagttcc tatttaatca aaacatcagg gacaacatga 1980caaatagtag tcaaatatcc aggcaagaaa aaaaaccata aacaatgaaa atactgatca 2040aaagtcctgt ttggatctcc taagaaaaat gagaatgaga tccaaacaat tggattctag 2100aatccagcta tctatcccaa acccattatt tggcgagatt ttcactatgc agaggcaatg 2160atcactataa gaataagatt caaacaccca cttattattt ttttaatcca gaaaccagat 2220tctacattca ctatagaatc cagaacttca atatgggaat gagatccaaa

tagaccctaa 2280gccaaaatga aattggtgag atgaagtggc tagttgtcat aacctcctgt aaagaagaca 2340gcggtttaca gtcccaactc ccaaataaac atatgacatt aatataatga ctacaactca 2400caacctaaac ctaaaccaat atacatccaa acataagaca aaaggagaac tgagttttat 2460atgatcacac tgatgaactg atgctgtagt ctagcattcc agtgtttaag atagttgact 2520ataaaccctt caccttgcag attacatgtg acagaaagat acctcttcct caagttgttt 2580tttacgcctt tcctcctcct cttgcttctg tttctcaaga acagcttctc tcgcagctgt 2640ttcttcaagg cgacggagct cagcctcctg tagggccttt aactcctttt cttgatcagc 2700ttgtagcgat gcaaggtact catcgtccta caaatttaaa atttataaaa gtgctcaccc 2760atagtggcaa ttatgagcaa tggataaatc ttagacctca aacctgctgc tctcgtaata 2820accgctgttc agttaatgct ggtgatggag aatgagatat tgggggataa taagtagagg 2880ttctgtgaga aggcatagag aaaggatatg ttggtccttc aggaactcca ccaaacattg 2940cagcctcaag cataacagct tcatcatgtt cctcagaaga aatgccaccc cactttattg 3000taaaaagacg tcagaaatta aacaaatcca tctaatgtct tagcgcacat tggaaccaca 3060gattatataa tacctcagat gggaaatcat ctccattata ctggtgattg ttcagaacag 3120ggctagcacc tgggtgcact atttttggta attcattgtc ctcagaaggg gcacgccgag 3180aacgacgcct aactaacggc tgttcttcta catcttcagc ctcctcctgg aagctctcgt 3240catctattgg ttgcctagat gttccagcct tttctgaagc tagtccttgc ctccaaaagg 3300aaattgcatg tataaggaat caaatgatac tgtagtaggg tagcctgagt gaagaggtgg 3360gtagtaaagt taacattcac ctctcaacta ttccatttgc ggtttccatg cctgctttat 3420cagaagaatg gtccctcaaa ttcacttcct cttgatgctg gcctccttgc tcgactatct 3480gatgattatt gaaaaattag agatgacatc aagataggtt caagtaagca tgttgggga 3539343540DNAArtificial SequenceSynthetic construct, WT_G (OB-4547, OB-4548) 34caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgaaatg 60ttgaaaaagg tacaaggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttttatgg 360gtgtctagat tcatcatcct ccatttgaat atagaaagaa aaatcataac taaaacgaat 420actatttggg aacggaggga gttactattt tgcccgaatg ctcccaggaa agcacgagtt 480tcagggctag tttggaaggc taaacctagg gagggaaaac cccccacatg taactaaata 540tcttattcaa atgttacccc tagggattac tcaccctggg aaatgagaag ggtcccaagg 600ggatttcggt ttctattatt ttttctgcaa accatttcag agcaatgata tgaaaccaac 660ctaactactt ataacatttc ttaagaatat cagacatagg aaagtgatgg cctggaagca 720aagtaagact gatagataaa tagatcacta gaataaaccc tgacagttca tagccttcat 780agaagcaaaa ggaaacacta cgggagcaat tggttgcttg cactagcaat tcactgcatt 840gggtctaatg caggatagac taagccagca taagtgtgca caatgtgttt gtgtttggtt 900gccatgttat aagtaagttg catttgctaa tataatgttt ggttagttgg ctgttttgta 960tttcttgtga tcacccatgc ttgttgtggt gagattaaac tctcacgttt aatgctacag 1020aagcatccat gagacaatga aacatcgctc aaaagccacg tagtagcata ccctgactta 1080tgaataaagc aactcgatct gatttatttg agaaaacagg aaactgacaa gttattttta 1140acacaaaatt tcattaaaaa cgaatggtag acaattacca atctgtaggt ccctggcttg 1200caagtcctcc caatgtctaa gaaatcaaat aggaactgca ggcaagccag caagaaagta 1260ttaatcactg gatataaaat ataaagaaaa aagaaggaaa gacggctact cggctagcat 1320atgtttttgt taggggtgaa aatggatact tattcagaaa tcattttttg atcttttttc 1380tttaattagg aataaatagg atatagaata tgctaagcaa attcatattc ttgttcttag 1440cattgggctt gtaaagattc ataaaaggta aatctcaaat ttatcatata tcttaaatgg 1500tagatataaa attcagatac aaatattttt caactttttt gctgtaggga acaaataata 1560taaaaaaatt tatgcacaat tctattctta tttgtaataa tgtgcttgat aacataataa 1620aagattacca tcaaatttca cacacacaca cacacacacc cacccccccc cacaccccca 1680cgcacgagcg cacacacaca ctatatgtgt gtatatatat taaatattga atttatccta 1740acaatttgga tacccacttt catccctagt ttttgttctg gattcaagca tctcaaaatt 1800gtttacctga agtttatcag ttttgagaaa gcggcgcccc tgtcgactac catcaggcat 1860tcggactaca actgtcacag caccctctgc gtctggagac ggttccggtg gtaatgatgc 1920ttgcttcgaa gtgagactgg actctagttc ctatttaatc aaaacatcag ggacaacatg 1980acaaatagta gtcaaatatc caggcaagaa aaaaaaccat aaacaatgaa aatactgatc 2040aaaagtcctg tttggatctc ctaagaaaaa tgagaatgag atccaaacaa ttggattcta 2100gaatccagct atctatccca aacccattat ttggcgagat tttcactatg cagaggcaat 2160gatcactata agaataagat tcaaacaccc acttattatt tttttaatcc agaaaccaga 2220ttctacattc actatagaat ccagaacttc aatatgggaa tgagatccaa atagacccta 2280agccaaaatg aaattggtga gatgaagtgg ctagttgtca taacctcctg taaagaagac 2340agcggtttac agtcccaact cccaaataaa catatgacat taatataatg actacaactc 2400acaacctaaa cctaaaccaa tatacatcca aacataagac aaaaggagaa ctgagtttta 2460tatgatcaca ctgatgaact gatgctgtag tctagcattc cagtgtttaa gatagttgac 2520tataaaccct tcaccttgca gattacatgt gacagaaaga tacctcttcc tcaagttgtt 2580ttttacgcct ttcctcctcc tcttgcttct gtttctcaag aacagcttct ctcgcagctg 2640tttcttcaag gcgacggagc tcagcctcct gtagggcctt taactccttt tcttgatcag 2700cttgtagcga tgcaaggtac tcatcgtcct acaaatttaa aatttataaa agtgctcacc 2760catagtggca attatgagca atggataaat cttagacctc aaacctgctg ctctcgtaat 2820aaccgctgtt cagttaatgc tggtgatgga gaatgagata ttgggggata ataagtagag 2880gttctgtgag aaggcataga gaaaggatat gttggtcctt caggaactcc accaaacatt 2940gcagcctcaa gcataacagc ttcatcatgt tcctcagaag aaatgccacc ccactttatt 3000gtaaaaagac gtcagaaatt aaacaaatcc atctaatgtc ttagcgcaca ttggaaccac 3060agattatata atacctcaga tgggaaatca tctccattat actggtgatt gttcagaaca 3120gggctagcac ctgggtgcac tatttttggt aattcattgt cctcagaagg ggcacgccga 3180gaacgacgcc taactaacgg ctgttcttct acatcttcag cctcctcctg gaagctctcg 3240tcatctattg gttgcctaga tgctccagcc ttttctgaag ctagtccttg cctccaaaag 3300gaaattgcat gtataaggaa tcaaatgata ctgtagtagg gtagcctgag tgaagaggtg 3360ggtagtaaag ttaacattca cctctcaact attccatttg cggtttccat gcctgcttta 3420tcagaagaat ggtccctcaa attcacttcc tcttgatgct ggcctccttg ctcgactatc 3480tgatgattat tgaaaaatta gagatgacat caagataggt tcaagtaagc atgttgggga 3540353540DNAArtificial SequenceSynthetic construct, Null_ BC2ES2_512x (OB-4578 to OB-4580) 35caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgaaatg 60ttgaaaaagg tacaagggct gggcggattt aaacagtaga atgaaaaggt gccacaagaa 120aactcgtcaa agaattgact acgcgtcaat gttccatagt taaaaagact tgaactctgg 180atcagggact ttcaaacaag gatagctgcc tggtcaccag tcattaactg taatgtaatg 240gccatagatg atgcatgagt acaataataa aaaaacacca tccagccaaa tatatactcc 300ctgtcacaaa tgaaaattcg ttttagataa ttagtggatt catacaatat ttgttttatg 360ggtgtctaga ttcatcatcc tccatttgaa tatagaaaga aaaatcataa ctaaaacgaa 420tactatttgg gaacggaggg agttactatt ttgcccgaat gctcccagga aagcacgagt 480ttcagggcta gtttggaagg ctaaacctag ggagggaaaa ccccccacat gtaactaaat 540atcttattca aatgttaccc ctagggatta ctcaccctgg gaaatgagaa gggtcccaag 600gggatttcgg tttctattat tttttctgca aaccatttca gagcaatgat atgaaaccaa 660cctaactact tataacattt cttaagaata tcagacatag gaaagtgatg gcctggaagc 720aaagtaagac tgatagataa atagatcact agaataaacc ctgacagttc atagccttca 780tagaagcaaa aggaaacact acgggagcaa ttggttgctt gcactagcaa ttcactgcat 840tgggtctaat gcaggataga ctaagccagc ataagtgtgc acaatgtgtt tgtgtttggt 900tgccatgtta taagtaagtt gcatttgcta atataatgtt tggttagttg gctgttttgt 960atttcttgtg atcacccatg cttgttgtgg tgagattaaa ctctcacgtt taatgctaca 1020gaagcatcca tgagacaatg aaacatcgct caaaagccac gtagtagcat accctgactt 1080atgaataaag caactcgatc tgatttattt gagaaaacag gaaactgaca agttattttt 1140aacacaaaat ttcattaaaa acgaatggta gacaattacc aatctgtagg tccctggctt 1200gcaagtcctc ccaatgtcta agaaatcaaa taggaactgc aggcaagcca gcaagaaagt 1260attaatcact ggatataaaa tataaagaaa aaagaaggaa agacggctac tcggctagca 1320tatgtttttg ttaggggtga aaatggatac ttattcagaa atcatttttt gatctttttt 1380ctttaattag gaataaatag gatatagaat atgctaagca aattcatatt cttgttctta 1440gcattgggct tgtaaagatt cataaaaggt aaatctcaaa tttatcatat atcttaaatg 1500gtagatataa aattcagata caaatatttt tcaacttttt tgctgtaggg aacaaataat 1560ataaaaaaat ttatgcacaa ttctattctt atttgtaata atgtgcttga taacataata 1620aaagattacc atcaaatttc acacacacac acacacacac ccaccccccc cacaccccca 1680cgcacgagcg cacacacaca ctatatgtgt gtatatatat taaatattga atttatccta 1740acaatttgga tacccacttt catccctagt ttttgttctg gattcaagca tctcaaaatt 1800gtttacctga agtttatcag ttttgagaaa gcggcgcccc tgtcgactac catcaggcat 1860tcggactaca actgtcacag caccctctgc gtctggagac ggttccggtg gtaatgatgc 1920ttgcttcgaa gtgagactgg actctagttc ctatttaatc aaaacatcag ggacaacatg 1980acaaatagta gtcaaatatc caggcaagaa aaaaaaccat aaacaatgaa aatactgatc 2040aaaagtcctg tttggatctc ctaagaaaaa tgagaatgag atccaaacaa ttggattcta 2100gaatccagct atctatccca aacccattat ttggcgagat tttcactatg cagaggcaat 2160gatcactata agaataagat tcaaacaccc acttattatt tttttaatcc agaaaccaga 2220ttctacattc actatagaat ccagaacttc aatatgggaa tgagatccaa atagacccta 2280agccaaaatg aaattggtga gatgaagtgg ctagttgtca taacctcctg taaagaagac 2340agcggtttac agtcccaact cccaaataaa catatgacat taatataatg actacaactc 2400acaacctaaa cctaaaccaa tatacatcca aacataagac aaaaggagaa ctgagtttta 2460tatgatcaca ctgatgaact gatgctgtag tctagcattc cagtgtttaa gatagttgac 2520tataaaccct tcaccttgca gattacatgt gacagaaaga tacctcttcc tcaagttgtt 2580ttttacgcct ttcctcctcc tcttgcttct gtttctcaag aacagcttct ctcgcagctg 2640tttcttcaag gcgacggagc tcagcctcct gtagggcctt taactccttt tcttgatcag 2700cttgtagcga tgcaaggtac tcatcgtcct acaaatttaa aatttataaa agtgctcacc 2760catagtggca attatgagca atggataaat cttagacctc aaacctgctg ctctcgtaat 2820aaccgctgtt cagttaatgc tggtgatgga gaatgagata ttgggggata ataagtagag 2880gttctgtgag aaggcataga gaaaggatat gttggtcctt caggaactcc accaaacatt 2940gcagcctcaa gcataacagc ttcatcatgt tcctcagaag aaatgccacc ccactttatt 3000gtaaaaagac gtcagaaatt aaacaaatcc atctaatgtc ttagcgcaca ttggaaccac 3060agattatata atacctcaga tgggaaatca tctccattat actggtgatt gttcagaaca 3120gggctagcac ctgggtgcac tatttttggt aattcattgt cctcagaagg ggcacgccga 3180gaacgacgcc taactaacgg ctgttcttct acatcttcag cctcctcctg gaagctctcg 3240tcatctattg gttgcctaga tgttccagcc ttttctgaag ctagtccttg cctccaaaag 3300gaaattgcat gtataaggaa tcaaatgata ctgtagtagg gtagcctgag tgaagaggtg 3360ggtagtaaag ttaacattca cctctcaact attccatttg cggtttccat gcctgcttta 3420tcagaagaat ggtccctcaa attcacttcc tcttgatgct ggcctccttg ctcgactatc 3480tgatgattat tgaaaaatta gagatgacat caagataggt tcaagtaagc atgttgggga 3540363542DNAArtificial SequenceSynthetic construct, Null_BC1GS2_518x (OB-4582 to OB-4584) 36caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgaaatg 60ttgaaaaagg tacaaggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttttatgg 360gtgtctagat tcatcatcct ccatttgaat atagaaagaa aaatcataac taaaacgaat 420actatttggg aacggaggga gttactattt tgcccgaatg ctcccaggaa agcacgagtt 480tcagggctag tttggaaggc taaacctagg gagggaaaac cccccacatg taactaaata 540tcttattcaa atgttacccc tagggattac tcaccctggg aaatgagaag ggtcccaagg 600ggatttcggt ttctattatt ttttctgcaa accatttcag agcaatgata tgaaaccaac 660ctaactactt ataacatttc ttaagaatat cagacatagg aaagtgatgg cctggaagca 720aagtaagact gatagataaa tagatcacta gaataaaccc tgacagttca tagccttcat 780agaagcaaaa ggaaacacta cgggagcaat tggttgcttg cactagcaat tcactgcatt 840gggtctaatg caggatagac taagccagca taagtgtgca caatgtgttt gtgtttggtt 900gccatgttat aagtaagttg catttgctaa tataatgttt ggttagttgg ctgttttgta 960tttcttgtga tcacccatgc ttgttgtggt gagattaaac tctcacgttt aatgctacag 1020aagcatccat gagacaatga aacatcgctc aaaagccacg tagtagcata ccctgactta 1080tgaataaagc aactcgatct gatttatttg agaaaacagg aaactgacaa gttattttta 1140acacaaaatt tcattaaaaa cgaatggtag acaattacca atctgtaggt ccctggcttg 1200caagtcctcc caatgtctaa gaaatcaaat aggaactgca ggcaagccag caagaaagta 1260ttaatcactg gatataaaat ataaagaaaa aagaaggaaa gacggctact cggctagcat 1320atgtttttgt taggggtgaa aatggatact tattcagaaa tcattttttg atcttttttc 1380tttaattagg aataaatagg atatagaata tgctaagcaa attcatattc ttgttcttag 1440cattgggctt gtaaagattc ataaaaggta aatctcaaat ttatcatata tcttaaatgg 1500tagatataaa attcagatac aaatattttt caactttttt gctgtaggga acaaataata 1560taaaaaaatt tatgcacaat tctattctta tttgtaataa tgtgcttgat aacataataa 1620aagattacca tcaaatttca cacacacaca cacacacaca cccacccccc cccacacccc 1680cacgcacgag cgcacacaca cactatatgt gtgtatatat attaaatatt gaatttatcc 1740taacaatttg gatacccact ttcatcccta gtttttgttc tggattcaag catctcaaaa 1800ttgtttacct gaagtttatc agttttgaga aagcggcgcc cctgtcgact accatcaggc 1860attcggacta caactgtcac agcaccctct gcgtctggag acggttccgg tggtaatgat 1920gcttgcttcg aagtgagact ggactctagt tcctatttaa tcaaaacatc agggacaaca 1980tgacaaatag tagtcaaata tccaggcaag aaaaaaaacc ataaacaatg aaaatactga 2040tcaaaagtcc tgtttggatc tcctaagaaa aatgagaatg agatccaaac aattggattc 2100tagaatccag ctatctatcc caaacccatt atttggcgag attttcacta tgcagaggca 2160atgatcacta taagaataag attcaaacac ccacttatta tttttttaat ccagaaacca 2220gattctacat tcactataga atccagaact tcaatatggg aatgagatcc aaatagaccc 2280taagccaaaa tgaaattggt gagatgaagt ggctagttgt cataacctcc tgtaaagaag 2340acagcggttt acagtcccaa ctcccaaata aacatatgac attaatataa tgactacaac 2400tcacaaccta aacctaaacc aatatacatc caaacataag acaaaaggag aactgagttt 2460tatatgatca cactgatgaa ctgatgctgt agtctagcat tccagtgttt aagatagttg 2520actataaacc cttcaccttg cagattacat gtgacagaaa gatacctctt cctcaagttg 2580ttttttacgc ctttcctcct cctcttgctt ctgtttctca agaacagctt ctctcgcagc 2640tgtttcttca aggcgacgga gctcagcctc ctgtagggcc tttaactcct tttcttgatc 2700agcttgtagc gatgcaaggt actcatcgtc ctacaaattt aaaatttata aaagtgctca 2760cccatagtgg caattatgag caatggataa atcttagacc tcaaacctgc tgctctcgta 2820ataaccgctg ttcagttaat gctggtgatg gagaatgaga tattggggga taataagtag 2880aggttctgtg agaaggcata gagaaaggat atgttggtcc ttcaggaact ccaccaaaca 2940ttgcagcctc aagcataaca gcttcatcat gttcctcaga agaaatgcca ccccacttta 3000ttgtaaaaag acgtcagaaa ttaaacaaat ccatctaatg tcttagcgca cattggaacc 3060acagattata taatacctca gatgggaaat catctccatt atactggtga ttgttcagaa 3120cagggctagc acctgggtgc actatttttg gtaattcatt gtcctcagaa ggggcacgcc 3180gagaacgacg cctaactaac ggctgttctt ctacatcttc agcctcctcc tggaagctct 3240cgtcatctat tggttgccta gatgttccag ccttttctga agctagtcct tgcctccaaa 3300aggaaattgc atgtataagg aatcaaatga tactgtagta gggtagcctg agtgaagagg 3360tgggtagtaa agttaacatt cacctctcaa ctattccatt tgcggtttcc atgcctgctt 3420tatcagaaga atggtccctc aaattcactt cctcttgatg ctggcctcct tgctcgacta 3480tctgatgatt attgaaaaat tagagatgac atcaagatag gttcaagtaa gcatgttggg 3540ga 3542373542DNAArtificial SequenceSynthetic construct, WT_B73Chr7_141681606-141685147 37caccctcgct gttggtaaac gtgcgccttg ggtatgtcct cacctgcatg atacgaaatg 60ttgaaaaagg tacaaggctg ggcggattta aacagtagaa tgaaaaggtg ccacaagaaa 120actcgtcaaa gaattgacta cgcgtcaatg ttccatagtt aaaaagactt gaactctgga 180tcagggactt tcaaacaagg atagctgcct ggtcaccagt cattaactgt aatgtaatgg 240ccatagatga tgcatgagta caataataaa aaaacaccat ccagccaaat atatactccc 300tgtcacaaat gaaaattcgt tttagataat tagtggattc atacaatatt tgttttatgg 360gtgtctagat tcatcatcct ccatttgaat atagaaagaa aaatcataac taaaacgaat 420actatttggg aacggaggga gttactattt tgcccgaatg ctcccaggaa agcacgagtt 480tcagggctag tttggaaggc taaacctagg gagggaaaac cccccacatg taactaaata 540tcttattcaa atgttacccc tagggattac tcaccctggg aaatgagaag ggtcccaagg 600ggatttcggt ttctattatt ttttctgcaa accatttcag agcaatgata tgaaaccaac 660ctaactactt ataacatttc ttaagaatat cagacatagg aaagtgatgg cctggaagca 720aagtaagact gatagataaa tagatcacta gaataaaccc tgacagttca tagccttcat 780agaagcaaaa ggaaacacta cgggagcaat tggttgcttg cactagcaat tcactgcatt 840gggtctaatg caggatagac taagccagca taagtgtgca caatgtgttt gtgtttggtt 900gccatgttat aagtaagttg catttgctaa tataatgttt ggttagttgg ctgttttgta 960tttcttgtga tcacccatgc ttgttgtggt gagattaaac tctcacgttt aatgctacag 1020aagcatccat gagacaatga aacatcgctc aaaagccacg tagtagcata ccctgactta 1080tgaataaagc aactcgatct gatttatttg agaaaacagg aaactgacaa gttattttta 1140acacaaaatt tcattaaaaa cgaatggtag acaattacca atctgtaggt ccctggcttg 1200caagtcctcc caatgtctaa gaaatcaaat aggaactgca ggcaagccag caagaaagta 1260ttaatcactg gatataaaat ataaagaaaa aagaaggaaa gacggctact cggctagcat 1320atgtttttgt taggggtgaa aatggatact tattcagaaa tcattttttg atcttttttc 1380tttaattagg aataaatagg atatagaata tgctaagcaa attcatattc ttgttcttag 1440cattgggctt gtaaagattc ataaaaggta aatctcaaat ttatcatata tcttaaatgg 1500tagatataaa attcagatac aaatattttt caactttttt gctgtaggga acaaataata 1560taaaaaaatt tatgcacaat tctattctta tttgtaataa tgtgcttgat aacataataa 1620aagattacca tcaaatttca cacacacaca cacacacaca cccacccccc cccacacccc 1680cacgcacgag cgcacacaca cactatatgt gtgtatatat attaaatatt gaatttatcc 1740taacaatttg gatacccact ttcatcccta gtttttgttc tggattcaag catctcaaaa 1800ttgtttacct gaagtttatc agttttgaga aagcggcgcc cctgtcgact accatcaggc 1860attcggacta caactgtcac agcaccctct gcgtctggag acggttccgg tggtaatgat 1920gcttgcttcg aagtgagact ggactctagt tcctatttaa tcaaaacatc agggacaaca 1980tgacaaatag tagtcaaata tccaggcaag aaaaaaaacc ataaacaatg aaaatactga 2040tcaaaagtcc tgtttggatc tcctaagaaa aatgagaatg agatccaaac aattggattc 2100tagaatccag ctatctatcc caaacccatt atttggcgag attttcacta tgcagaggca 2160atgatcacta taagaataag attcaaacac ccacttatta tttttttaat ccagaaacca 2220gattctacat tcactataga atccagaact tcaatatggg aatgagatcc aaatagaccc 2280taagccaaaa tgaaattggt gagatgaagt ggctagttgt cataacctcc tgtaaagaag 2340acagcggttt acagtcccaa ctcccaaata aacatatgac attaatataa tgactacaac 2400tcacaaccta aacctaaacc aatatacatc caaacataag acaaaaggag aactgagttt 2460tatatgatca cactgatgaa ctgatgctgt agtctagcat tccagtgttt aagatagttg 2520actataaacc cttcaccttg cagattacat gtgacagaaa gatacctctt cctcaagttg 2580ttttttacgc ctttcctcct cctcttgctt ctgtttctca agaacagctt ctctcgcagc 2640tgtttcttca aggcgacgga gctcagcctc ctgtagggcc tttaactcct tttcttgatc 2700agcttgtagc gatgcaaggt actcatcgtc ctacaaattt aaaatttata aaagtgctca 2760cccatagtgg caattatgag

caatggataa atcttagacc tcaaacctgc tgctctcgta 2820ataaccgctg ttcagttaat gctggtgatg gagaatgaga tattggggga taataagtag 2880aggttctgtg agaaggcata gagaaaggat atgttggtcc ttcaggaact ccaccaaaca 2940ttgcagcctc aagcataaca gcttcatcat gttcctcaga agaaatgcca ccccacttta 3000ttgtaaaaag acgtcagaaa ttaaacaaat ccatctaatg tcttagcgca cattggaacc 3060acagattata taatacctca gatgggaaat catctccatt atactggtga ttgttcagaa 3120cagggctagc acctgggtgc actatttttg gtaattcatt gtcctcagaa ggggcacgcc 3180gagaacgacg cctaactaac ggctgttctt ctacatcttc agcctcctcc tggaagctct 3240cgtcatctat tggttgccta gatgttccag ccttttctga agctagtcct tgcctccaaa 3300aggaaattgc atgtataagg aatcaaatga tactgtagta gggtagcctg agtgaagagg 3360tgggtagtaa agttaacatt cacctctcaa ctattccatt tgcggtttcc atgcctgctt 3420tatcagaaga atggtccctc aaattcactt cctcttgatg ctggcctcct tgctcgacta 3480tctgatgatt attgaaaaat tagagatgac atcaagatag gttcaagtaa gcatgttggg 3540ga 35423823DNAArtificial SequenceSynthetic construct, primer 509 (A) 38gaattgttca tcataaggcg tga 233922DNAArtificial SequenceSynthetic construct, primer 516 (B) 39aacgtgactc ccttaattct cc 224024DNAArtificial SequenceSynthetic construct, probe PB5 40aaactgaagg cgggaaacga caac 244123DNAArtificial SequenceSynthetic construct, primer 750 (A) 41gagatgcttg aatccagaac aaa 234222DNAArtificial SequenceSynthetic construct, primer 751 (B) 42ttgtcttggt tgtgatgatg tg 224325DNAArtificial SequenceSynthetic construct, primer 749 (C) 43gattaccatc aaatttcaca cacac 254422DNAArtificial SequenceSynthetic construct, probe PB17 44tagaacgacc gcccaaccag ac 224521DNAArtificial SequenceSynthetic construct, primer 513 (B) 45aaacgtccgc aatgtgttat t 214619DNAArtificial SequenceSynthetic construct, primer 608 (C) 46tcatgcaatt gtgccaacc 194723DNAArtificial SequenceSynthetic construct, primer 609 (A) 47acatagtcaa cctaacggct tat 234822DNAArtificial SequenceSynthetic construct, primer 371 (X) 48ggttataagc ccggttgaag ta 224921DNAArtificial SequenceSynthetic construct, primer 525 (Y) 49ctattccttg ctcggactga c 215028DNAArtificial SequenceSynthetic construct, probe PB2 50cacctgatat gccagatgtt ctgtctca 2851137DNAArtificial SequenceSynthetic construct, OB-2880_4588.259_locus_PCR 51gaattgttca tcataaggcg tgatgaattc atagcgtcat ggaatattat gaaatcacat 60gctcaaacac tgatagttta aactgaaggc gggaaacgac aacctgatca tgagcggaga 120attaagggag tcacgtt 13752107DNAArtificial SequenceSynthetic construct, OB-4451_4588.652_locus_PCR 52ttgtcttggt tgtgatgatg tggtctggtt gggcggtcgt tctagatcgg agtagaattc 60tgttacccac tttcatccct agtttttgtt ctggattcaa gcatctc 10753174DNAArtificial SequenceSynthetic construct, 4588.652_wt_zygosity_PCR 53gattaccatc aaatttcaca cacacacaca cacacacacc cacccccccc cacaccccca 60cgcacgagcg cacacacaca ctatatgtgt gtatatatat taaatattga atttatccta 120acaatttgga tacccacttt catccctagt ttttgttctg gattcaagca tctc 17454100DNAArtificial SequenceSynthetic construct, OB-3237_4588.757_locus_PCR 54aaacgtccgc aatgtgttat taagttgtct aagcgtcaat ttgtttacac cacaatataa 60aatctacctg ttcgctgata agccgttagg ttgactatgt 10055218DNAArtificial SequenceSynthetic construct, _B73ref_4588.757_wt_zygosity_PCR 55tcatgcaatt gtgccaacca aaatatctaa atgagatatt caagactcaa cagaattaaa 60gtatggaata gggtgtatat acactcaacc attattaaat ggtataatca tctatctata 120tcactataaa atctaccagt ttaaacttca caaaactcat ctagctaatg gagtagaggt 180agcatggcct agctgataag ccgttaggtt gactatgt 21856208DNAArtificial SequenceSynthetic construct, ZmGWDref 56ggttataagc ccggttgaag tatcaggtta tgtggttgtg gttgatgagt tacttgctgt 60ccagaacaaa tcttatgata aaccaaccat ccttgtggca aagagtgtca agggagagga 120agaaatacca gatggagtag ttggtgtaat tacacctgat atgccagatg ttctgtctca 180tgtgtcagtc cgagcaagga atagcaag 208

Claims

1. A transgenic plant, part or seed thereof comprising at least one synthetic nucleic acid encoding a glucanase and at least one synthetic polynucleotide producing a diagnostic amplicon for identifying event 4588.652, wherein the at least one nucleic acid comprises a sequence with at least 90% identity to the sequence set forth in SEQ ID NO: 8, and the at least one synthetic polynucleotide comprises a sequence with at least 90% identity to the sequence selected from the group consisting of SEQ ID NOS: 29-31.

2. The transgenic plant or part thereof of claim 1, wherein the glucanase is capable of degrading one or more polysaccharides selected from the group consisting of beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.

3. The transgenic plant or part thereof of claim 1, wherein the glucanase is active upon expression in the plant and exposure to a pH in the range from 5.0 to 10.0.

4. The transgenic plant or part thereof of claim 1, wherein the glucanase is active upon expression in the plant and exposure to a temperature in the range from 25.degree. C. to 130.degree. C.

5. The transgenic plant or part thereof of claim 1, wherein the glucanase activity has improved stability upon expression in the plant compared to the activity of a glucanase having an identical amino acid sequence and expressed in a bacterial cell.

6. The transgenic plant or part thereof of claim 1, wherein a plant is selected from the group consisting of: wheat, maize, barley, and sorghum.

7. An animal feedstock comprising the transgenic plant, part or seed thereof of claim 1.

8. The animal feedstock of claim 7 further comprising a feed supplement.

9. The animal feedstock of claim 8, wherein the feed supplement is plant material selected from the group consisting of: a non-transgenic plant, another transgenic, mutant and engineered plant.

10. The animal feedstock of claim 8, wherein the feed supplement comprises one or more exogenous enzymes selected from the group consisting of: a hydrolytic enzyme selected from the group consisting of: xylanase, endoglucanase, cellulase, protease, phytase, amylase and mannanase.

11. The animal feedstock of claim 8, wherein the feed supplement comprises at least one component selected from the group consisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, barley grain, barley pellets, soybean meal, soybean oilcake, sorghum grain and sorghum pellets.

12. The animal feedstock of claim 8, wherein the feed supplement comprises at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN.RTM., vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, and mineral premix.

13. A method of producing an animal feedstock comprising mixing the transgenic plant, part or seed thereof of claim 1 with other plant material to form a mixture.

14. The method of claim 13, wherein the method further comprises pelletizing the mixture.

15. A method of increasing metabolizable energy of a diet, wherein the method comprises mixing the transgenic plant, part, or seed thereof of claim 1 with a feed ingredient.

16. The method of 15, wherein the feed ingredient comprises at least one component selected from the group consisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheat grain, wheat middlings, barley grain, barley pellets, soybean meal, soy hulls, dried distillers grain, soybean oilcake, sorghum grain and sorghum pellets.

17. The method of claim 15, wherein the feed ingredient comprises at least one component selected from the group consisting of: soluble solids, fat and vermiculite, limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN.RTM., vitamin premix, dicalcium phosphate, selenium premix, choline chloride, sodium chloride, mineral premix, and one or more exogenous enzymes.