# Patent

## Title
Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them

## Abstract

Transgenic plants that produce high levels of polyhydroxybutyrate and methods of producing them are provided. In a preferred embodiment the transgenic plants are produced using plastid transformation technologies and utilize genes which are codon optimized. Stably transformed plants able to produce greater than 10% dwt PHS in tissues are also provided.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No. 61/157,809, filed on Mar. 5, 2009. The entire disclosure of the above application is incorporated herein by reference.


STATEMENT REGARDING FEDERAL FUNDING OR SUPPORT
This work was supported in part by a Department of Energy Industry of the Future Award (DE-FC07-011D14214) and a grant from the United States Department of Agriculture (USDA-68-3A75-3-142). Therefore the government has certain rights in the invention.


FIELD OF THE INVENTION
The invention is generally related to the field of polymer production in transgenic plants. Methods for producing stable, high polyhydroxyalkanoate producing transgenic plants via plastid transformation technologies are also provided.
BACKGROUND OF THE INVENTION
Fuels, plastics, and chemicals derived from agricultural feedstocks are receiving considerable attention as the world looks for alternatives to petroleum. Production of polyhydroxyalkanoates (PHAs), a family of naturally renewable and biodegradable plastics, in crops has the potential of providing a renewable source of polymers and bio-energy from one crop if plant residues remaining after polymer isolation are converted to liquid fuels and/or energy. PHAs can provide an additional revenue stream that would make crops including bioenergy crops more economically viable.
PHAs are a natural component of numerous organisms in multiple ecosystems and accumulate in a wide range of bacteria as a granular storage material when the microbes are faced with an unfavorable growth environment, such as a limitation in an essential nutrient (Madison et al., Microbiol. Mol. Biol. Rev. 63:21-53 (1999); Suriyamongkol et al. Biotechnol Adv. 25:148-75 (2007)). The monomer unit composition of these polymers is largely dictated by available carbon source as well as the native biochemical pathways present in the organism. PHAs can be produced industrially from renewable resources in bacterial fermentations providing an alternative to plastics derived from fossil fuels. PHAs possess properties enabling their use in a variety of applications currently served by petroleum-based plastics and are capable of matching or exceeding the performance characteristics of fossil fuel derived plastics with a broad spectrum of properties that can be obtained by varying the monomer composition of homo- and co-polymers, or by manipulating properties such as molecular weight (Sudesh et al., Prog. Polym. Sci. 25:1503-1555 (2000)).
SUMMARY OF THE INVENTION
Transgenic plants, plant material, and plant cells for synthesis of biopolymers, for example polyhydroxyalkanoates (“PHA”) are provided. In one embodiment, the transgenic plants synthesize polyhydroxybutyrate (“PHB”). Host plants, plant tissue, and plant material have been engineered to express genes encoding enzymes in the biosynthetic pathway for PHB production from the plastid genome to produce PHB. These genes include phaA, phaB, and phaC, all of which are known in the art. Preferably, native genes are selected based on their similarity in codon usage to the host plastome. Alternatively, genes are codon optimized. The genes can be introduced in the plant, plant tissue, or plant cell using conventional plant molecular biology techniques. Plants with recombinant plastids are also referred to as transplastomic plants. In certain embodiments, the transplastomic plants are fertile.
Provided herein is a transplastomic plant having one or more plastids engineered to express enzymes for the production of PHA, wherein the transgenic plant produces greater than 10%, 12%, 15% or more polyhydroxyalkanoate per unit dry cell weight in the plant tissue. For instance, the transplastomic plant can produce greater than 10% PHA per unit dry cell weight (dwt) in leaves. The transplastomic plant can produce greater than about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% (dwt) or more in the leaves of the plant The PHA can be poly(3-hydroxybutyrate) (PHB).
The genes encoding enzymes for the production of PHA can be selected to have codon usage similar to the host plastome of the transplastomic plant. The genes encoding enzymes for the production of PHA can be codon optimized for expression in the transplastomic plant.
The transplastomic plants can be dicots or monocots. The transplastomic plant can be a biomass crop plant. Preferred host plants include, but are not limited to members of the Brassica family including B. napus, B. rappa, B. carinata and B. juncea; industrial oilseeds such as Camelina sativa, Crambe, jatropha, castor; Ambidopsis thaliana; maize; soybean; cottonseed; sunflower; palm; algae; coconut; safflower; peanut; mustards including Sinapis alba; sugarcane; silage corn; alfalfa; switchgrass; miscanthus; sorghum; and tobacco.
In certain embodiments, the transplastomic plants have delayed flowering relative to wild-type plants. The typical flowering time of a transplastomic plant producing more than 14% dwt PHB in parts of its leaves is no more than 100%, 110%, 120%, 130% of flowering time of a wild type plant. The final height of a transplastomic plant producing more than 16% dwt PHB in parts of its leaves is no less than 100%, 90%, 80% of the final height of a wild type plant. Other embodiments provide plant material and plant parts of the transplastomic plants including seeds, flowers, stems, and leaves. The plant material and plant parts can be used to produce a feedstock for industrial use in for example a biorefinery.
Still another embodiment provides a method for producing a transgenic plant including selecting a host plant and transfecting one or more plastids of the host plant with a vector having genes whose codon usage avoids the use of codons with a low frequency of use (<10/1000) in the host plastome and whose GC content is <50%. In one embodiment, untranslated regions (UTRs) of the vector allow high level expression of the genes wherein the sequence length of the UTRs is minimal (≦55 nucleotides) and the total amount of plastidial derived DNA in the vector is <3% (excluding sequences of the left and right flanks) such that recombination with the host plastome is limited. Preferably, the genes encode enzymes for producing polyhydroxyalkanoate.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1(a)-(c) show diagrams of plastid transformation vectors (a) pCAB(2); (b) pCA(2); and (c) pUCaadA. The following abbreviations are used in the maps: psbA/left flank, DNA homologous to the reverse complement of nucleotides 536 to 1597 of the N. tabacum plastome [EMBL accession no. Z00044, (Shinozaki et al., EMBO J. 5: 2043-2049 (1986))], contains the complete coding sequence of psbA encoding the D1 protein of photosystem II; 5′ UTR T7g10, 5′ UTR of gene 10 of bacteriophage T7, DNA homologous to nucleotides 22904 to 22969 of the bacteriophage T7 genome [EMBL accession no. V01146 (Dunn et al., J. Mol. Biol. 166:477-535 (1983))]; phaC, gene encoding PHB synthase from Acinetobacter sp., homologous to nucleotides 2351 to 4123 of the Acinetobacter sp. PHA biosynthetic gene locus [EMBL accession no. L37761 (Schembri et al., J. Bacterial. 177:4501-7 (1995))]; rps19/rpl22 spacer, DNA homologous to nucleotides 86353 to 86399 of the N. tabacum plastome, contains intergenic region between ribosomal protein S19 (rps19) and ribosomal protein L22 (rpl22); phaA, gene encoding thiolase from Acinetobacter sp., homologous to nucleotide 4206 to 5384 of the Acinetobacter sp. PHA biosynthetic gene locus; psbD/C spacer, DNA homologous to nucleotides 35463 to 35517 of the N. tabacum plastome, contains intergenic region between photosystem II D2 protein (psbD) and photosystem II 44kd protein (psbC); phaB, gene encoding acetoacetyl-CoA reductase from Bacillus megaterium, homologous to nucleotide 4758 to 5501 of the Bacillus megaterium PHA gene cluster [EMBL accession no. AF109909 (McCool et al., J. Bacterial. 181:585-592 (1999))]; 5′ UTR rbcL, 15 nucleotides of the 5′ untranslated leader sequence of the gene encoding the large subunit of rubisco, homologous to nucleotides 57580 to 57594 of the N. tabacum plastome; aadA, gene encoding aminoglycoside 3′-adenyltransferase from E. coli, spectinomycin/streptomycin resistance marker (Svab et at, Proc. Natl. Acad. Sci. USA 90:913-917 (1993)); right flank, contains 3′ UTR of psbA, trnH (tRNA-Histidine), and part of ribosomal protein L2 (rpl2), right flank DNA is homologous to the reverse complement of nucleotides 155398 to 155943 and 1 to 530 of the N. tabacum plastome; P(LAC), lac promoter of parent vector pUC19; ORI, origin of replication of vector pUC19; Apr, gene within pUC19 vector sequence encoding β-lactamase conferring resistance to ampicillin; P(BLA), promoter driving expression of gene encoding β-lactamase.
FIG. 2 shows a diagram of the insertion site of plastid transformation vector pCAB(2). The binding sites of primers KMB 77 (upstream of the transgenic DNA insert) and KMB 41 (within the transgenic DNA insert) are used to verify the correct insertion of the transgenic DNA at the left flank. The binding sites of primers KMB 153 (downstream of the transgenic DNA insert) and KMB 36 (within the transgenic DNA insert) are used to verify the correct insertion of the transgenic DNA at the right flank. Size of predicted PCR products are shown.
FIG. 3(a) shows a diagram of the wild-type locus showing the regions around sequences of the left and right flanks used in plastid transformation vectors. The left flank consists of the psbA coding region (see FIG. 1), the right flank consists of the 3′ UTR of psbA, trnH, and a partial fragment of rpl2 (see FIG. 1). FIG. 3(b) shows a diagram of the expected integration for plasmid pUCaadA. FIG. 3(c) shows a diagram of the expected integration for plasmid pCA(2). FIG. 3(d) shows a diagram of the expected integration site for plasmid pCAB(2). The expected size of southern fragments when genomic DNA is digested with Pst I and probed with Probe I are shown in FIGS. 3(a) to 3(d) with a dashed line.
FIG. 4 shows a bar graph of PHB production in leaves of transplastomic PHB producing plants over three regeneration cycles. P1 lines were obtained after plastid transformation of pCAB(2) and isolation of regenerant. P2 lines were subjected to one additional cycle of shoot regeneration from an excised leaf. P3 lines were subjected to another additional cycle of shoot regeneration from an excised leaf.
FIG. 5 shows a bar graph comparing height of wild-type and transgenic plants. Plants were grown in the green house until seed set and the height of each plant was measured. Data was obtained from 8 plants of each line. Lines are as follows: wt, wild-type; PBCAB2 and PBCAB6, these lines were obtained from seed of lines that were obtained after plastid transformation of pCAB(2), isolation of regenerant, and performance of one additional cycle of shoot regeneration from excised leaf.
FIG. 6 shows a bar graph of the comparison of days to flowering of wild-type and transgenic plants. Plants were grown in the green house until flowers started to appear. Data was obtained from 8 plants of each line. Lines are as follows: wt, wild-type; PBCAB2, line pCAB P2 T1 #2; PBCAB6, line pCAB P2 T1 #6. Lines pCAB P2 T1 #2 and pCAB P2 T1 #6 were obtained from seed of lines that were obtained after plastid transformation of pCAB(2), isolation of regenerant, and performance of one additional cycle of shoot regeneration from excised leaf.



DETAILED DESCRIPTION OF THE INVENTION
Provided herein are transplastomic plants that produce greater than 10% polyhydroxyalkanoate per unit dry cell weight (dwt) in leaves. The plants include one or more plastids engineered to express genes encoding enzymes for the production of polyhydroxyalkanoate (PHA). Also provided are methods for making such plants.
Industrial production of PHAs in crop plants would provide a low cost, renewable source of plastics. Production of PHAs in plants has been previously demonstrated in a number of crops [for review, see (Suriyamongkol et al., Biotechnol. Adv. 25:148-75 (2007)) and references within], including maize (Poirier et al., 2002, Polyhydroxyalkanoate production in transgenic plants, in Biopolymers, Vol 3a, Steinbuchel, A. (ed), Wiley-VHC Verlag GmbH, pgs 401-435), sugarcane (Petrasovits et al., Plant Biotechnol. J. 5:162-172 (2007); Purnell et al., Plant Biotechnol. J. 5:173-184 (2007)), switchgrass (Somleva et al., Plant Biotechnol. J. 6:663-678 (2008)), flax (Wrobel et al., J. Biotechnol. 107:41-54 (2004); Wrobel-Kwiatkowski et al., Biotechnol. Prog. 23:269-277 (2007)), cotton (John et al., Proc. Natl. Acad. Sci. USA 93:12768-12773 (1996)), alfalfa (Saruul et al., Crop Sci. 42:919-927 (2002)), tobacco (Arai et al., Plant Biotechnol. 18:289-293 (2001); Bohmert et al., Plant Physiol. 128:1282-1290 (2002); Lössl et al., Plant Cell Rep. 21:891-899 (2003); Lössl et al., Plant Cell Physiol. 46:1462-1471 (2005)), potato (Bohmert et al., Plant Physiol. 128:1282-1290 (2002)), and oilseed rape (Valentin et al., Int. J. Biol. Macromol. 25:303-306 (1999); Slater et al., Nat. Biotechnol. 17:1011-1016 (1999)) (U.S. Pat. Nos. 5,663,063 and 5,534,432) resulting in the production of a range of polymer levels depending on the crop and mode of transformation as well as the polymer composition. Most of the efforts to produce PHAs in plants have focused on production of the homopolymer poly-3-hydroxybutyrate (P3HB) or the copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HBV). Other researchers have studied the production of PHAs having higher carbon chain lengths in the monomers (Romano et al., Planta 220:455-464 (2005); Mittendorf et al., Proc. Natl. Acad. Sci. USA 95:13397-13402 (1998); Poirier et al., Plant Physiol. 121:1359-1366 (1999); Matsumoto, J. Polym. Environ. 14:369-374 (2006); Wang et al., Chinese Sci. Bull. 50:1113-1120 (2005)).
To date, the highest levels of polymer have been obtained when P3HB is produced in plastids by targeting the three enzymes encoded by transgenes in the plant nucleus into the plastid using plastid targeting sequences (Suriyamongkol et al., Biotechnol. Adv. 25:148-75 (2007); Bohmert et al., Molecular Biology and Biotechnology of Plant Organelles, pp. 559-585 (2004); van Beilen et al., Plant J. 54:684-701 (2008)). This is likely due to the high flux of carbon through substrate acetyl-CoA in these organdies during fatty acid biosynthesis (Bohmert et al., Molecular Biology and Biotechnology of Plant Organelles, pp. 559-585 (2004)). Expression of three genes encoding β-keto thiolase, aceto-acetyl CoA reductase, and PHA synthase, allows the conversion of acetyl-CoA within the plastid to PHB. Levels of PHA production greater than 10% have only been demonstrated in the model plant Arabidopsis (Bohmert et al., Planta 211:841-845 (2000); Kourtz et al., Transgenic Res. 16:759-769 (2007); Nawrath et al., Proc. Natl. Acad. Sci. USA 91:12760-12764 (1994)) and not in any crops of industrial relevance.
One way to potentially increase product yield is to increase expression of the PHB transgenes. Plastid-encoded expression can potentially yield high levels of expression due to the multiple copies of the plastome within a plastid and the presence of multiple plastids within the cell. Transgenic proteins have been observed to accumulate to 45% (De Cosa et al., Nat. Biotechnol. 19:71-74 (2001)) and >70% (Oey et al., Plant J. 57:436-445 (2009)) of the plant's total soluble protein. Since plastid DNA is maternally inherited in most plants, the presence of plastid-encoded transgenes in pollen is significantly reduced or eliminated, providing some level of gene containment in plants created by plastid transformation.
Previous researchers have attempted PUB production via plastid-encoded expression of transgenes in tobacco with only limited success (Lössl et al., Plant Cell Rep. 21:891-899 (2003); Lössl et al., Plant Cell Physiol. 46:1462-1471 (2005); Arai et al., Plant Cell Physiol. 45:1176-1184 (2004); Nakashita et al., Biosci. Biotechnol. Biochem. 65:1688-1691 (2001)). The highest levels, up to 1.7% dry weight (dwt) PHB, were observed in leaves of tobacco plantlets after regeneration from callus (Lössl et al., Plant Cell Rep. 21:891-899 (2003)) but product levels dropped significantly during a subsequent three week in vitro culture growth period yielding an average PHB content of only 20 ppm of polymer (Lössl et al., Plant Cell Rep. 21:891-899 (2003)). In addition, PHB producing plants were found to be sterile, eliminating or severely limiting their utility for PHB crop production.
Researchers have also engineered plants to produce medium chain length PHAs via plastid transformation technologies using potato (Romano et al., Planta 220:455-464 (2005)) and tobacco (Wang et al., Chinese Sci. Bull. 50:1113-1120 (2005)). Levels of 0.026 and 0.48% dwt medium chain length PHA, respectively, were observed in these studies.
Provided herein are stable, fertile, transgenic plants engineered by plastid transformation technologies for the production of unexpectedly high levels of polyhydroxyalkanoates.
Also provided are transgenic plants producing ultra high levels (>10% of dry cell weight) of polymer in tissues.
I. Definitions
Unless otherwise indicated, the disclosure encompasses conventional techniques of plant breeding, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology ((F. M. Ausubel, et al. eds., (1987)); Plant Breeding: Principles and Prospects (Plant Breeding, Vol 1) (1993), M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall; Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology, PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995), Academic Press, Inc.).
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Wiley-Interscience, 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, VCH Publishers, Inc., 1995; Ausubel et al., 1987, Current Protocols in Molecular Biology, Green Publishing; Sambrook and Russell, 2001, Molecular Cloning: A Laboratory Manual (3rd. edition).
A number of terms used herein are defined and clarified in the following section.
The term “PHA copolymer” refers to a polymer composed of at least two different hydroxyalkanoic acid monomers.
The term “PHA homopolymer” refers to a polymer that is composed of a single hydroxyalkanoic acid monomer.
As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.
As used herein, an “expression vector” is a vector that includes one or more expression control sequences.
As used herein, an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
As used herein, “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest in the host plant.
As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid into a cell by a number of techniques known in the art.
“Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers.
As used herein the term “heterologous” means from another host. The other host can be the same or different species.
As used herein the term “improving codon utilization” means changing one or more codons in the transgene such that the codons in the transgene more closely resemble those used by the plastome encoded genes of the host plant.
The term “cell” refers to a membrane-bound biological unit capable of replication or division.
The term “construct” refers to a recombinant genetic molecule including one or more isolated polynucleotide sequences.
Genetic constructs used for plastid-encoded transgene expression in a host organism typically comprise in the 5′-3′ direction, a left flank which mediates—together with the right flank—integration of the genetic construct into the target plastome; a promoter sequence; a sequence encoding a 5′ untranslated region (5′ UTR containing a ribosome binding site; a sequence encoding a gene of interest, such as the genes disclosed herein; a 3′ untranslated region (3′ UTR); and a right flank. Plastid gene expression is regulated to a large extent at the post-transcriptional level and 5′ and 3′ UTRs have been shown to impact RNA stability and translation efficiency (Eibl et al., Plant J 19, 333-345 (1999)). Due to the prokaryotic nature of plastid expression systems, one or more transgenes may be arranged in an operon such that multiple genes are expressed from the same promoter. The promoter driving transcription of the operon may be located within the genetic construct, or alternatively, an endogenous promoter in the host plastome upstream of the transgene insertion site may drive transcription. In addition, the 3′UTR may be part of the right flank. The open reading frame may be orientated in either a sense or anti-sense direction. The construct may also comprise selectable marker gene(s) and other regulatory elements for expression.
The term “plant” is used in it broadest sense. It includes, but is not limited to, any species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and photosynthetic green algae (e.g., Chlamydomonas reinhardtii). It also refers to a plurality of plant cells that are largely differentiated into a structure that is present at any stage of a plant's development. Such structures include, but are not limited to, a fruit, shoot, stem, leaf, flower petal, etc. The term “plant tissue” includes differentiated and undifferentiated tissues of plants including those present in roots, shoots, leaves, pollen, seeds and tumors, as well as cells in culture (e.g., single cells, protoplasts, embryos, callus, etc.). Plant tissue may be in planta, in organ culture, tissue culture, or cell culture. The term “plant part” as used herein refers to a plant structure, a plant organ, or a plant tissue.
A non-naturally occurring plant refers to a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants produced by non-transgenic means such as plant breeding.
With regard to plants, the term “fertile” refers to a plant producing seeds that are able to germinate and to produce viable plants.
The term “days to flowering” refers to the day of seed imbibition until opening of the first flower of the first inflorescence.
The term “plant cell” refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.
The term “plant cell culture” refers to cultures of plant units such as, for example, protoplasts, cells in cell culture, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
The term “plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
A “plant organ” refers to a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
“Plant tissue” refers to a group of plant cells organized into a structural and functional unit. Any tissue of a plant whether in a plant or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
II. Transgenic Plants
Transgenic plants, in particular, transplastomic plants, have been developed that produce increased levels of biopolymers such as polyhydroxyalkanoates (PHAs). Methods and constructs for engineering plant plastids with genes for high level, stable PHA, in particular PHB, production are described. One embodiment provides transgenic plants for the direct, large scale production of PHAs in crop plants or in energy crops where a plant by-product, such as biomass can be used for production of energy. Proof of concept studies for polyhydroxybutyrate (PHB) synthesis in switchgrass (Somleva et al., Plant Biotechnol. J. 6:663-678 (2008)), sugarcane (Petrasovits et al., Plant Biotechnol. J. 5:162-172 (2007); Purnell et al., Plant Biotechnol. J. 5:173-184 (2007)), canola (Valentin et al., Int. J. Biol. Macromol. 25:303-306 (1999); Slater et al., Nat. Biotechnol. 17:1011-1016 (1999); Houmiel et al., Planta 209:547-550 (1999)), and corn stover (Poirier et al., 2002, Polyhydroxyalkanoate production in transgenic plants, in Biopolymers, Vol 3a, Steinbuchel, A. (ed), Wiley-VHC Verlag GmbH, pgs 401-435), have been reported. While these studies have yielded significant scientific results (Slater et al., Nat. Biotechnol. 17:1011-1016 (1999)), higher yields will enhance overall economics of polymer produced in a crop platform.
As shown herein, fertile transgenic plants that produced elevated levels of PHAs, i.e., at least 10% dwt in plant tissues, were produced using plastid-encoded gene expression. Genes were selected whose codon usage and GC content were similar to the host plant's native plastome, avoiding the use of genes with codons with a low frequency of use (<10/1000) in the host plastome and whose GC content is <50%. In one embodiment, untranslated regions (UTRs) of the vector allow high level expression of the genes wherein the sequence length of the UTRs is minimal (≦55 nucleotides) and the total amount of plastidial derived DNA in the vector is <3% (excluding sequences of the left and right flanks) such that recombination with the host plastome is limited. This strategy allowed significantly increased PHB production in both hetero- and autotrophically grown plants compared to previously published results (>11 fold higher) (Lössl et al., Plant Cell Rep. 21:891-899 (2003); Lössl et al., Plant Cell Physiol. 46:1462-1471 (2005); Arai et al., Plant Cell Physiol. 45:1176-1184 (2004); Nakashita et al., Biosci. Biotechnol. Biochem. 65:1688-1691 (2001)).
In another embodiment, plastid encoded constructs are disclosed for optimized expression in monocots or dicots.
In yet another embodiment, constructs are disclosed for enhanced expression of PHA, preferably PHB, in algae. Preferred species of algae include, but are not limited to Emiliana huxleyi, Arthrospira platensis (Spirulina), Haematococcus pluvialis, Dunaliella salina, and Chlamydomanas reinhardtii. 
A. Genetic Constructs for Transformation
Suitable genetic constructs include expression cassettes for plastid-encoded expression of enzymes for the production of polyhydroxyalkanoates, in particular from the polyhydroxybutyrate biosynthetic pathway. In one embodiment, the construct contains operatively linked in the 5′ to 3′ direction, a promoter that directs transcription of a nucleic acid sequence in the plastid; a 5′ UTR that increases levels of expression of transgenes; a nucleic acid sequence encoding one of the PHB biosynthetic enzymes; and a 3′ UTR that increases levels of expression of transgenes relative to expression if the UTR were not there.
In an alternative embodiment, expression of the PHB biosynthetic pathway is initiated by a promoter that is native to the host plastome and is outside of the DNA insertion.
In another embodiment, multiple genes are expressed from one promoter by creating a synthetic operon.
DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes into plants. As used herein, “transgenic” refers to an organism in which a nucleic acid fragment containing a heterologous nucleotide sequence has been introduced. The transgenes in the transgenic organism are preferably stable and inheritable. The heterologous nucleic acid fragment may or may not be integrated into the host genome.
Traditional methods and vector options for transformation of the nuclear genome are available, including those described in “Gene Transfer to Plants” (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in Plant Molecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995). A preferred transformation approach is to use a vector to specifically transform the plant plastid chromosome by homologous recombination (as described in U.S. Pat. No. 5,545,818). Plastid transformation vectors typically include one or more coding sequences of interest whose expression is controlled by a promoter and 5′ and 3′ regulatory sequences, and a selectable or screenable marker gene. With plastid transformation procedures, it is possible to take advantage of the prokaryotic nature of the plastid genome and insert a number of transgenes as an operon.
A transgene may be constructed to encode a multifunctional enzyme through gene fusion techniques in which the coding sequences of different genes are fused with or without linker sequences to obtain a single gene encoding a single protein with the activities of the individual genes. Such synthetic fusion gene/enzyme combinations can be further optimized using molecular evolution technologies.
1. Genes Involved in Polyhydroxyalkanoate Synthesis
In a preferred embodiment, the products of the transgenes are enzymes and other factors required for production of a biopolymer, such as a polyhydroxyalkanoate (PHA).
For PHA production, transgenes must encode enzymes such as beta-ketothiolase, acetoacetyl-CoA reductase, PHB (“short chain”) synthase, PHA (“long chain”) synthase, threonine dehydratase, dehydratases such as 3-OH acyl ACP, isomerases such as Δ3-cis, Δ2-trans isomerase, propionyl-CoA synthetase, hydroxyacyl-CoA synthetase, hydroxyacyl-CoA transferase, thioesterase, fatty acid synthesis enzymes and fatty acid beta-oxidation enzymes. Useful genes are well known in the art, and are disclosed for example by Snell and Peoples Metab. Eng. 4:29-40 (2002) and Bohmert et al. in: Molecular Biology and Biotechnology of Plant Organelles, H. Daniell, C. D. Chase Eds. (Kluwer Academic Publishers, Netherlands, 2004, pp. 559-585).
PHA Synthases
Examples of PHA synthases include a synthase with medium chain length substrate specificity, such as phaC1 from Pseudomonas oleovorans (WO 91/00917; Huisman, et al., J. Biol. Chem. 266:2191-2198 (1991)) or Pseudomonas aeruginosa (Timm, A. & Steinbuchel, A., Eur. J. Biochem. 209:15-30 (1992)), the synthase from Alcaligenes eutrophus with short chain length specificity (Peoples, O. P. & Sinskey, A. J., J. Biol. Chem. 264:15298-15303 (1989)), or a two subunit synthase such as the synthase from Thiocapsa pfennigii encoded by phaE and phaC (U.S. Pat. No. 6,011,144). Other useful PHA synthase genes have been isolated from, for example, Aeromonas caviae (Fukui & Doi, J. Bacterial. 179:4821-30 (1997)), Rhodospirillum rubrum (U.S. Pat. No. 5,849,894), Rhodococcus ruber (Pieper & Steinbuechel, FEMS Microbiol. Lett. 96(1):73-80 (1992)), and Nocardia corallina (Hall et al., Can. J. Microbial. 44:687-91 (1998)). PHA synthases with broad substrate specificity useful for producing copolymers of 3-hydroxybutyrate and longer chain length (from 6 to 14 carbon atoms) hydroxyacids have also been isolated from Pseudomonas sp. A33 (Appl. Microbial. Biotechnol. 42:901-909 (1995)) and Pseudomonas sp. 61-3 (Kato, et al., Appl. Microbiol. Biotechnol. 45:363-370 (1996)).
A range of PHA synthase genes and genes encoding additional metabolic steps useful in PHA biosynthesis are described by Madison and Huisman (Microbiology and Molecular biology Reviews 63:21-53 (1999)).
Hydratases
The transgene can encode a hydratase, such as the (R)-specific enoyl-CoA hydratase (PhaJ) from Aeromonas caviae (Fukui, T. et al., J. Bacteriol. 180, 667-673 (1998)) or the PhaJ1 and PhaJ2 (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa (Tsuge, T. et al., FEMS Microbiol. Lett, 184, 193-198 (1999)). These hydratases catalyze the formation of R-3-hydroxyacyl-CoA from an enoyl-CoA.).
Reductases
The transgene can encode a reductase. A reductase refers to an enzyme that can reduce β-ketoacyl CoAs to R-3-OH-acyl CoAs, such as the NADH dependent reductase from Chromatium vinosum (Liebergesell, M., & Steinbuchel, A., Eur. J. Biochem. 209:135-150 (1992)), the NADPH dependent reductase from Alcaligenes eutrophus (Peoples, O. P. & Sinskey, A. J., J. Biol. Chem. 264:15293-15297 (1989)), the NADPH reductase from Zoogloea ramigera (Peoples, O. P. & Sinskey, A. J., Molecular Microbiology 3:349-357 (1989)) or the NADPH reductase from Bacillus megaterium (U.S. Pat. No. 6,835,820).
Thiolases
The transgene can encode a thiolase. A beta-ketothiolase refers to an enzyme that can catalyze the conversion of acetyl CoA and an acyl CoA to a β-ketoacyl CoA, a reaction that is reversible. An example of such thiolases are PhaA from Alcaligenes eutropus (Peoples, O. P. & Sinskey, A. J., J. Biol. Chem. 264:15293-15297 (1989)), and BktB from Alcaligenes eutrophus (Slater et al., J Bacteriol. 180(8):1979-87 (1998)).
R-3-Hydroxyacyl-ACP:CoA Transferases
The transgene can encode an R-3-hydroxyacyl-ACP:CoA transferase (PhaG), an enzyme that can convert R-3-hydroxyacyl-ACP, an intermediate in fatty acid biosynthesis, to R-3-hydroxyacyl-CoA, the monomer unit for PHA synthase and thus PHA synthesis. Genes encoding PhaG enzymes have been isolated from a range of Pseudomads, including Pseudomonas putida (Rehm et al., J. Biol. Chem., 273, 24044-24051 (1998)), Pseudomonas aeruginosa (Hoffmann et al., FEMS Microbiology Letters, 184, 253-259 (2000)), and Pseudomonas sp. 61-3 (Matsumoto et al., Biomacromolecules, 2, 142-147 (2001)). While it has been reported that PhaG can catalyze the complete conversion of R-3-hydroxyacyl-ACP to R-3-hydroxyacyl-CoA in Pseudomonads, in E. coli it has been shown that an additional acyl CoA synthetase activity is needed to accumulate medium chain length PHAs from simple carbon sources in strains engineered to express a medium chain length synthase (US Patent Application 2003/0017576).
Acyl-CoA Synthetase
An acyl-CoA synthetase refers to an enzyme that can convert free fatty acids, including R-3-hydroxyalkanoic acids, to the corresponding acyl-CoA. Genes encoding acyl CoA synthetases have been isolated from a range of organisms, including the alkK gene from Pseudomonas oleovorans (van Beilen, J. et al. Mol Microbiol, 6, 3121-36 (1992)), the fadD gene from E. coli (Black, P. et al., Biol. Chem. 267, 25513-25520 (1992)), and the ydiD gene from E. coli (Campbell et al., Mol Microbiol. 47, 793-805 (2003)).
2. Promoters
Plant promoters can be selected to control the expression of the transgene in different plant tissues or organdies, for all of which methods are known to those skilled in the art (Gasser & Fraley, Science 244:1293-99 (1989)). In a preferred embodiment, promoters are selected from those of plant or prokaryotic origin that are known to yield high expression in plastids. In certain embodiments the promoters are inducible. Inducible plant promoters are known in the art.
As shown below, the transgenes can be inserted into an existing transcription unit (such as, but not limited to, psbA) to generate an operon. However, other insertion sites can be used to add additional expression units as well, such as existing transcription units and existing operons (e.g., atpE, accD). Such methods are described in, for example, U.S. Pat. App. Pub. 2004/0137631, which is incorporated herein by reference in its entirety. For an overview of other insertion sites used for integration of transgenes into the tobacco plastome, see Staub (Staub, J. M., “Expression of Recombinant Proteins via the Plastid Genome,” in: Vinci V A, Parekh S R (eds.) Handbook of Industrial Cell Culture: Mammalian, and Plant Cells, pp. 259-278, Humana Press Inc., Totowa, N.J. (2002)).
In general, the promoter from any class I, II or III gene can be utilized in the invention. For example, any of the following plastidial promoters and/or transcription regulation elements can be used for expression in plastids. Sequences can be derived from the same species as that used for transformation. Alternatively, sequences can be derived from other species to decrease homology and to prevent homologous recombination with endogenous sequences.
For instance, the following plastidial promoters can be used for expression in plastids.

    
    
        PrbcL promoter (Allison L A, Simon L D, Maliga P, EMBO J, 15:2802-2809 (1996); Shiina T, Allison L, Maliga P, Plant Cell 10:1713-1722 (1998));
        PpsbA promoter (Agrawal G K, Kato H, Asayama M, Shirai M, Nucleic Acids Research 29:1835-1843 (2001));
        Prrn 16 promoter (Svab Z, Maliga P, Proc. Natl. Acad. Sci. USA 90:913-917 (1993); Allison L A, Simon L D, Maliga P, EMBO J. 15:2802-2809 (1996));
        PaccD promoter (Hajdukiewicz P T J, Allison L A, Maliga P, EMBO J. 16:4041-4048 (1997); WO 97/06250);
        PclpP promoter (Hajdukiewicz P T J, Allison L A, Maliga P, EMBO J. 16:4041-4048 (1997); WO 99/46394);
        PatpB, PatpI, PpsbB promoters (Hajdukiewicz P T J, Allison L A, Maliga P, EMBO J. 16:4041-4048 (1997));
        PrpoB promoter (Liere K, Maliga P, EMBO J. 18:249-257 (1999));
        PatpB/E promoter (Kapoor S, Suzuki J Y, Sugiura M, Plant J 11:327-337 (1997)).
    
    


In addition, prokaryotic promoters (such as those from, e.g., E. coli or Synechocystis) or synthetic promoters can also be used.
3. Intergenic and Untranslated Sequences
Intergenic sequences can be used in the invention to control expression of genes.
For instance, the intergenic sequences rps19/rpl22, psbD/C, and psaA/B can be used (Herz S, Füβl M, Steiger S, Koop H-U, Transgenic Research 14:969-982 (2005)).
Intact or truncated 5′ UTRs of highly expressed plastid genes such as psbA, atpB or rbcL have been used to regulate transgene expression in plastids at the post-transcriptional level (Staub J M, Maliga P, EMBO J. 12:601-606 (1993); Kuroda H, Maliga P, Plant Physiology 125:430-436 (2001)). The following 5′UTRs can be used in the invention.

    
    
        5′ UTR rbcL (Shiina T, Allison L, Maliga P, Plant Cell 10:1713-1722 (1998);
        5′ UTR psbA (Agrawal G K, Kato H, Asayama M, Shirai M, Nucleic Acids Research 29:1835-1843 (2001));
        5′ UTR of gene 10 from bacteriophage T7 has also been shown to mediate very high expression in plastids (Kuroda H, Maliga P, Nucleic Acid Research 29:970-975 (2001)).
    
    


The following 3′ UTRs can be used to stabilize transcripts.

    
    
        3′ UTR rbcL (Shinozaki K, Sugiura M, Gene 20:91-102 (1982));
        3′ UTR psbA from tobacco, Chlamydomonas, or Synechocystis. 
    
    


Modifications or extensions of the N-terminus of a desired protein have also been shown to increase transgene expression level (Kuroda H, Maliga P, Nucleic Acid Research 29:970-975 (2001); Kuroda H, Maliga P, Plant Physiology 125:430-436 (2001)). These sequences immediately downstream of the start codon have been called downstream boxes (DB). Examples of downstream boxes that can be used in the invention include, but are not limited to, the sequence ATG GCT AGC ATT TCC (SEQ ID NO: 1) (Herz S, Füβl M, Steiger S, Koop H-U, Transgenic Research 14:969-982 (2005), those listed in international application publication no. WO 00/07431, and the wild type downstream box of the T7 bacteriophage gene 10 (see, international application publication no. WO 01/21782).
4. Selectable Markers
Genetic constructs may encode a selectable marker to enable selection of plastid transformation events. There are many methods that have been described for the selection of transformed plants in traditional nuclear plant transformation methods [for review see (Mild et al., Journal of Biotechnology 107:193-232 (2004)) and references incorporated within].
A preferred selectable marker for plastid transformation is the bacterial aadA gene that encodes aminoglycoside 3′-adenyltransferase (AadA) conferring spectinomycin and streptomycin resistance (Svab et al., Proc. Natl. Acad. Sci. USA 90:913-917 (1993)). Other selectable markers that have been successfully used in plastid transformation include the spectinomycin-resistant allele of the plastid 16S ribosomal RNA gene (Staub J M, Maliga P, Plant Cell 4:39-45 (1992); Svab Z, Hajdukiewicz P, Maliga P, Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990)), nptII that encodes aminoglycoside phosphotransferase for selection on kanamycin (Carrer H, Hockenberry T N, Svab Z, Maliga P., Mol. Gen. Genet. 241:49-56 (1993); Lutz K A, et al., Plant J. 37:906-913 (2004); Lutz K A, et al., Plant Physiol. 145:1201-1210 (2007)), and aphA6, another aminoglycoside phosphotransferase (Huang F-C, et al., Mol. Genet. Genomics 268:19-27 (2002)). Another selection scheme has been reported that uses a chimeric betaine aldehyde dehydrogenase gene (BADH) capable of converting toxic betaine aldehyde to nontoxic glycine betaine (Daniell H, et al., Curr. Genet. 39:109-116 (2001)).
In addition methods described for selection of nuclear transformants can be used after initial selection of transplastomic lines with plastidial selection markers. Methods have been described using e.g. herbicide markers as the bar gene encoding phosphinothricin acetyltransferase or glyphosate resistant forms of the 5-enolpyruvylshikimate-3-phosphate synthase genes (US Patent Application 2002/0042934 A1; Ye et al., Plant Physiology 133(1): 402-410 (2003)).
Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., EMBO J. 6:3901-3907 (1987); U.S. Pat. No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., Trends Biochem. Sci. 20:448-455 (1995); Pan et al., Plant Physiol. 112:893-900 (1996)). Both genes have been used in combination with the aadA gene or the spectinomycin-resistant allele of the plastid 16S ribosomal RNA gene for plastid transformation ((Hibberd et al., Plant Journal 16(5): 627-632 (1998); Sidorov, et al., Plant Journal 19(2): 209-216 (1999); Khan and Maliga, Nature Biotechnology 17(9): 910-915 (1999); Staub and Maliga; EMBO J. 12(2): 601-606 (1993)).
B. Exemplary Host Plants
Plants transformed in accordance with the present disclosure may be monocots or dicots. The transformation of suitable agronomic plant hosts using vectors for direct plastid transformation can be accomplished with a variety of methods and plant tissues. Representative plants useful in the methods disclosed herein include the Brassica family including B. napus, B. rappa, B. carinata and B. juncea; industrial oilseeds such as Camelina saliva, Crambe, jatropha, castor; Arabidopsis thaliana; maize; soybean; cottonseed; sunflower; palm; coconut; safflower; peanut; mustards including Sinapis alba; sugarcane and flax. Crops harvested as biomass, such as silage corn, alfalfa, switchgrass, miscanthus, sorghum or tobacco, also are useful with the methods disclosed herein. Representative tissues for transformation using these vectors include protoplasts, cells, callus tissue, leaf discs, pollen, and meristems. Representative transformation procedures include biolistics, microinjection, electroporation, polyethylene glycol-mediated protoplast transformation, liposome-mediated transformation, and silicon fiber-mediated transformation (U.S. Pat. No. 5,464,765; “Gene Transfer to Plants” (Potrykus, et al., eds.) Springer-Verlag Berlin Heidelberg New York (1995); “Transgenic Plants: A Production System for Industrial and Pharmaceutical Proteins” (Owen, et al., eds.) John Wiley & Sons Ltd. England (1996); and “Methods in Plant Molecular Biology: A Laboratory Course Manual” (Maliga, et al. eds.) Cold Spring Laboratory Press, New York (1995)). There has been one report using Agrobacterium-mediated transformation for plastid transformation (De Block et al., The EMBO Journal 4, 1367-1372 (1985)).
C. Methods of Plant Transformation
Methods for transformation of plastids such as chloroplasts are known in the art. See, for example, Svab et al., Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990); Svab and Maliga, Proc. Natl. Acad. Sci. USA 90:913-917 (1993); Svab and Maliga, EMBO J. 12:601-606 (1993). The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation may be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al., Proc. Natl. Acad. Sci. USA 91:7301-7305 (1994).
The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin were utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P., Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990); Staub, J. M., and Maliga, P., Plant Cell 4:39-45 (1992)). The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign DNA molecules (Staub, J. M., and Maliga, P., EMBO J. 12:601-606 (1993)). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P., Proc. Natl. Acad. Sci. USA 90:913-917 (1993)). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M., Nucl. Acids Res. 19:4083-4089 (1991)).
The nucleic acids of interest to be targeted to the plastid may be optimized for expression in the plastid to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using plastid-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.
Recombinase technologies which are useful for producing the disclosed transgenic plants include the cre-lox, FLP/FRT and Gin systems. Methods by which these technologies can be used for the purpose described herein are described for example in U.S. Pat. No. 5,527,695, Dale And Ow, Proc. Natl. Acad. Sci. USA 88:10558-10562 (1991), and Medberry et al., Nucleic Acids Res. 23:485-490 (1995). Another useful approach is the utilization of phiC31 phage integrase (Lutz K A, Corneille S, Azhagiri A K, Svab Z, Maliga P, Plant J. 37:906-913 (2004)).
D. Methods for Reproducing Transgenic Plants
Following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.
Further rounds of regeneration of plants from explants of a transformed plant or tissue can be performed to increase the number of transgenic plastids such that the transformed plant reaches a state of homoplasmy where all plastids contain uniform plastomes containing the transgene insert.
II. Methods for Use
The disclosed vectors can be used to produce transplastomic plants that produce at least 10%, 12%, 15% PHA in regions of leaves. For the whole plant, at least about 8% or more per unit dry weight of polyhydroxyalkanoate, preferably polyhydroxybutyrate, or a co-polymer thereof can be produced.
The transplastomic plants can also produce greater than 10%, 12%, 15%, or 20% in leaves or more dwt in regions of leaves of the plant. In certain embodiments, the transplastomic plants have delayed flowering relative to wild-type plants. The transplastomic plants typically are delayed by flowering compared to the wild-type by 10%, 20%, 30% or more of the total flowering time.
The transplastomic plants can be grown and harvested. The polyhydroxyalkanoate can be isolated from the plants and the remaining plant material can be used as a feedstock for industrial use, preferably for the production of energy. The polyhydroxyalkanoate harvested from the plants can then be used to produce plastics for use in a wide range of applications such as injection molded goods, films, fibers and non-woven articles, foams, bottles and other containers and coating materials such as paper coatings and paints. PHA also can be converted to a range of chemical intermediates and has several medical applications.
The present invention will be further understood by reference to the following non-limiting examples.
EXAMPLES
Example 1
Design and Construction of Plastid Transformation Vectors
The plastome of Nicotiana tabacum contains 23 codons with a low frequency of use (<10/1000) (http://www.kazusa.or.jp/codon/). The presence of these codons in PHB pathway genes from various natural PHA producers as well as the overall GC content of the genes was compared to data available for the N. tabacum plastome. Genes from Acinetobacter sp. (Schembri et al., J. Bacteria 177:4501-7 (1995)) and Bacillus megaterium (McCool et al., J. Bacteriol. 181:585-592 (1999)) were chosen for use in plastid transformation vectors based on the similarity of GC content and codon usage to the N. tabacum plastome. These genes contain few codons with a low frequency of use (<10/1000) and posses GC content<50%. Detailed descriptions of the plastid transformation vectors used in this study as well as references to pertinent DNA sequences are available in FIGS. 1(a)-(c). Plasmid pJKD1425 (Schembri et al., J. Bacterial. 177:4501-7 (1995)) was used as the source of the Acinetobacter sp. PHA operon. Plasmid pGM10 (McCool et al., J. Bacteria 181:585-592 (1999)) was used as the source of PHB genes from B. megaterium. 
Plastid transformation vectors were designed to yield both high level expression and limited homology to the host plastome to prevent recombination. For example the sequence length of the UTRs is minimal (≦55 nucleotides) and the total amount of plastidial derived DNA in the vector is <3% (excluding sequences of the left and right flanks) such that recombination with the host plastome is limited.
Example 2
Plastid Transformation with Constructs for High Level PHB Production
Seeds of tobacco (Nicotiana tabacum L. cv Petite Havana SR1) were obtained from Lehle Seeds (Round Rock, Tex.). Plants in tissue culture were grown (16 h light period, 20 to 30 μmol photons m−2 s−1, 23° C.; 8 h dark period, 20° C.) on Murashige and Skoog medium (Murashige et al., Physiol. Plant. 15:473-497 (1962)) containing 2% (w/v) sucrose. Plastid transformation was performed using a PDS 1000 System (BIO RAD, Hercules, Calif., USA) and 0.6 μm gold particles as previously described (Svab et al., Proc. Natl. Acad. Sci. USA 87:8526-8530 (1990); Daniell, Methods in Molecular Biology 62:463-489 (1997)). Selection of transplastomic lines was performed on Murashige and Skoog/sucrose medium supplemented with 500 mg/L spectinomycin. Once transferred to soil, plants were grown in growth chambers (16 h light period, 40 to 80 μmol photons m−2 s−1, 23° C.; 8 h dark period, 20° C.) or in a greenhouse with supplemental lighting (16 h light period, minimum 150 μmol photons m−2 s−1, 23-25° C.; 8 h dark period, 20-22° C.).
Successful integration into the host plastome was verified by PCR using primers KMB41, KMB77, KMB153, and KMB36. Binding sites of these primers are shown in FIG. 2.







Primer KMB 41







(SEQ ID NO: 2)







Sequence: 5′-ttgagctgcgccaaagcctc-3′


 


Primer KMB 77







(SEQ ID NO: 3)







Sequence: 5′-cttgtgctagaactttagctcg-3′


 


Primer KMB 153







(SEQ ID NO: 4)







Sequence: 5′-cca ccc atg tgg tac ttc att cta cg-3′


 


Primer KMB 36







(SEQ ID NO: 5)







Sequence: 5′-gag ttg tag gga ggc aac cat ggc ag-3′






PCR analysis of plants was performed using 10-12 ng of total DNA with PCR Supermix Kit (Invitrogen). Total DNA was isolated from in vitro or green house derived tobacco leaves using the DNeasy kit (Qiagen, Santa Clarita, Calif.).
Confirmation of correct integration at the left flank was performed with primer pair KMB77 and KMB41 using conventional PCR procedures and an annealing temperature of 57° C. The expected 2.02 kb PCR product was observed in reactions with DNA from candidate plants 2, 3, 6, and 8 but not in reactions containing wild-type DNA (FIG. 2).
Confirmation of correct integration at the right flank was used with primer pair KMB153 and KMB36 using an annealing temperature of 52° C. The expected 1.81 kb PCR product was observed in reactions with DNA from candidate plants 2, 3, 6, and 8 but not in reactions containing wild-type DNA (FIG. 2).
Example 3
PHB Analysis of Transplastomic Plants
The amount of PHB present in plant tissue was measured by gas chromatography/mass spectroscopy (GC/MS) as previously described (Kourtz et al., Transgenic Research 16:759-769 (2007)) using 30-150 mg of lyophilized leaf material. The highest levels of PHB observed were 20.6% dwt PHB in leaf tissues of line pCAB2P3T0 and 19.6% dwt PHB in leaf tissues of line pCAB6P3T0. These lines were obtained after plastid transformation of pCAB(2), isolation of regenerant, and performance of two additional cycles of shoot regeneration from excised leaf.
Accumulation of PHB in leaves and stems were measured in plant line pCAB2.2m P2T1 and the percent dry weight accumulation throughout the plant was calculated (Table 1). Line pCAB2.2m P2T1 was obtained from seed of a plant that was obtained after plastid transformation of pCAB(2), isolation of regenerant, and performance of one additional cycle of shoot regeneration from excised leaf. In general, leaf tissue of this line contained more PHB than stem tissue. The total PHB production was 8.78% dwt of the total plant. The leaf tissue from plant pCAB2.2m P2T1 was a greater percent of the total plant biomass (71%) than leaf tissue from wild-type plants (54%) (Table 1).












TABLE 1







Mass of wild-
Mass of CAB(2)
Mass Ratio




type tissue*
tissue**
CAB(2)/Wild-
PHB



[g dwt]
[g dwt]
type
[% dwt]




















Total
33.5 ± 3.5
21.06
0.64
8.78


biomass


Leaf
18.4 ± 2.3
14.98
0.81
11.18


biomass


Stem
15.1 ± 1.8
6.08
0.40
2.87


biomass





*Data for wild-type tissue is an average of 5 plants.


**Data for CAB(2) tissue was obtained from a single plant that was grown from T1 seed. T1 seed was produced by a plastid transformed regenerant that was subjected to one additional cycle of shoot regeneration from an excised leaf.


dwt, dry weight. Inflorescences and seeds were not included in the measurements.






Example 4
Determination of Extent of Homoplasmy in Transformed Lines
Correct integration of the transgenes and the extent of homoplasmy of transgenic lines was analyzed by Southern analysis. Total DNA was isolated from in vitro or green house derived tobacco leaves using the DNeasy kit (Qiagen, Santa Clarita, Calif.). Aliquots of total DNA containing 2.5 to 7.5 μg were digested with the restriction enzyme Pst I and blotted onto positively charged nylon membranes (Roche Molecular Biochemicals, Indianapolis). A 0.61 kb digoxigenin-labeled hybridization probe (Probe I) for detection of genetic elements were prepared using conventional PCR procedures with the DIG probe synthesis kit (Roche Molecular Biochemicals) and oligonucleotides KMB96 and KMB97 using a primer annealing temperature of 64° C.








Primer KMB 96



5′-cttctgtaactggataactagcactg-3′
(SEQ ID NO: 6)


 


Primer KMB 97


5′-gttaccaaggaaccatgcatagcactg-3′
(SEQ ID NO: 7)






Hybridization signals were detected with alkaline-phosphatase conjugated anti-digoxigenin antibody and chemoluminescent detection (CDP-Star, Roche Molecular Biochemicals). DNA from a wild-type plant yielded a 2.4 kb fragment as expected for the wild-type plastome. DNA from a plant transformed with plasmid pUCaadA (FIG. 1(c)) yielded a 3.28 kb fragment as expected for insertion of a transgenic fragment containing the aadA gene. Plants from transformations of pCAB(2) yielded a prominent 4.12 kb fragment as expected for correct integration of the transgenic DNA into the plastome. Little, if any, 2.40 kb fragment was observed in pCAB(2) samples suggesting that little, if any, wild-type plastome was still present in these plants.
A method more sensitive than Southern analysis to determine the presence of wild type copies in transplastomic plants is to screen large numbers of seeds/descendants of transplastomic lines on media containing the selection agent. Segregation of copies of the plastome will lead to plants sensitive to the selection agent if wild type copies are still present. Seeds of three pCAB(2) lines (pCABP2T1 #2, #6 and #3) were therefore germinated on media containing 500 mg/ml spectinomycin and on control plates without selection agent. The germination rate and phenotype of seedlings were evaluated three weeks after plating (Table 2). Germination rates of 19, 13, and 55% were observed for pCABP2T1 transgenic lines 2, 3, and 6, respectively. Some of the germinating seeds showed mosaic white patches on their cotyledons suggesting possible sensitivity to spectinomycin. The percent of seedlings with mosaic cotyledons was 2.3, 11.6, and 0.6 for lines 2, 3, and 6, respectively. None of the seedlings derived from seeds plated on media without spectinomycin showed any mosaic patterns (Table 2) suggesting that the mosaic patterns were indeed due to a lack of spectinomycin resistance rather than other recombination events that might lead to variegated patterns. In conclusion the most stable and highest PHB producing lines #2 and #6 were shown to have small amounts of wild type copies left (2.3% and 0.6% of the seeds capable of germinating) while a less stable line (#3) showed up to 11.6% of at least partial resistance to spectinomycin. Interestingly the real leaves of the seedlings did not show any mosaic patterns on medium containing selection agent.










TABLE 2









Seeds Plated on Media With




Spectinomycin










Seedlings
Seeds Plated on Media Without



With
Spectinomycin













pCAB

Seeds
Mosaic

Seeds
Seedlings


P2T1
Seeds
Germinated
Cotelydons
Seeds
Germinated
With 
















Line #
Plated
Total
%
Total
%
Plated
Total
%
Mosaic Cotelydons



















2
11095
2140
19.3
49
2.3
1783
1219
68.4
0


3
10445
1357
13
158 
11.6 
1790
655
36.6
0


6
7656
4241
55.4
28
0.6
1823
1366
74.9
0


wt
1648
1237
75.1
 0*
0* 
1369
1025
74.9
0





*cotelydons were completely white/bleached indicating they are dying.






Example 5
Average Days to Flowering
In order to determine the average days to flowering, wild type seeds of Nicotiana tabacum L. cv Petite Havana SR1 were germinated on Murashige and Skoog medium (Murashige et al., Physiol. Plant. 15:473-497 (1962)) containing 2% (w/v) sucrose and T1 seeds of lines pCAB P2T1 #2 and #6, were germinated on the same media supplemented with 500 mg/ml spectinomycin. pCAB P2T1 seeds are seeds obtained from lines that were obtained after plastid transformation of pCAB(2), isolation of regenerant, and performance of one additional cycle of shoot regeneration from excised leaf. Plants in tissue culture were grown with a 16 h light period (20 to 30 μmol photons m−2 s−1, 23° C.) and an 8 h dark period at 20° C. Three weeks after seed imbibition germinated seedlings were transferred to tissue culture vessels and maintained on the media described above. Six weeks after seed imbibitions, six wild type plants and eight plants of lines pCAB P2T1 #2 and #6, respectively, were transferred to a greenhouse with supplemental lighting (16 h light period, minimum 150 μmol photons m−2 s−1, 23-25° C.; 8 h dark period, 20-22° C.). The onset of formation of inflorescences was monitored (see FIG. 6). Days until flowering was calculated from the day of seed imbibition until opening of the first flower of the first inflorescence.
Additional wild type plants were grown to compare the phenotypes of wild type plants and pCAB P2T1 plants in comparable developmental stages. These additional wild type plants were plated 12 and 22 days after imbibition of pCAB P2T1 seeds. Further transfers of plants to tissue culture vessels and to the green house were performed as described above. Pictures were taken from a plant of pCAB P2T1 line #2 together with a wild type plant that was 12 days younger. The photo was taken 44 days after seed imbibition of pCAB P2T1 seeds and 32 days after seed imbibition of the wild type. To document the phenotype of a plant of pCAB P2T1 line #2 together with a wild type plant at a later developmental stage (when plants had already reached their final height), a picture was taken from a plant of pCAB P2T1 line #2 together with a wild type plant that was 22 days younger. The photo was taken 85 days after seed imbibition of pCAB P2T1 seeds and 63 days after seed imbibition of the wild type.
FIG. 6 shows a bar graph of the comparison of days to flowering of wild-type and transgenic plants. Plants were grown in the green house until flowers started to appear. Data was obtained from 8 plants of each line. Lines are as follows: wt, wild-type; PBCAB2, line pCABP2 T1 #2; and PBCAB6, line pCAB P2T1 #6. These lines were obtained from seed of lines that were obtained after plastid transformation of pCAB(2), isolation of regenerant, and performance of one additional cycle of shoot regeneration from excised leaf.
Example 6
Analysis of Chloroplasts
Leaf samples were prepared for analysis by transmission electron microscopy (TEM) by fixing in 2% paraformaldehyde, 2% glutaraldehyde, 4% sucrose, 1 mM CaCl2, 2 mM MgCl2 in 50 mM sodium cacodylate buffer, pH 7.2. One cm square leaf pieces were cut from the mid-blade blade area and cut into strips 0.5-1.0 mm wide while submerged in the fixative. The fixative was vacuum infiltrated into the leaf tissue at  ̃70 kPa for several cycles until most pieces sank. The fixation was conducted for 2 hr at room temperature. Tissue was rinsed in 3 changes of 50 mM sodium cacodylate buffer containing 4% sucrose, and post-fixed in the same buffer with 1% osmium tetroxide for 8 hr at 4° C. Tissue was rinsed in several changes of distilled water and dehydrated in acetone by 10% increments to 100% acetone, and gradually infiltrated (1:3, 1:2, 1:1, 2:1, 3:1, 100%) with Ellis low-viscosity epoxy resin formulation (Ellis, E., Ann. Microscopy Today 14:32-33 (2006)), an update of the Spurr's resin mixture (Spurr, A. R., J., Ultrastructure Res. 26:31 (1969)). The samples received 3 changes of 100% resin at 2 hr intervals, were embedded in the same and polymerized 16 hr at 70° C. Sections were cut at 60 nm thickness, mounted on copper grids, and stained 20 minutes at room temperature with uranyl acetate (uranyl acetate solution was saturated at 4° C. in 50% ethanol), and 3 minutes in lead citrate (2.5 mg/ml in 0.1 N NaOH). Sections were observed at 80 kV in a JEOL JEM-100S transmission electron microscope and photographed with a CCD camera (SIA, Model 7C).
The absence of starch granules, the smaller size of plastoglobuli, and the presence of PHB granules in transplastomic chloroplasts was noted in line pCAB2.7 P2 T1 producing 5.4% dwt PHB and line pCAB2.1 P2 T1 producing 6.3% dwt PHB. PHB analysis was performed using the tip of each leaf sampled for TEM analysis.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10. The terms “one,” “a,” or “an” as used herein are intended to include “at least one” or “one or more,” unless otherwise indicated.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.








Sequence of pUCaadA











1
TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT
(SEQ ID NO: 8)




AACTTCGTAA ATAGTCCCAA TAACAGAGTA CTCGCCTATG TATAAACTTA
(SEQ ID NO: 9)


 


51
GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA



CATAAATCTT TTTATTTGTT TATCCCCAAG GCGCGTGTAA AGGGGCTTTT


 


101
GTGCCACCTG ACGTCTAAGA AACCATTATT ATCATGACAT TAACCTATAA



CACGGTGGAC TGCAGATTCT TTGGTAATAA TAGTACTGTA ATTGGATATT


 


151
AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC GGTGATGACG



TTTATCCGCA TAGTGCTCCG GGAAAGGAGA GCGCGCAAAG CCACTACTGC


 


201
GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG



CACTTTTGGA GACTGTGTAC GTCGAGGGCC TCTGCCAGTG TCGAACAGAC


 


251
TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT



ATTCGCCTAC GGCCCTCGTC TGTTCGGGCA GTCCCGCGCA GTCGCCCACA


 


301
TGGCGGGTGT CGGGGCTGGC TTAACTATGC GGCATCAGAG CAGATTGTAC



ACCGCCCACA GCCCCGACCG AATTGATAGG CCGTAGTCTC GTCTAACATG


 


351
TGAGAGTGCA CCATATGCGG TGTGAAATAC CGCACAGATG CGTAAGGAGA



ACTCTCACGT GGTATACGCC ACACTTTATG GCGTGTCTAC GCATTCCTCT


 


401
AAATACCGCA TCAGGCGCCA TTCGCCATTC AGGCTGCGCA ACTGTTGGGA



TTTATGGCGT AGTCCGCGGT AAGCGGTAAG TCCGACGCGT TGACAACCCT


 


451
AGGGCGATCG GTGCGGGCCT CTTCGCTATT ACGCCAGCTG GCGAAAGGGG



TCCCGCTAGC CACGCCCGGA GAAGCGATAA TGCGGTCGAC CGCTTTCCCC


 


501
GATGTGCTGC AAGGCGATTA AGTTGGGTAA CGCCAGGGTT TTCCCAGTCA



CTACACGACG TTCCGCTAAT TCAACCCATT GCGGTCCCAA AAGGGTCAGT


 


551
CGACGTTGTA AAACGACGGC CAGTGAATTC ATGACTGCAA TTTTAGAGAG



GCTGCAACAT TTTGCTGCCG GTCACTTAAG TACTGACGTT AAAATCTCTC


 


601
ACGCGAAAGC GAAAGCCTAT GGGGTCGCTT CTGTAACTGG ATAACTAGCA



TGCGCTTTCG CTTTCGGATA CCCCAGCGAA GACATTGACC TATTGATCGT


 


651
CTGAAAACCG TCTTTACATT GGATGGTTTG GTGTTTTGAT GATCCCTACC



GACTTTTGGC AGAAATGTAA CCTACCARAC CACAAAACTA CTAGGGATGG


 


701
TTATTGACGG CAACTTCTGT ATTTATTATT GCCTTCATTG CTGCTCCTCC



AATAACTGCC GTTGAAGACA TAAATAATAA CGGAAGTAAC GACGAGGAGG


 


751
AGTAGACATT GATGGTATTC GTGAACCTGT TTCAGGGTCT CTACTTTACG



TCATCTGTAA CTACCATAAG CACTTGGACA AAGTCCCAGA GATGAAATGC


 


801
GAAACAATAT TATTTCCGGT GCCATTATTC CTACTTCTGC AGCTATAGGT



CTTTGTTATA ATAAAGGCCA CGGTAATAAG GATGAAGACG TCGATATCCA


 


851
TTACATTTTT ACCCAATCTG GGAAGCGGCA TCCGTTGATG AATGGTTATA



AATGTAAAAA TGGGTTAGAC CCTTCGCCGT AGGCAACTAC TTACCAATAT


 


901
CAACGGTGGT CCTTATGAAC TAATTGTTCT ACACTTCTTA CTTGGCGTAG



GTTGCCACCA GGAATACTTG ATTAACAAGA TGTGAAGAAT GAACCGCATC


 


951
CTTGTTACAT GGGTCGTGAG TGGGAGCTTA GTTTCCGTCT GGGTATGCGA



GAACAATGTA CCCAGCACTC ACCCTCGAAT CAAAGGCAGA CCCATACGCT


 


1001
CCTTGGATTG CTGTTGCATA TTCAGCTCCT GTTGCAGCTG CTACCGCAGT



GGAACCTAAC GACAACGTAT AAGTCGAGGA CAACGTCGAC GATGGCGTCA


 


1051
TTTCTTGATC TACCCAATTG GTCAAGGAAG TTTTTCTGAT GGTATGCCTC



AAAGAACTAG ATGGGTTAAC CAGTTCCTTC AAAAAGACTA CCATACGGAG


 


1101
TAGGAATCTC TGGTACTTTC AATTTCATGA TTGTATTCCA GGCTGAGCAC



ATCCTTAGAG ACCATGAAAG TTAAAGTACT AACATAAGGT CCGACTCGTG


 


1151
AACATCCTTA TGCACCCATT TCACATGTTA GGCGTAGCTG GTGTATTCGG



TTGTAGGAAT ACGTGGGTAA AGTGTACAAT CCGCATCGAC CACATAAGCC


 


1201
CGGCTCCCTA TTCAGTGCTA TGCATGGTTC CTTGGTAACT TCTAGTTTGA



GCCGAGGGAT AAGTCACGAT ACGTACCAAG GAACCATTGA AGATCAAACT


 


1251
TCAGGGAAAC CACAGAAAAT GAATCTGCTA ATGAAGGTTA CAGATTCGGT



AGTCCCTTTG GTGTCTTTTA CTTAGACGAT TACTTCCAAT GTCTAAGCCA


 


1301
CAAGAGGAAG AAACTTATAA CATCGTAGCC GCTCATGGTT ATTTTGGCCG



GTTCTCCTTC TTTGAATATT GTAGCATCGG CGAGTRCCAA TAAAACCGGC


 


1351
ATTGATCTTC CAATATGCTA GTTTCAACAA CTCTCGTTCG TTACACTTCT



TAACTAGAAG GTTATACGAT CAAAGTTGTT GAGAGCAAGC AATGTGAAGA


 


1401
TCCTAGCTGC TTGGCCTGTA GTAGGTATCT GGTTTACCGC TTTAGGTATC



AGGATCGACG AACCGGACAT CATCCATAGA CCAAATGGCG AAATCCATAG


 


1451
AGCACTATGG CTTTCAACCT AAATGGTTTC AATTTCAACC AATCTGTAGT



TCGTGATACC GAAAGTTGGA TTTACCAAAG TTAAAGTTGG TTAGACATCA


 


1501
TGACAGTCAA GGCCGTGTAA TTAATACTTG GGCTGATATC ATTAACCGTG



ACTGTCAGTT CCGGCACATT AATTATGAAC CCGACTATAG TAATTGGCAC


 


1551
CTAACCTTGG TATGGAAGTT ATGCATGAAC GTAATGCTCA CAACTTCCCT



GATTGGAACC ATACCTTCAA TACGTACTTG CATTACGAGT GTTGAAGGGA


 


1601
CTAGACCTAG CTGCTATCGA AGCTCCATCT ACAAATGGAT AAGTCGACAA



GATCTGGATC GACGATAGCT TCGAGGTAGA TGTTTACCTA TTCAGCTGTT


 


1651
GTGTTTGCGG CCGCGAGCTC GGACTCGAGT TTGGATCCAA TCGATACAAG



CACAAACGCC GGCGCTCGAG CCTGAGCTCA AACCTAGGTT AGCTATGTTC


 


1701
TGAGTTGTAG GGAGGCAACC ATGGCAGAAG CGGTGATCGC CGAAGTATCG



ACTCAACATC CCTCCGTTGG TACCGTCTTC GCCACTAGCG GCTTCATAGC


 


1751
ACTCAACTAT CAGAGGTAGT TGGCGTCATC GAGCGCCATC TCGAACCGAC



TGAGTTGATA GTCTCCATCA ACCGCAGTAG CTCGCGGTAG AGCTTGGCTG


 


1801
GTTGCTGGCC GTACATTTGT ACGGCTCCGC AGTGGATGGC GGCCTGAAGC



CAACGACCGG CATGTAAACA TGCCGAGGCG TCACCTACCG CCGGACTTCG


 


1851
CACACAGTGA TATTGATTTG CTGGTTACGG TGACCGTAAG GCTTGATGAA



GTGTGTCACT ATAACTAAAC GACCAATGCC ACTGGCATTC CGAACTACTT


 


1901
ACAACGCGGC GAGCTTTGAT CAACGACCTT TTGGAAACTT CGGCTTCCCC



TGTTGCGCCG CTCGAAACTA GTTGCTGGAA AACCTTTGAA GCCGAAGGGG


 


1951
TGGAGAGAGC GAGATTCTCC GCGCTGTAGA AGTCACCATT GTTGTGCACG



ACCTCTCTCG CTCTAAGAGG CGCGACATCT TCAGTGGTAA CAACACGTGC


 


2001
ACGACATCAT TCCGTGGCGT TATCCAGCTA AGCGCGAACT GCAATTTGGA



TGCTGTAGTA AGGCACCGCA ATAGGTCGAT TCGCGCTTGA CGTTAARCCT


 


2051
GAATGGCAGC GCAATGACAT TCTTGCAGGT ATCTTCGAGC CAGCCACGAT



CTTACCGTCG CGTTACTGTA AGAACGTCCA TAGAAGCTCG GTCGGTGCTA


 


2101
CGACATTGAT CTGGCTATCT TGCTGACAAA AGCAAGACAA CATAGCGTTG



GCTGTAACTA GACCGATAGA ACGACTGTTT TCGTTCTCTT GTATCGCAAC


 


2151
CCTTGGTAGG TCCAGCGGCG GAGGAACTCT TTGATCCGGT TCCTGAACAG



GGAACCATCC AGGTCGCCGC CTCCTTGAGA AACTAGGCCA AGGACTTGTC


 


2201
GATCTATTTG AGGCGCTAAA TGAAACCTTA ACGCTATGGA ACTCGCCGCC



CTAGATAAAC TCCGCGATTT ACTTTGGAAT TGCGATACCT TGAGCGGCGG


 


2251
CGACTGGGCT GGCGATGAGC GAAATGTAGT GCTTACGTTG TCCCGCATTT



GCTGACCCGA CCGCTACTCG CTTTACATCA CGAATGCAAC AGGGCGTAAA


 


2301
GGTACAGCGC AGTAACCGGC AAAATCGCGC CGAAGGATGT CGCTGCCGAC



CCATGTCGCG TCATTGGCCG TTTTAGCGCG GCTTCCTACA GCGACGGCTG


 


2351
TGGGCAATGG AGCGCCTGCC GGCCCAGTAT CAGCCCGTCA TACTTGAAGC



ACCCGTTACC TCGCGGACGG CCGGGTCATA GTCGGGCAGT ATGAACTTCG


 


2401
TAGACAGGCT TATCTTGGAC AAGAAGAAGA TCGCTTGGCC TCGCGCGCAG



ATCTGTCCGA ATAGAACCTG TTCTTCTTCT AGCGAACCGG AGCGCGCGTC


 


2451
ATCAGTTGGA AGAATTTGTC CACTACGTGA AAGGCGAGAT CACCAAGGTA



TAGTCAACCT TCTTAAACAG GTGATGCACT TTCCGCTCTA GTGGTTCCAT


 


2501
GTCGGCAAAT AAATCTAAGC CGAATTGGGC CTAGTCTATA GGAGGTTTTG



CAGCCGTTTA TTTAGATTCG GCTTAACCCG GATCAGATAT CCTCCAAAAC


 


2551
AAAAGAAAGG AGCAATAATC ATTTTCTTGT TCTATCAAGA GGGTGCTATT



TTTTCTTTCC TCGTTATTAG TAAAAGAACA AGATAGTTCT CCCACGATAA


 


2601
GCTCCTTTCT TTTTTTCTTT TTATTTATTT ACTAGTATTT TACTTACATA



CGAGGAAAGA AAAAAAGAAA AATAAATAAA TGATCATAAA ATGAATGTAT


 


2651
GACTTTTTTG TTTACATTAT AGAAAAAGAA GGAGAGGTTA TTTTCTTGCA



CTGAAAAAAC AAATGTAATA TCTTTTTCTT CCTCTCCAAT AAAAGAACGT


 


2701
TTTATTCATG ATTGAGTATT CTATTTTGAT TTTGTATTTG TTTAAAATTG



AAATAAGTAC TAACTCATAA GATAAAACTA AAACATAAAC AAATTTTAAC


 


2751
TAGAAATAGA ACTTGTTTCT CTTCTTGCTA ATGTTACTAT ATCTTTTTGA



ATCTTTATCT TGAACAAAGA GAAGAACGAT TACAATGATA TAGAAAAACT


 


2801
TTTTTTTTTT CCAAAAAAAA AATCAAATTT TGACTTCTTC TTATCTCTTA



AAAAAAAAAA GGTTTTTTTT TTAGTTTAAA ACTGAAGAAG AATAGAGAAT


 


2851
TCTTTGAATA TCTCTTATCT TTGAAATAAT AATATCATTG AAATAAGAAA



AGAAACTTAT AGAGAATAGA AACTTTATTA TTATAGTAAC TTTATTCTTT


 


2901
GAAGAGCTAT ATTCGAACTT GAATCTTTTG TTTTCTAATT TAAATAATGT



CTTCTCGATA TAAGCTTGAA CTTAGAAAAC AAAAGATTAA ATTTATTACA


 


2951
AAAAACGGAA TGTAAGTAGG CGAGGGGGCG GATGTAGCCA AGTGGATCAA



TTTTTGCCTT ACATTCATCC GCTCCCCCGC CTACATCGGT TCACCTAGTT


 


3001
GGCAGTGGAT TGTGAATCCA CCATGCGCGG GTTCAATTCC CGTCGTTCGC



CCGTCACCTA ACACTTAGGT GGTACGCGCC CAAGTTAAGG GCAGCAAGCG


 


3051
CCATAATTAC TCCTATTTTT TTTTTTTTTG TAAAAACGAA GAATTTAATT



GGTATTAATG AGGATAAAAA AAAAAAAAAC ATTTTTGCTT CTTAAATTAA


 


3101
CGATTTTCTC TCCTATTTAC TACGGCGACG AAGAATCAAA TTATCACTAT



GCTAAAAGAG AGGATAAATG ATGCCGCTGC TTCTTAGTTT AATAGTGATA


 


3151
ATTTATTCCT TTTTCTACTT CTTCTTCCAA GTGCAGGATA ACCCCAAGGG



TAAATAAGGA AAAAGATGAA GAAGAAGGTT CACGTCCTAT TGGGGTTCCC


 


3201
GTTGTGGGTT TTTTTCTACC AATTGGGGCT CTCCCTTCAC CACCCCCATG



CAACACCCAA AAAAAGATGG TTAACCCCGA GAGGGAAGTG GTGGGGGTAC


 


3251
GGGATGGTCT ACAGGGTTCA TAACTACTCC TCTTACTACA GGACGCTTAC



CCCTACCAGA TGTCCCAAGT ATTGATGAGG AGAATGATGT CCTGCGAATG


 


3301
CTAGCCAACG CTTAGATCCG GCTCTACCCA AACTTTTCTG GTTCACCCCA



GATCGGTTGC GAATCTAGGC CGAGATGGGT TTGAAAAGAC CAAGTGGGGT


 


3351
ACATTCCCCA CTTGTCCGAC TGTTGCTGAG CAGTTTTTGG ATATCAAACG



TGTAAGGGGT GAACAGGCTG ACAACGACTC GTCAAAAACC TATAGTTTGC


 


3401
GACCTCCCCA GAAGGTAATT TTAATGTGGC CGATTTCCCC TCTTTTGCAA



CTGGAGGGGT CTTCCATTAA AATTACACCG GCTAAAGGGG AGAAAACGTT


 


3451
TCAGTTTCGC TACAGCACCC GCTGCTCTAG CTAATTGTCC ACCCTTTCCA



AGTCAAAGCG ATGTCGTGGG CGACGAGATC GATTAACAGG TGGGAAAGGT


 


3501
AGTGTGATTT CTATGTTATG TATGGCCGTG CCTAAGGGCA TATCGGTTGA



TCACACTAAA GATACAATAC ATACCGGCAC GGATTCCCGT ATAGCCAACT


 


3551
AGTAGATTCT TCTTTTGATC AATCAAAACC CCTTCCCAAA CTGTACAAGC



TCATCTAAGA AGAAAACTAG TTAGTTTTGG GGAAGGGTTT GACATGTTCG


 


3601
TTGGCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT GTTATCCGCT



AACCGCATTA GTACCAGTAT CGACAAAGGA CACACTTTAA CAATAGGCGA


 


3651
CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG



GTGTTAAGGT GTGTTGTATG CTCGGCCTTC GTATTTCACA TTTCGGACCC


 


3701
GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC



CACGGATTAC TCACTCGATT GAGTGTAATT AACGCAACGC GAGTGACGGG


 


3751
GCTTTCCAGT CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA



CGAAAGGTCA GCCCTTTGGA CAGCACGGTC GACGTAATTA CTTAGCCGGT


 


3801
ACGCGCGGGG AGAGGCGGTT TGCGTATTGG GCGCTCTTCC GCTTCCTCGC



TGCGCGCCCC TCTCCGCCAA ACGCATAACC CGCGAGAAGG CGAAGGAGCG


 


3851
TCACTGACTC GCTGCGCTCG GTCGTTCGGC TGCGGCGAGC GGTATCAGCT



AGTGACTGAG CGACGCGAGC CAGCAAGCCG ACGCCGCTCG CCATAGTCGA


 


3901
CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG ATAACGCAGG



GTGAGTTTCC GCCATTATGC CAATAGGTGT CTTAGTCCCC TATTGCGTCC


 


3951
AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG



TTTCTTGTAC ACTCGTTTTC CGGTCGTTTT CCGGTCCTTG GCATTTTTCC


 


4001
CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC



GGCGCAACGA CCGCAAAAAG GTATCCGAGG CGGGGGGACT GCTCGTAGTG


 


4051
AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG



TTTTTAGCTG CGAGTTCAGT CTCCACCGCT TTGGGCTGTC CTGATATTTC


 


4101
ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA



TATGGTCCGC AAAGGGGGAC CTTCGAGGGA GCACGCGAGA GGACAAGGCT


 


4151
CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG



GGGACGGCGA ATGGCCTATG GACAGGCGGA AAGAGGGAAG CCCTTCGCAC


 


4201
GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT



CGCGAAAGAG TATCGAGTGC GACATCCATA GAGTCAAGCC ACATCCAGCA


 


4251
TCGCTCCARG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT



AGCGAGGTTC GACCCGACAC ACGTGCTTGG GGGGCAAGTC GGGCTGGCGA


 


4301
GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC



CGCGGAATAG GCCATTGATA GCAGAACTCA GGTTGGGCCA TTCTGTGCTG


 


4351
TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA



AATAGCGGTG ACCGTCGTCG GTGACCATTG TCCTAATCGT CTCGCTCCAT


 


4401
TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA



ACATCCGCCA CGATGTCTCA AGAACTTCAC CACCGGATTG ATGCCGATGT


 


4451
CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC



GATCTTCCTG TCATAAACCA TAGACGCGAG ACGACTTCGG TCAATGGAAG


 


4501
GGAAARAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG



CCTTTTTCTC AACCATCGAG AACTAGGCCG TTTGTTTGGT GGCGACCATC


 


4551
CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT



GCCACCAAAA AAACAAACGT TCGTCGTCTA ATGCGCGTCT TTTTTTCCTA


 


4601
CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC



GAGTTCTTCT AGGAAACTAG AAAAGATGCC CCAGACTGCG AGTCACCTTG


 


4651
GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT



CTTTTGAGTG CAATTCCCTA AAACCAGTAC TCTAATAGTT TTTCCTAGAA


 


4701
CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA



GTGGATCTAG GAAAATTTAR TTTTTACTTC AAAATTTAGT TAGATTTCAT


 


4751
TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA



ATATACTCAT TTGAACCAGA CTGTCAATGG TTACGAATTA GTCACTCCGT


 


4801
CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC



GGATAGAGTC GCTAGACAGA TAAAGCAAGT AGGTATCAAC GGACTGAGGG


 


4851
CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG



GCAGCACATC TATTGATGCT ATGCCCTCCC GAATGGTAGA CCGGGGTCAC


 


4901
CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA



GACGTTACTA TGGCGCTCTG GGTGCGAGTG GCCGAGGTCT AAATAGTCGT


 


4951
ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT



TATTTGGTCG GTCGGCCTTC CCGGCTCGCG TCTTCACCAG GACGTTGAAA


 


5001
ATCCGCCTCC ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA



TAGGCGGAGG TAGGTCAGAT AATTAACAAC GGCCCTTCGA TCTCATTCAT


 


5051
GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC TACAGGCATC



CAAGCGGTCA ATTATCAAAC GCGTTGCAAC AACGGTAACG ATGTCCGTAG


 


5101
GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA



CACCACAGTG CGAGCAGCAA ACCATACCGA AGTAAGTCGA GGCCAAGGGT


 


5151
ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA RAAGCGGTTA



TGCTAGTTCC GCTCAATGTA CTAGGGGGTA CAACACGTTT TTTCGCCAAT


 


5201
GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA



CGAGGAAGCC AGGAGGCTAG CAACAGTCTT CATTCAACCG GCGTCACAAT


 


5251
TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC



AGTGAGTACC AATACCGTCG TGACGTATTA AGAGAATGAC AGTACGGTAG


 


5301
CGTAAGATGC TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG



GCATTCTACG AAAAGACACT GACCACTCAT GAGTTGGTTC AGTAAGACTC


 


5351
AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC AATACGGGAT



TTATCACATA CGCCGCTGGC TCAACGAGAA CGGGCCGCAG TTATGCCCTA


 


5401
AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG



TTATGGCGCG GTGTATCGTC TTGAAATTTT CACGAGTAGT AACCTTTTGC


 


5451
TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT



AAGAAGCCCC GCTTTTGAGA GTTCCTAGAA TGGCGACAAC TCTAGGTCAA


 


5501
CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC



GCTACATTGG GTGAGCACGT GGGTTGACTA GAAGTCGTAG AAAATGAAAG


 


5551
ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA



TGGTCGCAAA GACCCACTCG TTTTTGTCCT TCCGTTTTAC GGCGTTTTTT


 


5601
GGGAATAAGG GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC



CCCTTATTCC CGCTGTGCCT TTACAACTTA TGAGTATGAG AAGGAAAAAG


 


5651
AATATTA



TTATAAT


 








Sequence of p(CA)2











1
AAGCATTTAT CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA
(SEQ ID NO: 10)




TTCGTAAATA GTCCCAATAA CAGAGTACTC GCCTATGTAT AAACTTACAT
(SEQ ID NO: 11)


 


51
TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC CCGAAAAGTG



AAATCTTTTT ATTTGTTTAT CCCCAAGGCG CGTGTAAAGG GGCTTTTCAC


 


101
CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA



GGTGGACTGC AGATTCTTTG GTRATAATAG TACTGTAATT GGATATTTTT


 


151
TAGGCGTATC ACGAGGCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG



ATCCGCATAG TGCTCCGGGA AAGCAGAGCG CGCAAAGCCA CTACTGCCAC


 


201
RAAACCTCTG ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA



TTTTGGAGAC TGTGTACGTC GAGGGCCTCT GCCAGTGTCG AACAGACATT


 


251
GCGGATGCCG GGAGCAGACA AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG



CGCCTACGGC CCTCGTCTGT TCGGGCAGTC CCGCGCAGTC GCCCACAACC


 


301
CGGGTGTCGG GGCTGGCTTA ACTATGCGGC ATCAGAGCAG ATTGTACTGA



GCCCACAGCC CCGACCGAAT TGATACGCCG TAGTCTCGTC TAACATGACT


 


351
GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAAAA



CTCACGTGGT ATACGCCACA CTTTATGGCG TGTCTACGCA TTCCTCTTTT


 


401
TACCGCATCA GGCGCCATTC GCCATTCAGG CTGCGCAACT GTTGGGAAGG



ATGGCGTAGT CCGCGGTAAG CGGTAAGTCC GACGCGTTGA CAACCCTTCC


 


451
GCGATCGGTG CGGGCCTCTT CGCTATTACG CCAGCTGGCG AAAGGGGGAT



CGCTAGCCAC GCCCGGAGAA GCGATAATGC GGTCGACCGC TTTCCCCCTA


 


501
GTGCTGCAAG GCGATTAAGT TGGGTAACGC CAGGGTTTTC CCAGTCACGA



CACGACGTTC CGCTAATTCA ACCCATTGCG GTCCCAAAAG GGTCAGTGCT


 


551
CGTTGTAAAA CGACGGCCAG TGAATTCATG ACTGCAATTT TAGAGAGACG



GCAACATTTT GCTGCCGGTC ACTTAAGTAC TGACGTTAAA ATCTCTCTGC


 


601
CGAAAGCGAA AGCCTATGGG GTCGCTTCTG TAACTGGATA ACTAGCACTG



GCTTTCGCTT TCGGATACCC CAGCGAAGAC ATTGACCTAT TGATCGTGAC


 


651
AAAACCGTCT TTACATTGGA TGGTTTGGTG TTTTGATGAT CCCTACCTTA



TTTTGGCAGA AATGTAACCT ACCAAACCAC AAAACTACTA GGGATGGAAT


 


701
TTGACGGCAA CTTCTGTATT TATTATTGCC TTCATTGCTG CTCCTCCAGT



AACTGCCGTT GAAGACATAA ATAATAACGG AAGTAACGAC GAGGAGGTCA


 


751
AGACATTGAT GGTATTCGTG AACCTGTTTC AGGGTCTCTA CTTTACGGAA



TCTGTAACTA CCATAAGCAC TTGGACAAAG TCCCAGAGAT GAAATGCCTT


 


801
ACAATATTAT TTCCGGTGCC ATTATTCCTA CTTCTGCAGC TATAGGTTTA



TGTTATAATA AAGGCCACGG TAATAAGGAT GAAGACGTCG ATATCCAAAT


 


851
CATTATTATC CAATCTGGGA AGCGGCATCC GTTGATGAAT GGTTATACAA



GTAAAAATGG GTTAGACCCT TCGCCGTAGG CAACTACTTA CCAATATGTT


 


901
CGGTGGTCCT TATGAACTAA TTGTTCTACA CTTCTTACTT GGCGTAGCTT



GCCACCAGGA ATACTTGATT AACAAGATGT GAAGAATGAA CCGCATCGAA


 


951
GTTACATGGG TCGTGAGTGG GAGCTTAGTT TCCGTCTGGG TATGCGACCT



CAATGTACCC AGCACTCACC CTCGAATCAA AGGCAGACCC ATACGCTGGA


 


1001
TGGATTGCTG TTGCATATTC AGCTCCTGTT GCAGCTGCTA CCGCAGTTTT



ACCTAACGAC AACGTATAAG TCGAGGACAA CGTCGACGAT GGCGTCAAAA


 


1051
CTTGATCTAC CCAATTGGTC AAGGAAGTTT TTCTGATGGT ATGCCTCTAG



GAACTAGATG GGTTAACCAG TTCCTTCAAA AAGACTACCA TACGGAGATC


 


1101
GAATCTCTGG TACTTTCART TTCATGATTG TATTCCAGGC TGAGCACAAC



CTTAGAGACC ATGAAAGTTA AAGTACTAAC ATAAGGTCCG ACTCGTGTTG


 


1151
ATCCTTATGC ACCCATTTCA CATGTTAGGC GTAGCTGGTG TATTCGGCGG



TAGGAATACG TGGGTAAAGT GTACAATCCG CATCGACCAC ATAAGCCGCC


 


1201
CTCCCTATTC AGTGCTATGC ATGGTTCCTT GGTAACTTCT AGTTTGATCA



GAGGGATAAG TCACGATACG TACCAAGGAA CCATTGAAGA TCAAACTAGT


 


1251
GGGAAACCAC AGAAAATGAA TCTGCTAATG AAGGTTACAG ATTCGGTCAA



CCCTTTGGTG TCTTTTACTT AGACGATTAC TTCCAATGTC TAAGCCAGTT


 


1301
GAGGAAGAAA CTTATAACAT CGTAGCCGCT CATGGTTATT TTGGCCGATT



CTCCTTCTTT GAATATTGTA GCATCGGCGA GTACCAATAA AACCGGCTAA


 


1351
GATCTTCCAA TATGCTAGTT TCAACAACTC TCGTTCGTTA CACTTCTTCC



CTAGAAGGTT ATACGATCAA AGTTGTTGAG AGCAAGCAAT GTGAAGAAGG


 


1401
TAGCTGCTTG GCCTGTAGTA GGTATCTGGT TTACCGCTTT AGGTATCAGC



ATCGACGAAC CGGACATCAT CCATAGACCA AATGGCGAAR TCCATAGTCG


 


1451
ACTATGGCTT TCAACCTAAA TGGTTTCAAT TTCAACCAAT CTGTAGTTGA



TGATACCGAA AGTTGGATTT ACCAAAGTTA AAGTTGGTTA GACATCAACT


 


1501
CAGTCAAGGC CGTGTAATTA ATACTTGGGC TGATATCATT AACCGTGCTA



GTCAGTTCCG GCACATTAAT TATGAACCCG ACTATAGTAA TTGGCACGAT


 


1551
ACCTTGGTAT GGAAGTTATG CATGAACGTA ATGCTCACAA CTTCCCTCTA



TGGAACCATA CCTTCAATAC GTACTTGCAT TACGAGTGTT GAAGGGAGAT


 


1601
GACCTAGCTG CTATCGAAGC TCCATCTACA AATGGATAAG TCGACGGTAT



CTGGATCGAC GATAGCTTCG AGGTAGATGT TTACCTATTC AGCTGCCATA


 


1651
CGATAAGCTT CCCCGGGAGA CCACAACGGT TTCCCTCTAG AAATAATTTT



GCTATTCGAA GGGGCCCTCT GGTGTTGCCA AAGGGAGATC TTTATTAAAA


 


1701
GTTTAACTTT AAGAAGGAGA TGTACTACTG AACCCGAACT CATTTCAATT



CAAATTGAAA TTCTTCCTCT ATATGTATAC TTGGGCTTGA GTAAAGTTAA


 


1751
CAAAGAAAAC ATACTACAAT TTTTTTCTGT ACATGATGAC ATCTGGAAAA



GTTTCTTTTG TATGATGTTA AAAAAAGACA TGTACTACTG TAGACCTTTT


 


1801
AATTACAAGA ATTTTATTAT GGGCAAAGCC CAATTAATGA GGCTTTGGCG



TTAATGTTCT TAAAATAATA CCCGTTTCGG GTTAATTACT CCGAAACCGC


 


1851
CAGCTCAACA AAGAAGATAT GTCTTTGTTC TTTGAAGCAC TATCTAAAAA



GTCGAGTTGT TTCTTCTATA CAGAAACAAG AAACTTCGTG ATAGATTTTT


 


1901
CCCAGCTCGC ATGATGGAAA TGCAATGGAG CTGGTGGCAA GGTCAAATAC



GGGTCGAGCG TACTACCTTT ACGTTACCTC GACCACCGTT CCAGTTTATG


 


1951
AAATCTACCA AAATGTGTTG ATGCGCAGCG TGGCCAAAGA TGTAGCACCA



TTTAGATGGT TTTACACAAC TACGCGTCGC ACCGGTTTCT ACATCGTGGT


 


2001
TTTATTCAGC CTGAAAGTGG TGATCGTCGT TTTAACAGCC CATTATGGCA



AAATAAGTCG GACTTTCACC ACTAGCAGCA AAATTGTCGG GTAATACCGT


 


2051
AGAACACCCA AATTTTGACT TGTTGTCACA GTCTTATTTA CTGTTTAGCC



TCTTQTGGGT TTAAAACTGA ACAACAGTGT CAGAATAAAT GACAAATCGG


 


2101
AGTTAGTGCA AAACATGGTA GATGTGGTCG AAGGTGTTCC AGACAAAGTT



TCAATCACGT TTTGTACCAT CTACACCAGC TTCCACAAGG TCTGTTTCAA


 


2151
CGCTATCGTA TTCACTTCTT TACCCGCCAA ATGATCAATG CGTTATCTCC



GCGATAGCAT AAGTGAAGAA ATGGGCGGTT TACTAGTTAC GCAATAGAGG


 


2201
AAGTAACTTT CTGTGGACTA ACCCAGAAGT GATTCAGCAA ACTGTAGCTG



TTCATTGAAA GACACCTGAT TGGGTCTTCA CTAAGTCGTT TGACATCGAC


 


2251
AACAAGGTGA AAACTTAGTC CGTGGCATGC AAGTTTTCCA TGATGATGTC



TTCTTCCTCT TTTGAATCAG GCACCGTACG TACACAAGGT ACTACTACAG


 


2301
ATGAATAGCG GCAAGTATTT ATCTATTCGC ATGGTGAATA GCGACTCTTT



TACTTATCGC CGTTCATAAA TAGATAAGCG TACCACTTAT CGCTGAGAAA


 


2351
CAGCTTGGGC AAAGATTTAG CTTACACCCC TGGTGCAGTC GTCTTTGAAA



GTCGAACCCG TTTCTAAATC GAATGTGGGG ACCACGTCAG CAGAAACTTT


 


2401
ATGACATTTT CCAATTATTG CAATATGAAG CAACTACTGA AAATGTGTAT



TACTGTAAAA GGTTAATAAC GTTATACTTC GTTGATGACT TTTACACATA


 


2451
CAAACCCCTA TTCTAGTCGT ACCACCGTTT ATCRATAAAT ATTATGTGCT



GTTTGGGGAT AAGATCAGCA TGGTGGCAAA TAGTTATTTA TAATACACGA


 


2501
GGATTTACGC GAACAAAACT CTTTAGTGAA CTGGTTGCGC CAGCAAGGTC



CCTAAATGCG CTTGTTTTGA GAAATCACTT GACCAACGCG GTCGTTCCAG


 


2551
ATACAGTCTT TTTAATGTCA TGGCGTAACC CAAATGCCGA ACAGAAAGAA



TATGTCAGAA AAATTACAGT ACCGCATTGG GTTTACGGCT TGTCTTTCTT


 


2601
TTGACTTTTG CCGATCTCAT TACACAAGGT TCAGTGGAAG CTTTGCGTGT



AACTGAAAAC GGCTAGAGTA ATGTGTTCCA AGTCACCTTC GAAACGCACA


 


2651
AATTGAAGAA ATTACCGGTG AAAAAGAGGC CAACTGCATT GGCTACTGTA



TTAACTTCTT TAATGGCCAC TTTTTCTCCG GTTGACGTAA CCGATGACAT


 


2701
TTGGTGGTAC GTTACTTGCT GCGACTCAAG CCTATTACGT GGCAAAACGC



AACCACCATG CAATGAACGA CGCTGAGTTC GGATAATGCA CCGTTTTGCG


 


2751
CTGAAAAATC ACGTAAAGTC TGCGACCTAT ATGGCCACCA TTATCGACTT



GACTTTTTAG TGCATTTCAG ACGCTGGATA TACCGGTGGT AATAGCTGAA


 


2801
TGAAAACCCA GGCAGCTTAG GTGTATTTAT TAATGAACCT GTAGTGAGCG



ACTTTTGGGT CCGTCGAATC CACATAAATA ATTACTTGGA CATCACTCGC


 


2851
GTTTAGAAAA CCTGAACAAT CAATTGGGTT ATTTCGATGG TCGTCAGTTG



CAAATCTTTT GGACTTGTTA GTTAACCCAA TAAAGCTACC AGCAGTCAAC


 


2901
GCAGTTACCT TCAGTTTACT GCGTGAAAAT ACGCTGTACT GGAATTACTA



CGTCAATGGA AGTCAAATGA CGCACTTTTA TGCGACATGA CCTTAATGAT


 


2951
CATCGACAAC TACTTAAAAG GTAAAGAACC TTCTGATTTT GATATTTTAT



GTAGCTGTTG ATGAATTTTC CATTTCTTGG AAGACTAAAA CTATAAAATA


 


3001
ATTGGAACAG CGATGGTACG AATATCCCTG CCAAAATTCA TAATTTCTTA



TAACCTTGTC GCTACCATGC TTATAGGGAC GGTTTTAAGT ATTAAAGAAT


 


3051
TTGCGCAATT TGTATTTGAA CAATGAATTG ATTTCACCAA ATGCCGTTAA



AACGCGTTAA ACATAAACTT GTTACTTAAC TAAAGTGGTT TACGGCAATT


 


3101
GGTTAACGGT GTGGGCTTGA ATCTATCTCG TGCTAAAGAA CCAAGCTTCT



CCAATTGCCA CACCCGAACT TAGATAGAGC ACATTTTTGT GGTTCGAAGA


 


3151
TTATTGCGAC GCAGGAAGAC CATATCGCAC TTTGGGATAC TTGTTTCCGT



AATAACGCTG CGTCCTTCTG GTATAGCGTG AAACCCTATG AACAAAGGCA


 


3201
GGCGCAGATT ACTTGGGTGG TGAATCAACC TTGGTTTTAG GTGAATCTGG



CCGCGTCTAA TGAACCCACC ACTTAGTTGG AACCAAAATC CACTTAGACC


 


3251
ACACGTAGCA GGTATTGTCA ATCCTCCAAG CCGTAATAAA TACGGTTGCT



TGTGCATCGT CCATAACAGT TAGGAGGTTC GGCATTATTT ATGCCAACGA


 


3301
ACACCAATGC TGCCAAGTTT GAAAATACCA AACAATGGCT AGATGGCGCA



TGTGGTTACG ACGGTTCAAA CTTTTATGGT TTGTTACCGA TCTACCGCGT


 


3351
GAATATCACC CTGAATCTTG GTGGTTGCGC TGGCAGGCAT GGGTCACACC



CTTATAGTGG GACTTAGAAC CACCAACGCG ACCGTCCGTA CCCAGTGTGG


 


3401
GTACACTGGT GAACAAGTCC CTGCCCGCAA CTTGGGTAAT GCGCAGTATC



CATGTGACCA CTTGTTCAGG GACGGGCGTT GAACCCATTA CGCGTCATAG


 


3451
CAAGCATTGA AGCGGCACCG GGTCGCTATG TTTTGGTAAA TTTATTCTAA



GTTCGTAACT TCGCCGTGGC CCAGCGATAC AAAACCATTT AAATAAGATT


 


3501
GCGGCCGCCA CCGCGGTGGA GCTCAATAAA AAAAATCTAG ATGCTTATGA



CGCCGGCGGT GGCGCCACCT CGAGTTATTT TTTTTAGATC TACGAATACT


 


3551
TTCAGTAGTA GGAGGCAAAC CATATGAAAG ATGTTGTGAT TGTTGCAGCA



AAGTCATCAT CCTCCGTTTG GTATACTTTC TACAACACTA ACAACGTCGT


 


3601
AAACGTACTG CGATTGGTAG CTTTTTAGGT AGTCTTGCAT CTTTATCTGC



TTTGCATGAC GCTAACCATC GAAAAATCCA TCAGAACGTA GAAATAGACG


 


3651
ACCACAGTTG GGGCAAACAG CAATTCGTGC AGTTTTAGAC AGCGCTAATG



TGGTGTCAAC CCCGTTTGTC GTTAAGCACG TCAAAATCTG TCGCGATTAC


 


3701
TAAAACCTGA ACAAGTTGAT CAGGTGATTA TGGGCAACGT ACTCACGACA



ATTTTGGACT TGTTCAACTA GTCCACTAAT ACCCGTTGCA TGAGTGCTGT


 


3751
GGCGTGGGAC AAAACCCTGC ACGTCAGGCA GCAATTGCTG CTGGTATTCC



CCGCACCCTG TTTTGGGACG TGCAGTCCGT CGTTAACGAC GACCATAAGG


 


3801
AGTACAAGTG CCTGCATCTA CGCTGAATGT CGTCTGTGGT TCAGGTTTGC



TCATGTTCAC GGACGTAGAT GCGACTTACA GCAGACACCA AGTCCAAACG


 


3851
GTGCGGTACA TTTGGCAGCA CAAGCCATTC AATGCGATGA AGCCGACATT



CACGCCATGT AAACCGTCGT GTTCGGTAAG TTACGCTACT TCGGCTGTAA


 


3901
GTGGTCGCAG GTGGTCAAGA ATCTATGTCA CAAAGTGCGC ACTATATGCA



CACCAGCGTC CACCAGTTCT TAGATACAGT GTTTCACGCG TGATATACGT


 


3951
GCTGCGTAAT GGGCAAAAAA TGGGTAATGC ACAATTGGTG GATAGCATGG



CGACGCATTA CCCGTTTTTT ACCCATTACG TGTTAACCAC CTATCGTACC


 


4001
TGGCTGATGG TTTAACCGAT GCCTATAACC AGTATCAAAT GGGTATTACC



ACCGACTACC AAATTGGCTA CGGATATTGG TCATAGTTTA CCCATAATGG


 


4051
GCAGAAAATA TTGTAGAAAA ACTGGGTTTA AACCGTGAAG AACAAGATCA



CGTCTTTTAT AACATCTTTT TGACCCAAAT TTGGCACTTC TTGTTCTAGT


 


4101
ACTTGCATTG ACTTCACAAC AACGTGCTGC GGCAGCTCAG GCAGCTGGCA



TGAACGTAAC TGAAGTGTTG TTGCACGACG CCGTCGAGTC CGTCGACCGT


 


4151
AGTTTAAAGA TGAAATTGCC GTAGTCAGCA TTCCACAACG TAAAGGTGAG



TCAAATTTCT ACTTTAACGG CATCAGTCGT AAGGTGTTGC ATTTCCACTC


 


4201
CCTGTTGTAT TTGCTGAAGA TGAATACATT AAAGCCAATA CCAGCCTTGA



GGACAACATA AACGACTTCT ACTTATGTAA TTTCGGTTAT GGTCGGAACT


 


4251
AAGCCTCACA AAACTACGCC CAGCCTTTAA AAAAGATGGT AGCGTAACCG



TTCGGAGTGT TTTGATGCGG GTCGGAAATT TTTTCTACCA TCGCATTGGC


 


4301
CAGGTAATGC TTCAGGCATT AATGATGGTG CAGCAGCAGT ACTGATGATG



GTCCATTACG AAGTCCGTAA TTACTACCAC GTCGTCGTCA TGACTACTAC


 


4351
AGTGCGGACA AAGCAGCAGA ATTAGGTCTT AAGCCATTGG CACGTATTAA



TCACGCCTGT TTCGTCGTCT TAATCCAGAA TTCGGTAACC GTGCATAATT


 


4401
AGGCTATGCC ATGTCTGGTA TTGAGCCTGA AATTATGGGG CTTGGTCCTG



TCCGATACGG TACAGACCAT AACTCGGACT TTAATACCCC GAACCAGGAC


 


4451
TCGATGCAGT AAAGAAAACC CTCAACAAAG CAGGCTGGAG CTTAGATCAG



AGCTACGTCA TTTCTTTTGG GAGTTGTTTC GTCCGACCTC GAATCTAGTC


 


4501
GTTGATTTGA TTGAAGCCAA TGAAGCATTT GCTGCACAGG CTTTGGGTGT



CAACTAAACT AACTTCGGTT ACTTCGTAAA CGACGTGTCC GAAACCCACA


 


4551
TGCTAAAGAA TTAGGCTTAG ACCTGGATAA AGTCAACGTC AATGGCGGTG



ACGATTTCTT AATCCGAATC TGGACCTATT TCAGTTGCAG TTACCGCCAC


 


4601
CAATTGCATT GGGTCACCCA ATTGGGGCTT CAGGTTGCCG TATTTTGGTG



GTTAACGTAA CCCAGTGGGT TAACCCCGAA GTCCAACGGC ATAAAACCAC


 


4651
ACTTTATTAC ATGAAATGCA GCGCCGTGAT GCCAAGAAAG GCATTGCAAC



TGAAATAATG TACTTTACGT CGCGGCACTA CGGTTCTTTC CGTAACGTTG


 


4701
CCTCTGTGTT GGCGGTGGTA TGGGTGTTGC ACTTGCAGTT GAACGTGACT



GGAGACACAA CCGCCACCAT ACCCACAACG TGAACGTCAA CTTGCACTGA


 


4751
AAGCGGCCGC TCGAGTTTGG ATCCAATCGA TACAAGTGAG TTGTAGGGAG



TTCGCCGGCG AGCTCAAACC TAGGTTAGCT ATGTTCACTC AACATCCCTC


 


4801
GCAACCATGG CAGAAGCGGT GATCGCCGAA GTATCGACTC AACTATCAGA



CGTTGGTACC GTCTTCGCCA CTAGCGGCTT CATAGCTGAG TTCATAGTCT


 


4851
GGTAGTTGGC GTCATCGAGC GCCATCTCGA ACCGACGTTG CTGGCCGTAC



CCATCAACCG CAGTAGCTCG CGGTAGAGCT TGGCTGCAAC GACCGGCATG


 


4901
ATTTGTACGG CTCCGCAGTG GATGGCGGCC TGAAGCCACA CAGTGATATT



TAAACATGCC GAGGCGTCAC CTACCGCCGG ACTTCGGTGT GTCACTATAA


 


4951
GATTTGCTGG TTACGGTGAC CGTAAGGCTT GATGAAACAA CGCGGCGAGC



CTAAACGACC AATGCCACTG GCATTCCGAA CTACTTTGTT GCGCCGCTCG


 


5001
TTTGATCAAC GACCTTTTGG AAACTTCGGC TTCCCCTGGA GAGAGCGAGA



AAACTAGTTG CTGGAAAACC TTTGAAGCCG AAGGGGACCT CTCTCGCTCT


 


5051
TTCTCCGCGC TGTAGAAGTC ACCATTGTTG TGCACGACGA CATCATTCCG



AAGAGGCGCG ACATCTTCAG TGGTAACAAC ACGTGCTGCT GTAGTAAGGC


 


5101
TGGCGTTATC CAGCTAAGCG CGAACTGCAA TTTGGAGAAT GGCAGCGCAA



ACCGCAATAG GTCGATTCGC GCTTGACGTT AAACCTCTTA CCGTCGCGTT


 


5151
TGACATTCTT GCAGGTATCT TCGAGCCAGC CACGATCGAC ATTGATCTGG



ACTGTAAGAA CGTCCATAGA AGCTCGGTCG GTGCTAGCTG TAACTAGACC


 


5201
CTATCTTGCT GACAAAAGCA AGAGAACATA GCGTTGCCTT GGTAGGTCCA



GATAGAACGA CTGTTTTCGT TCTCTTGTAT CGCAACGGAA CCATCCAGGT


 


5251
GCGGCGGAGG AACTCTTTGA TCCGGTTCCT GAACAGGATC TATTTGAGGC



CGCCGCCTCC TTGAGAAACT AGGCCAAGGA CTTGTCCTAG ATAAACTCCG


 


5301
GCTAAATGAA ACCTTAACGC TATGGAACTC GCCGCCCGAC TGGGCTGGCG



CGATTTACTT TGGAATTGCG ATACCTTGAG CGGCGGGCTG ACCCGACCGC


 


5351
ATGAGCGAAA TGTAGTGCTT ACGTTGTCCC GCATTTGGTA CAGCGCAGTA



TACTCGCTTT ACATCACGAA TGCAACAGGG CGTAAACCAT GTCGCGTCAT


 


5401
ACCGGCAAAA TCGCGCCGAA GGATGTCGCT GCCGACTGGG CAATGGAGCG



TGGCCGTTTT AGCGCGGCTT CCTACAGCGA CGGCTGACCC GTTACCTCGC


 


5451
CCTGCCGGCC CAGTATCAGC CCGTCATACT TGAAGCCACA CAGGCTTATC



GGACGGCCGG GTCATAGTCG GGCAGTATGA ACTTCGATCT GTCCGAATAG


 


5501
TTGGACAAGA AGAAGATCGC TTGGCCTCGC GCGCAGATCA GTTGGAAGAA



AACCTGTTCT TCTTCTAGCG AACCGGAGCG CGCGTCTAGT CAACCTTCTT


 


5551
TTTGTCCACT ACGTGAAAGG CGAGATCACC AAGGTAGTCG GCAAATAAAT



AAACAGGTGA TGCACTTTCC GCTCTAGTGG TTCCATCAGC CGTTTATTTA


 


5601
CTAAGCCGAA TTGGGCCTAG TCTATAGGAG GTTTTGAAAA GAAAGGAGCA



GATTCGGCTT AACCCGGATC AGATATCCTC CAAAACTTTT CTTTCCTCGT


 


5651
ATAATCATTT TCTTGTTCTA TCAAGAGGGT GCTATTGCTC CTTTCTTTTT



TATTAGTAAA AGAACAAGAT AGTTCTCCCA CGATAACGAG GAAAGAAAAA


 


5701
TTCTTTTTAT TTATTTACTA GTATTTTACT TACATAGACT TTTTTGTTTA



AAGAAAAATA AATAAATGAT CATAAAATGA ATGTATCTGA AAAAACAAAT


 


5751
CATTATAGAA AAAGAAGGAG AGGTTATTTT CTTGCATTTA TTCATGATTG



GTAATATCTT TTTCTTCCTC TCCAATAAAA GAACGTAAAT AAGTACTAAC


 


5801
AGTATTCTAT TTTGATTTTG TATTTGTTTA AAATTGTAGA AATAGAACTT



TCATAAGATA AAACTAAAAC ATAAACAAAT TTTAACATCT TTATCTTTGA


 


5851
GTTTCTCTTC TTGCTAATGT TACTATATCT TTTTGATTTT TTTTTTCCAA



CAAAGAGAAG AACGATTACA ATGATATAGA AAAACTAAAA AAAAAAGGTT


 


5901
AAAAAAAATC AAATTTTGAC TTCTTCTTAT CTCTTATCTT TGAATATCTC



TTTTTTTTAG TTTAAAACTG AAGAAGAATA GAGAATAGAA ACTTATAGAG


 


5951
TTATCTTTGA AATAATAATA TCATTGAAAT AAGAAAGAAG AGCTATATTC



AATAGAAACT TTATTATTAT AGTAACTTTA TTCTTTCTTC TCGATATAAG


 


6001
GAACTTGAAT CTTTTGTTTT CTAATTTAAA TAATGTAAAA ACGGAATGTA



CTTGAACTTA GAAAACAAAA GATTAAATTT ATTACATTTT TGCCTTACAT


 


6051
AGTAGGCGAG GGGGCGGATG TAGCCAAGTG GATCAAGGCA GTGGATTGTG



TCATCCGCTC CCCCGCCTAC ATCGGTTCAC CTAGTTCCGT CACCTAACAC


 


6101
AATCCACCAT GCGCGGGTTC AATTCCCGTC GTTCGCCCAT AATTACTCCT



TTAGGTGGTA CGCGCCCAAG TTAAGGGCAG CAAGCGGGTA TTAATGAGGA


 


6151
ATTTTTTTTT TTTTTGTAAA AACGAAGAAT TTAATTCGAT TTTCTCTCCT



TAAAAAAAAA AAAAACATTT TTGCTTCTTA AATTAAGCTA AAAGAGAGGA


 


6201
ATTTACTACG GCGACGAAGA ATCAAATTAT CACTATATTT ATTCCTTTTT



TAAATGATGC CGCTGCTTCT TAGTTTAATA GTGATATAAA TAAGGAAAAA


 


6251
CTACTTCTTC TTCCAAGTGC AGGATAACCC CAAGGGGTTG TGGGTTTTTT



GATGAAGAAG AAGGTTCACG TCCTATTGGG GTTCCCCAAC ACCCAAAAAA


 


6301
TCTACCAATT GGGGCTCTCC CTTCACCACC CCCATGGGGA TGGTCTACAG



AGATGGTTAA CCCCGAGAGG GAAGTGGTGG GGGTACCCCT ACCAGATGTC


 


6351
GGTTCATAAC TACTCCTCTT ACTACAGGAC GCTTACCTAG CCAACGCTTA



CCAAGTATTG ATGAGGAGAA TGATGTCCTG CGAATGGATC GGTTGCGAAT


 


6401
GATCCGGCTC TACCCAAACT TTTCTGGTTC ACCCCAACAT TCCCCACTTG



CTAGGCCGAG ATGGGTTTGA AAAGACCAAG TGGGGTTGTA AGGGGTGAAC


 


6451
TCCGACTGTT GCTGAGCAGT TTTTGGATAT CAAACGGACC TCCCCAGAAG



AGGCTGACAA CGACTCGTCA AAAACCTATA GTTTGCCTGG AGGGGTCTTC


 


6501
GTAATTTTAA TGTGGCCGAT TTCCCCTCTT TTGCAATCAG TTTCGCTACA



CATTAAAATT ACACCGGCTA AAGGGGAGAA AACGTTAGTC AAAGCGATGT


 


6551
GCACCCGCTG CTCTAGCTAA TTGTCCACCC TTTCCAAGTG TGATTTCTAT



CGTGGGCGAC GAGATCGATT AACAGGTGGG AAAGGTTCAC ACTAAAGATA


 


6601
GTTATGTATG GCCGTGCCTA AGGGCATATC GGTTGAAGTA GATTCTTCTT



CAATACATAC CGGCACGGAT TCCCGTATAG CCAACTTCAT CTAAGAAGAA


 


6651
TTGATCAATC AAAACCCCTT CCCAAACTGT ACAAGCTTGG CGTAATCATG



AACTAGTTAG TTTTGGGGAA GGGTTTGACA TGTTCGAACC GCATTAGTAC


 


6701
GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA



CAGTATCGAC AAAGGACACA CTTTAACAAT AGGCGAGTGT TAAGGTGTGT


 


6751
ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG



TGTATGCTCG GCCTTCGTAT TTCACATTTC GGACCCCACG GATTACTCAC


 


6801
AGCTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG



TCGATTGAGT GTAATTAACG CAACGCGAGT GACGGGCGAA AGGTCAGCCC


 


6851
AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GCGGGGAGAG



TTTGGACAGC ACGGTCGACG TAATTACTTA GCCGGTTGCG CGCCCCTCTC


 


6901
GCGGTTTGCG TATTGGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG



CGCCAAACGC ATAACCCGCG AGAAGGCGAA GGAGCGAGTG ACTGAGCGAC


 


6951
CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT



GCGAGCCAGC AAGCCGACGC CGCTCGCCAT AGTCGAGTGA GTTTCCGCCA


 


7001
AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG



TTATGCCAAT AGGTGTCTTA GTCCCCTATT GCGTCCTTTC TTGTACACTC


 


7051
CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG



GTTTTCCGGT CGTTTTCCGG TCCTTGGCAT TTTTCCGGCG CAACGACCGC


 


7101
TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC



AAAAAGGTAT CCGAGGCGGG GGGACTGCTC GTAGTGTTTT TAGCTGCGAG


 


7151
AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC



TTCAGTCTCC ACCGCTTTGG GCTGTCCTGA TATTTCTATG GTCCGCAAAG


 


7201
CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC



GGGGACCTTC GAGGGAGCAC GCGAGAGGAC AAGGCTGGGA CGGCGAATGG


 


7251
GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG



CCTATGGACA GGCGGAAAGA GGGAAGCCCT TCGCACCGCG AAAGAGTATC


 


7301
CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG



GAGTGCGACA TCCATAGAGT CAAGCCACAT CCAGCAAGCG AGGTTCGACC


 


7351
GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT



CGACACACGT GCTTGGGGGG CAAGTCGGGC TGGCGACGCG GAATAGGCCA


 


7401
AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC



TTGATAGCAG AACTCAGGTT GGGCCATTCT GTGCTGAATA GCGGTGACCG


 


7451
AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA



TCGTCGGTGA CCATTGTCCT AATCGTCTCG CTCCATACAT CCGCCACGAT


 


7501
CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA



GTCTCAAGAA CTTCACCACC GGATTGATGC CGATGTGATC TTCCTGTCAT


 


7551
TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG



AAACCATAGA CGCGAGACGA CTTCGGTCAA TGGAAGCCTT TTTCTCAACC


 


7601
TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG



ATCGAGAACT AGGCCGTTTG TTTGGTGGCG ACCATCGCCA CCAAAAAAAC


 


7651
TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT



AAACGTTCGT CGTCTAATGC GCGTCTTTTT TTCCTAGAGT TCTTCTAGGA


 


7701
TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA



AACTAGAAAA GATGCCCCAG ACTGCGAGTC ACCTTGCTTT TGAGTGCAAT


 


7751
AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT



TCCCTAAAAC CAGTACTCTA ATAGTTTTTC CTAGAAGTGG ATCTAGGAAA


 


7801
TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA TGAGTAAACT



ATTTAATTTT TACTTCAAAA TTTAGTTAGA TTTCATATAT ACTCATTTGA


 


7851
TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT



ACCAGACTGT CAATGGTTAC GAATTAGTCA CTCCGTGGAT AGAGTCGCTA


 


7901
CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA



GACAGATAAA GCAAGTAGGT ATCAACGGAC TGAGGGGCAG CACATCTATT


 


7951
CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG



GATGCTATGC CCTCCCGAAT GGTAGACCGG GGTCACGACG TTACTATGGC


 


8001
CGAGACCCAC GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC



GCTCTGGGTG CGAGTGGCCG AGGTCTAAAT AGTCGTTATT TGGTCGGTCG


 


8051
CGGAAGGGCC GAGCGCAGAA GTGGTCCTGC AACTTTATCC GCCTCCATCC



GCCTTCCCGG CTCGCGTCTT CACCAGGACG TTGAAATAGG CGGAGGTAGG


 


8101
AGTCTATTAA TTGTTGCCGG GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT



TCAGATAATT AACAACGGCC CTTCGATCTC ATTCATCAAG CGGTCAATTA


 


8151
AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG TGTCACGCTC



TCAAACGCGT TGCAACAACG GTAACGATGT CCGTAGCACC ACAGTGCGAG


 


8201
GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG



CAGCAAACCA TACCGAAGTA AGTCGAGGCC AAGGGTTGCT AGTTCCGCTC


 


8251
TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT



AATGTACTAG GGGGTACAAC ACGTTTTTTC GCCAATCGAG GAAGCCAGGA


 


8301
CCGATCGTTG TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT



GGCTAGCAAC AGTCTTCATT CAACCGGCGT CACAATAGTG AGTACCAATA


 


8351
GGCAGCACTG CATAATTCTC TTACTGTCAT GCCATCCGTA AGATGCTTTT



CCGTCGTGAC GTATTAAGAG AATGACAGTA CGGTAGGCAT TCTACGAAAA


 


8401
CTGTGACTGG TGAGTACTCA ACCAAGTCAT TCTGAGAATA GTGTATGCGG



GACACTGACC ACTCATGAGT TGGTTCAGTA AGACTCTTAT CACATACGCC


 


8451
CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA CCGCGCCACA



GCTGGCTCAA CGAGAACGGG CCGCAGTTAT GCCCTATTAT GGCGCGGTGT


 


8501
TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA



ATCGTCTTGA AATTTTCACG AGTAGTAACC TTTTGCAAGA AGCCCCGCTT


 


8551
AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT



TTGAGAGTTC CTAGAATGGC GACAACTCTA GGTCAAGCTA CATTGGGTGA


 


8601
CGTGCACCCA ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG



GCACGTGGGT TGACTAGAAG TCGTAGAAAA TGAAAGTGGT CGCAAAGACC


 


8651
GTGAGCAAAA ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA



CACTCGTTTT TGTCCTTCCG TTTTACGGCG TTTTTTCCCT TATTCCCGCT


 


8701
CACGGAAATG TTGAATACTC ATACTCTTCC TTTTTCAATA TTATTG



GTGCCTTTAC AACTTATGAG TATGAGAAGG AAAAAGTTAT AATAAC


 








Sequence of p(CAB)2











1
TGAAGCATTT ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG
(SEQ ID NO: 12)




ACTTCGTAAA TAGTCCCAAT AACAGAGTAC TCGCCTATGT ATAAACTTAC
(SEQ ID NO: 13)


 


51
TATTTAGAAA AATAAACAAA TAGGGGTTCC GCGCACATTT CCCCGAAAAG



ATAAATCTTT TTATTTGTTT ATCCCCAAGG CGCGTGTAAA GGGGCTTTTC


 


101
TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT AACCTATAAA



ACGGTGGACT GCAGATTCTT TGGTAATAAT AGTACTGTAA TTGGATATTT


 


151
AATAGGCGTA TCACGAGGCC CTTTCGTCTC GCGCGTTTCG GTGATGACGG



TTATCCGCAT AGTGCTCCGG GAAAGCAGAG CGCGCAAAGC CACTACTGCC


 


201
TGAAAACCTC TGACACATGC AGCTCCCGGA GACGGTCACA GCTTGTCTGT



ACTTTTGGAG ACTGTGTACG TCGAGGGCCT CTGCCAGTGT CGAACAGACA


 


251
AAGCGGATGC CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT



TTCGCCTACG GCCCTCGTCT GTTCGGGCAG TCCCGCGCAG TCGCCCACAA


 


301
GGCGGGTGTC GGGGCTGGCT TAACTATGCG GCATCAGAGC AGATTGTACT



CCGCCCACAG CCCCGACCGA ATTGATACGC CGTAGTCTCG TCTAACATGA


 


351
GAGAGTGCAC CATATGCGGT GTGAAATACC GCACAGATGC GTAAGGAGAA



CTCTCACGTG GTATACGCCA CACTTTATGG CGTGTCTACG CATTCCTCTT


 


401
AATACCGCAT CAGGCGCCAT TCGCCATTCA GGCTGCGCAA CTGTTGGGAA



TTATGGCGTA GTCCGCGGTA AGCGGTAAGT CCGACGCGTT GACAACCCTT


 


451
GGGCGATCGG TGCGGGCCTC TTCGCTATTA CGCCAGCTGG CGAAAGGGGG



CCCGCTAGCC ACGCCCGGAG AAGCGATAAT GCGGTCGACC GCTTTCCCCC


 


501
ATGTGCTGCA AGGCGATTAA GTTGGGTAAC GCCAGGGTTT TCCCAGTCAC



TACACGACGT TCCGCTAATT CAACCCATTG CGGTCCCAAA AGGGTCAGTG


 


551
GACGTTGTAA AACGACGGCC AGTGAATTCA TGACTGCAAT TTTAGAGAGA



CTGCAACATT TTGCTGCCGG TCACTTAAGT ACTGACGTTA AAATCTCTCT


 


601
CGCGAAAGCG AAAGCCTATG GGGTCGCTTC TGTAACTGGA TAACTAGCAC



GCGCTTTCGC TTTCGGATAC CCCAGCGAAG ACATTGACCT ATTGATCGTG


 


651
TGAAAACCGT CTTTACATTG GATGGTTTGG TGTTTTGATG ATCCCTACCT



ACTTTTGGCA GAAATGTAAC CTACCAAACC ACAAAACTAC TAGGGATGGA


 


701
TATTGACGGC AACTTCTGTA TTTATTATTG CCTTCATTGC TGCTCCTCCA



ATAACTGCCG TTGAAGACAT AAATAATAAC GGAAGTAACG ACGAGGAGGT


 


751
GTAGACATTG ATGGTATTCG TGAACCTGTT TCAGGGTCTC TACTTTACGG



CATCTGTAAC TACCATAAGC ACTTGGACAA AGTCCCAGAG ATGAAATGCC


 


801
AAACAATATT ATTTCCGGTG CCATTATTCC TACTTCTGCA GCTATAGGTT



TTTGTTATAA TAAAGGCCAC GGTAATAAGG ATGAAGACGT CGATATCCAA


 


851
TACATTTTTA CCCAATCTGG GAAGCGGCAT CCGTTGATGA ATGGTTATAC



ATGTAAAAAT GGGTTAGACC CTTCGCCGTA GGCAACTACT TACCAATATG


 


901
AACGGTGGTC CTTATGAACT AATTGTTCTA CACTTCTTAC TTGGCGTAGC



TTGCCACCAG GAATACTTGA TTAACAAGAT GTGAAGAATG AACCGCATCG


 


951
TTGTTACATG GGTCGTGAGT GGGAGCTTAG TTTCCGTCTG GGTATGCGAC



AACAATGTAC CCAGCACTCA CCCTCGAATC AAAGGCAGAC CCATACGCTG


 


1001
CTTGGATTGC TGTTGCATAT TCAGCTCCTG TTGCAGCTGC TACCGCAGTT



GAACCTAACG ACAACGTATA AGTCGAGGAC AACGTCGACG ATGGCGTCAA


 


1051
TTCTTGATCT ACCCAATTGG TCAAGGAAGT TTTTCTGATG GTATGCCTCT



AAGAACTAGA TGGGTTAACC AGTTCCTTCA AAAAGACTAC CATACGGAGA


 


1101
AGGAATCTCT GGTACTTTCA ATTTCATGAT TGTATTCCAG GCTGAGCACA



TCCTTAGAGA CCATGAAAGT TAAAGTACTA ACATAAGGTC CGACTCGTGT


 


1151
ACATCCTTAT GCACCCATTT CACATGTTAG GCGTAGCTGG TGTATTCGGC



TGTAGGAATA CGTGGGTAAA GTGTACAATC CGCATCGACC ACATAAGCCG


 


1201
GGCTCCCTAT TCAGTGCTAT GCATGGTTCC TTGGTAACTT CTAGTTTGAT



CCGAGGGATA AGTCACGATA CGTACCAAGG AACCATTGAA GATCAAACTA


 


1251
CAGGGAAACC ACAGAAAATG AATCTGCTAA TGAAGGTTAC AGATTCGGTC



GTCCCTTTGG TGTCTTTTAC TTAGACGATT ACTTCCAATG TCTAAGCCAG


 


1301
AAGAGGAAGA AACTTATAAC ATCGTAGCCG CTCATGGTTA TTTTGGCCGA



TTCTCCTTCT TTGAATATTG TAGCATCGGC GAGTACCAAT AAAACCGGCT


 


1351
TTGATCTTCC AATATGCTAG TTTCAACAAC TCTCGTTCGT TACACTTCTT



AACTAGAAGG TTATACGATC AAAGTTGTTG AGAGCAAGCA ATGTGAAGAA


 


1401
CCTAGCTGCT TGGCCTGTAG TAGGTATCTG GTTTACCGCT TTAGGTATCA



GGATCGACGA ACCGGACATC ATCCATAGAC CAAATGGCGA AATCCATAGT


 


1451
GCACTATGGC TTTCAACCTA AATGGTTTCA ATTTCAACCA ATCTGTAGTT



CGTGATACCG AAAGTTGGAT TTACCAAAGT TAAAGTTGGT TAGACATCAA


 


1501
GACAGTCAAG GCCGTGTAAT TAATACTTGG GCTGATATCA TTAACCGTGC



CTGTCAGTTC CGGCACATTA ATTATGAACC CGACTATAGT AATTGGCACG


 


1551
TAACCTTGGT ATGGAAGTTA TGCATGAACG TAATGCTCAC AACTTCCCTC



ATTGGAACCA TACCTTCAAT ACGTACTTGC ATTACGAGTG TTGAAGGGAG


 


1601
TAGACCTAGC TGCTATCGAA GCTCCATCTA CAAAGGGAGA AGTCGACGGT



ATCTGGATCG ACGATAGCTT CGAGGTAGAT GTTTACCTAT TCAGCTGCCA


 


1651
ATCGATAAGC TTCCCCGGGA GACCACAACG GTTTCCCTCT AGAAATAATT



TAGCTATTCG AAGGGGCCCT CTGGTGTTGC CAAAGGGAGA TCTTTATTAA


 


1701
TTGTTTAACT TTAAGAAGGA GATATACATA TGAACCCGAA CTCATTTCAA



AACAAATTGA AATTCTTCCT CTATATGTAT ACTTGGGCTT GAGTAAAGTT


 


1751
TTCAAAGAAA ACATACTACA ATTTTTTTCT GTACATGATG ACATCTGGAA



AAGTTTCTTT TGTATGATGT TAAAAAAAGA CATGTACTAC TGTAGACCTT


 


1801
AAAATTACAA GAATTTTATT ATGGGCAAAG CCCAATTAAT GAGGCTTTGG



TTTTAATGTT CTTAAAATAA TACCCGTTTC GGGTTAATTA CTCCGAAACC


 


1851
CGCAGCTCAA CAAAGAAGAT ATGTCTTTGT TCTTTGAAGC ACTATCTAAA



GCGTCGAGTT GTTTCTTCTA TACAGAAACA AGAAACTTCG TGATAGATTT


 


1901
AACCCAGCTC GCATGATGGA AATGCAATGG AGCTGGTGGC AAGGTCAAAT



TTGGGTCGAG CGTACTACCT TTACGTTACC TCGACCACCG TTCCAGTTTA


 


1951
ACAAATCTAC CAAAATGTGT TGATGCGCAG CGTGGCCAAA GATGTAGCAC



TGTTTAGATG GTTTTACACA ACTACGCGTC GCACCGGTTT CTACATCGTG


 


2001
CATTTATTCA GCCTGAAAGT GGTGATCGTC GTTTTAACAG CCCATTATGG



GTAAATAAGT CGGACTTTCA CCACTAGCAG CAAAATTGTC GGGTAATACC


 


2051
CAAGAACACC CAAATTTTGA CTTGTTGTCA CAGTCTTATT TACTGTTTAG



GTTCTTGTGG GTTTAAAACT GAACAACAGT GTCAGAATAA ATGACAAATC


 


2101
CCAGTTAGTG CAAAACATGG TAGATGTGGT CGAAGGTGTT CCAGACAAAG



GGTCAATCAC GTTTTGTACC ATCTACACCA GCTTCCACAA GGTCTGTTTC


 


2151
TTCGCTATCG TATTCACTTC TTTACCCGCC AAATGATCAA TGCGTTATCT



AAGCGATAGC ATAAGTGAAG AAATGGGCGG TTTACTAGTT ACGCAATAGA


 


2201
CCAAGTAACT TTCTGTGGAC TAACCCAGAA GTGATTCAGC AAACTGTAGC



GGTTCATTGA AAGACACCTG ATTGGGTCTT CACTAAGTCG TTTGACATCG


 


2251
TGAACAAGGT GAAAACTTAG TCCGTGGCAT GCAAGTTTTC CATGATGATG



ACTTGTTCCA CTTTTGAATC AGGCACCGTA CGTTCAAAAG GTACTACTAC


 


2301
TCATGAATAG CGGCAAGTAT TTATCTATTC GCATGGTGAA TAGCGACTCT



AGTACTTATC GCCGTTCATA AATAGATAAG CGTACCACTT ATCGCTGAGA


 


2351
TTCAGCTTGG GCAAAGATTT AGCTTACACC CCTGGTGCAG TCGTCTTTGA



AAGTCGAACC CGTTTCTAAA TCGAATGTGG GGACCACGTC AGCAGAAACT


 


2401
AAATGACATT TTCCAATTAT TGCAATATGA AGCAACTACT GAAAATGTGT



TTTACTGTAA AAGGTTAATA ACGTTATACT TCGTTGATGA CTTTTACACA


 


2451
ATCAAACCCC TATTCTAGTC GTACCACCGT TTATCAATAA ATATTATGTG



TAGTTTGGGG ATAAGATCAG CATGGTGGCA AATAGTTATT TATAATACAC


 


2501
CTGGATTTAC GCGAACAAAA CTCTTTAGTG AACTGGTTGC GCCAGCAAGG



GACCTAAATG CGCTTGTTTT GAGAAATCAC TCGACCACCG CGGTCGTTCC


 


2551
TCATACAGTC TTTTTAATGT CATGGCGTAA CCCAAATGCC GAACAGAAAG



AGTATGTCAG AAAAATTACA GTACCGCATT GGGTTTACGG CTTGTCTTTC


 


2601
AATTGACTTT TGCCGATCTC ATTACACAAG GTTCAGTGGA AGCTTTGCGT



TTAACTGAAA ACGGCTAGAG TAATGTGTTC CAAGTCACCT TCGAAACGCA


 


2651
GTAATTGAAG AAATTACCGG TGAAAAAGAG GCCAACTGCA TTGGCTACTG



CATTAACTTC TTTAATGGCC ACTTTTTCTC CGGTTGACGT AACCGATGAC


 


2701
TATTGGTGGT ACGTTACTTG CTGCGACTCA AGCCTATTAC GTGGCAAAAC



ATAACCACCA TGCAATGAAC GACGCTGAGT TCGGATAATG CACCGTTTTG


 


2751
GCCTGAAAAA TCACGTAAAG TCTGCGACCT ATATGGCCAC CATTATCGAC



CGGACTTTTT AGTGCATTTC AGACGCTGGA TATACCGGTG GTAATAGCTG


 


2801
TTTGAAAACC CAGGCAGCTT AGGTGTATTT ATTAATGAAC CTGTAGTGAG



AAACTTTTGG GTCCGTCGAA TCCACATAAA TAATTACTTG GACATCACTC


 


2851
CGGTTTAGAA AACCTGAACA ATCAATTGGG TTATTTCGAT GGTCGTCAGT



GCCAAATCTT TTGGACTTGT TAGTTAACCC AATAAAGCTA CCAGCAGTCA


 


2901
TGGCAGTTAC CTTCAGTTTA CTGCGTGAAA ATACGCTGTA CTGGATTTAC



ACCGTCAATG GAAGTCAAAT GACGCACTTT TATGCGACAT GACCTAAATG


 


2951
TACATCGACA ACTACTTAAA AGGTAAAGAA CCTTCTGATT TTGATATTTT



ATGTAGCTGT TGATGAATTT TCCATTTCTT GGAAGACTAA AACTATAAAA


 


3001
ATATTCGAAC AGCGATGGTA CGAATATCCC TGCCAAAATT CATAATTTCT



TATAACCTTG TCGCTACCAT GCTTATAGGG ACGGTTTTAA GTATTAAAGA


 


3051
TATTGCGCAA TTTGTATTTG AACAATGAAT TGATTTCACC AAATGCCGTT



ATAACGCGTT AAACATAAAC TTGTTACTTA ACTAAAGTGG TTTACGGCAA


 


3101
AAGGTTAACG GTGTGGGCTT GAATCTATCT CGTGTAAAAA CACCAAGCTT



TTCCAATTGC CACACCCGAA CTTAGATAGA GCACATTTTT GTGGTTCGAA


 


3151
CTTTATTGCG ACGCAGGAAG ACCATATCGC ACTTTGGGAT ACTTGTTTCC



GAAATAACGC TGCGTCCTTC TGGTATAGCG TGAAACCCTA TGAACAAAGG


 


3201
GTGGCGCAGA TTACTTGGGT GGTGAATCAA CCTTGGTTTT AGGTGAATCT



CACCGCGTCT AATGAACCCA CCACTTAGTT GGAACCAAAA TCCACTTAGA


 


3251
GGACACGTAG CAGGTATTGT CAATCCTCCA AGCCGTAATA AATACGGTTG



CCTGTGCATC GTCCATAACA GTTAGGAGGT TCGGCATTAT TTATGCCAAC


 


3301
CTACACCAAT GCTGCCAAGT TTGAAAATAC CAAACAATGG CTAGATGGCG



GATGTGGTTA CGACGGTTCA AACTTTTATG GTTTGTTACC GATCTACCGC


 


3351
CAGAATATCA CCCTGAATCT TGGTGGTTGC GCTGGCAGGC ATGGGTCACA



GTCTTATAGT GGGACTTAGA ACCACCAACG CGACCGTCCG TACCCAGTGT


 


3401
CCGTACACTG GTGAACAAGT CCCTGCCCGC AACTTGGGTA ATGCGCAGTA



GGCATGTGAC CACTTGTTCA GGGACGGGCG TTGAACCCAT TACGCGTCAT


 


3451
TCCAAGCATT GAAGCGGCAC CGGGTCGCTA TGTTTTGGTA AATTTATTCT



AGGTTCGTAA CTTCGCCGTG GCCCAGCGAT ACAAAACCAT TTAAATAAGA


 


3501
AAGCGGCCGC CACCGCGGTG GAGCTCAATA AAAAAAATCT AGATGCTTAT



TTCGCCGGCG GTGGCGCCAC CTCGAGTTAT TTTTTTTACA TCTACGAATA


 


3551
GATTCAGTAG TAGGAGGCAA ACCATATGAA AGATGTTGTG ATTGTTGCAG



CTAAGTCATC ATCCTCCGTT TGGTATACTT TCTACAACAC TAACAACGTC


 


3601
CAAAACGTAC TGCGATTGGT AGCTTTTTAG GTAGTCTTGC ATCTTTATCT



GTTTTGCATG ACGCTAACCA TCGAAAAATC CATCAGAACG TAGAAATAGA


 


3651
GCACCACAGT TGGGGCAAAC AGCAATTCGT GCAGTTTTAG ACAGCGCTAA



CGTGGTGTCA ACCCCGTTTG TCGTTAAGCA CGTCAAAATC TGTCGCGATT


 


3701
TGTAAAACCT GAACAAGTTG ATCAGGTGAT TATGGGCAAC GTACTCACGA



ACATTTTGGA CTTGTTCAAC TAGTCCACTA ATACCCGTTG CATGAGTGCT


 


3751
CAGGCGTGGG ACAAAACCCT GCACGTCAGG CAGCAATTGC TGCTGGTATT



GTCCGCACCC TGTTTTGGGA CGTGCAGTCC GTCGTTAACG ACGACCATAA


 


3801
CCAGTACAAG TGCCTGCATC TACGCTGAAT GTCGTCTGTG GTTCAGGTTT



GGTCATGTTC ACGGACGTAG ATGCGACTTA CAGCAGACAC CAAGTCCAAA


 


3851
GCGTGCGGTA CATTTGGCAG CACAAGCCAT TCAATGCGAT GAAGCCGACA



CGCACGCCAT GTAAACCGTC GTGTTCGGTA AGTTACGCTA CTTCGGCTGT


 


3901
TTGTGGTCGC AGGTGGTCAA GAATCTATCT CACAAAGTGC GCACTATATG



AACACCAGCG TCCACCAGTT CTTAGATACA GTGTTTCACG CGTGATATAC


 


3951
CAGCTGCGTA ATGGGCAAAA AATGGGTAAT GCACAATTGG TGGATAGCAT



GTCGACGCAT TACCCGTTTT TTACCCATTA CGTGTTAACC ACCTATCGTA


 


4001
GGTGGCTGAT GGTTTAACCG ATGCCTATAA CCAGTATCAA ATGGGTATTA



CCACCGACTA CCAAATTGGC TACGGATATT GGTCATAGTT TACCCATAAT


 


4051
CCGCAGAAAA TATTGTAGAA AAACTGGGTT TAAACCGTGA AGAACAAGAT



GGCGTCTTTT ATAACATCTT TTTGACCCAA ATTTGGCACT TCTTGTTCTA


 


4101
CAACTTGCAT TGACTTCACA ACAACGTGCT GCGGCAGCTC AGGCAGCTGG



GTTGAACGTA ACTGAAGTGT TGTTGCACGA CGCCGTCGAG TCCGTCGACC


 


4151
CAAGTTTAAA GATGAAATTG CCGTAGTCAG CATTCCACAA CGTAAAGGTG



GTTCAAATTT CTACTTTAAC GGCATCAGTC GTAAGGTGTT GCATTTCCAC


 


4201
AGCCTGTTGT ATTTGCTGAA GATGAATACA TTAAAGCCAA TACCAGCCTT



TCGGACAACA TAAACGACTT CTACTTATGT AATTTCGGTT ATGGTCGGAA


 


4251
GAAAGCCTCA CAAAACTACG CCCAGCCTTT AAAAAAGATG GTAGCGTAAC



CTTTCGGAGT GTTTTGATGC GGGTCGGAAA TTTTTTCTAC CATCGCATTG


 


4301
CGCAGGTAAT GCTTCAGGCA TTAATGATGG TGCAGCAGCA GTACTGATGA



GCGTCCATTA CGAAGTCCGT AATTACTACC ACGTCGTCGT CATGACTACT


 


4351
TGAGTGCGGA CAAAGCAGCA GAATTAGGTC TTAAGCCATT GGCACGTATT



ACTCACGCCT GTTTCGTCGT CTTAATCCAG AATTCGGTAA CCGTGCATAA


 


4401
AAAGGCTATG CCATGTCTGG TATTGAGCCT GAAATTATGG GGCTTGGTCC



TTTCCGATAC GGTACAGACC ATAACTCGGA CTTTAATACC CCGAACCAGG


 


4451
TGTCGATGCA GTAAAGAAAA CCCTCAACAA AGCAGGCTGG AGCTTAGATC



ACAGCTACGT CATTTCTTTT GGGAGTTGTT TCGTCCGACC TCGAATCTAG


 


4501
AGGTTGATTT GATTGAAGCC AATGAAGCAT TTGCTGCACA GGCTTTGGGT



TCCAACTAAA CTAACTTCGG TTACTTCGTA AACGACGTGT CCGAAACCCA


 


4551
GTTGCTAAAG AATTAGGCTT AGACCTGGAT AAAGTCAACG TCAATGGCGG



CAACGATTTC TTAATCCGAA TCTGGACCTA TTTCAGTTGC AGTTACCGCC


 


4601
TGCAATTGCA TTGGGTCACC CAATTGGGGC TTCAGGTTGC CGTATTTTGG



ACGTTAACGT AACCCAGTGG GTTAACCCCG AAGTCCAACG GCATAAAACC


 


4651
TGACTTTATT ACATGAAATG CAGCGCCGTG ATGCCAAGAA AGGCATTGCA



ACTGAAATAA TGTACTTTAC GTCGCGGCAC TACGGTTCTT TCCGTAACGT


 


4701
ACCCTCTGTG TTGGCGGTGG TATGGGTGTT GCACTTGCAG TTGAACGTGA



TGGGAGACAC AACCGCCACC ATACCCACAA CGTGAACGTC AACTTGCACT


 


4751
CTAAGCGGCC GCTCGAGTGG CGGCTCAAGA TCAGCCTCAT CAAAACCTTA



GATTCGCCGG CGAGCTCACC GCCGAGTTCT AGTCGGAGTA GTTTTGGAAT


 


4801
TATTCCCTGA GGAGGTTCTA CCCATATGAC AACATTACAA GGTAAAGTAG



ATAAGGGACT CCTCCAAGAT GGGTATACTG TTGTAATGTT CCATTTCATC


 


4851
CAATCGTAAC AGGCGGATCT AAAGGTATCG GGGCAGCAAT TACACGTGAG



GTTAGCATTG TCCGCCTAGA TTTCCATAGC CCCGTCGTTA ATGTGCACTC


 


4901
CTTGCTTCTA ATGGAGTAAA AGTAGCAGTA AACTATAACA GCAGTAAAGA



GAACGAAGAT TACCTCATTT TCATCGTCAT TTGATATTGT CGTCATTTCT


 


4951
ATCTGCAGAA GCAATTGTAA AAGAAATTAA AGACAACGGC GGAGAAGCTA



TAGACGTCTT CGTTAACATT TTCTTTAATT TCTGTTGCCG CCTCTTCGAT


 


5001
TTGCGGTTCA AGCTGACGTG TCTTATGTAG ATCAAGCAAA ACACCTAATC



AACGCCAAGT TCGACTGCAC AGAATACATC TAGTTCGTTT TGTGGATTAG


 


5051
GAAGAAACAA AAGCTGCGTT TGGTCAATTA GACATTCTAG TAAACAATGC



CTTCTTTGTT TTCGACGCAA ACCAGTTAAT CTGTAAGATC ATTTGTTACG


 


5101
TGGAATTACG CGCGACCGTT CATTCAAGAA GTTAGGTGAA GAAGATTGGA



ACCTTAATGC GCGCTGGCAA GTAAGTTCTT CAATCCACTT CTTCTAACCT


 


5151
AAAAAGTAAT TGATGTAAAC TTACATAGCG TATACAACAC AACATCAGCT



TTTTTCATTA ACTACATTTG AATGTATCGC ATATGTTGTG TTGTAGTCGA


 


5201
GCGCTAACGC ACCTTTTAGA ATCTGAAGGT GGTCGTGTTA TCAATATTTC



CGCGATTGCG TGGAAAATCT TAGACTTCCA CCAGCACAAT AGTTATAAAG


 


5251
ATCAATTATT GGTCAAGCGG GCGGATTTGG TCAAACAAAC TACTCAGCTG



TAGTTAATAA CCAGTTCGCC CGCCTAAACC AGTTTGTTTG ATGAGTCGAC


 


5301
CTAAAGCAGG TATGCTAGGA TTCACTAAAT CATTAGCTCT TGAACTAGCT



GATTTCGTCC ATACGATCCT AAGTGATTTA GTAATCGAGA ACTTGATCGA


 


5351
AAGACAGGCG TAACGGTTAA TGCAATTTGC CCAGGATTTA TTGAAACGGA



TTCTGTCCGC ATTGCCAATT ACGTTAAACG GGTCCTAAAT AACTTTGCCT


 


5401
AATGGTGATG GCAATTCCTG AAGATGTTCG TGCAAAAATT GTTGCGAAAA



TTACCACTAC CGTTAAGGAC TTCTACAAGC ACGTTTTTAA CAACGCTTTT


 


5451
TTCCAACTCG TCGCTTAGGT CACGCTGAAG AAATTGCACG TGGAGTTGTT



AAGGTTGAGC AGCGAATCCA GTGCGACTTC TTTAACGTGC ACCTCAACAA


 


5501
TACTTAGCAA AAGACGGCGC GTACATTACA GGACAACAGT TAAACATTAA



ATGAATCGTT TTCTGCCGCG CATGTAATGT CCTGTTGTCA ATTTGTAATT


 


5551
CGGCGGCTTA TACATGTAAT GGATCCAATC GATACAAGTG AGTTGTAGGG



GCCGCCGAAT ATGTACATTA CCTAGGTTAG CTATGTTCAC TCAACATCCC


 


5601
AGGCAACCAT GGCAGAAGCG GTGATCGCCG AAGTATCGAC TCAACTATCA



TCCGTTGGTA CCGTCTTCGC CACTAGCGGC TTCATAGCTG AGTTGATAGT


 


5651
GAGGTAGTTG GCGTCATCGA GCGCCATCTC GAACCGACGT TGCTGGCCGT



CTCCATCAAC CGCAGTAGCT CGCGGTAGAG CTTGGCTGCA ACGACCGGCA


 


5701
ACATTTGTAC GGCTCCGCAG TGGATGGCGG CCTGAAGCCA CACAGTGATA



TGTAAACATG CCGAGGCGTC ACCTACCGCC GGACTTCGGT GTGTCACTAT


 


5751
TTGATTTGCT GGTTACGGTG ACCGTAAGGC TTGATGAAAC AACGCGGCGA



AACTAAACGA CCAATGCCAC TGGCATTCCG AACTACTTTG TTGCGCCGCT


 


5801
GCTTTGATCA ACGACCTTTT GGAAACTTCG GCTTCCCCTG GAGAGAGCGA



CGAAACTAGT TGCTGGAAAA CCTTTGAAGC CGAAGGGGAC CTCTCTCGCT


 


5851
GATTCTCCGC GCTGTAGAAG TCACCATTGT TGTGCACGAC GACATCATTC



CTAAGAGGCG CGACATCTTC AGTGGTAACA ACACGTGCTG CTGTAGTAAG


 


5901
CGTGGCGTTA TCCAGCTAAG CGCGAACTGC AATTTGGAGA ATGGCAGCGC



GCACCGCAAT AGGTCGATTC GCGCTTGACG TTAAACCTCT TACCGTCGCG


 


5951
AATGACATTC TTGCAGGTAT CTTCGAGCCA GCCACGATCG ACATTGATCT



TTACTGTAAG AACGTCCATA GAAGCTCGGT CGGTGCTAGC TGTAACTAGA


 


6001
GGCTATCTTG CTGACAAAAG CAAGAGAACA TAGCGTTGCC TTGGTAGGTC



CCGATAGAAC GACTGTTTTC GTTCTCTTGT ATCGCAACGG AACCATCCAG


 


6051
CAGCGGCGGA GGAACTCTTT GATCCGGTTC CTGAACAGGA TCTATTTGAG



GTCGCCGCCT CCTTGAGAAA CTAGGCCAAG GACTTGTCCT AGATAAACTC


 


6101
GCGCTAAATG AAACCTTAAC GCTATGGAAC TCGCCGCCCG ACTGGGCTGG



CGCGATTTAC TTTGGAATTG CGATACCTTG AGCGGCGGGC TGACCCGACC


 


6151
CGATGAGCGA AATGTAGTGC TTACGTTGTC CCGCATTTGG TACAGCGCAG



GCTACTCGCT TTACATCACG AATGCAACAG GGCGTAAACC ATGTCGCGTC


 


6201
TAACCGGCAA AATCGCGCCG AAGGATGTCG CTGCCGACTG GGCAATGGAG



ATTGGCCGTT TTAGCGCGGC TTCCTACAGC GACGGCTGAC CCGTTACCTC


 


6251
CGCCTGCCGG CCCAGTATCA GCCCGTCATA CTTGAAGCTA GACAGGCTTA



GCGGACGGCC GGGTCATAGT CGGGCAGTAT GAACTTCGAT CTGTCCGAAT


 


6301
TCTTGGACAA GAAGAAGATC GCTTGGCCTC GCGCGCAGAT CAGTTGGAAG



AAAACTAGTT CTTCTTCTAG CGAACCGGAG CGCGCGTCTA GTCAACCTTC


 


6351
AATTTGTCCA CTACGTGAAA GGCGAGATCA CCAAGGTAGT CGGCAAATAA



TTAAACAGGT GATGCACTTT CCGCTCTAGT GGTTCCATCA GCCGTTTATT


 


6401
ATCTAGGCCG AATTGGGCCT AGTCTATAGG AGGTTTTGAA AAGAAAGGAG



TAGATCCGGC TTAACCCGGA TCAGATATCC TCCAAAACTT TTCTTTCCTC


 


6451
CAATAATCAT TTTCTTGTTC TATCAAGAGG GTGCTATTGC TCCTTTCTTT



GTTATTAGTA AAAGAACAAG ATAGTTCTCC CACGATAACG AGGAAAGAAA


 


6501
TTTTCTTTTT ATTTATTTAC TAGTATTTTA CTTACATAGA CTTTTTTGTT



AAAAGAAAAA TAAATAAATG ATCATAAAAT GAATGTATCT GAAAAACTAA


 


6551
TACATTATAG AAAAAGAAGG AGAGGTTATT TTCTTGCATT TATTCATGAT



ATGTAATATC TTTTTCTTCC TCTCCAATAA AAGAACGTAA ATAAGTACTA


 


6601
TGAGTATTCT ATTTTGATTT TGTATTTGTT TAAAATTGTA GAAATAGAAC



ACTCATAAGA TAAAACTAAA ACATAAACAA ATTTTAACAT CTTTATCTTG


 


6651
TTGTTTCTCT TCTTGCTAAT GTTACTATAT CTTTTTTGTT TTTTTTTTCC



AACAAAGAGA AGAACGATTA CAATGATATA GAAAAACTAA AAAAAAAAGG


 


6701
AAAAAAAAAA TCAAATTTTG ACTTCTTCTT ATCTCTTATC TTTGAATATC



TTTTTTTTTT AGTTTAAAAC TGAAGAAGAA TAGAGAATAG AAACTTATAG


 


6751
TCTTATCTTT GAAATAATAA TATCATTGAA ATAAGAAAGA AGAGCTATAT



AGAATAGAAA CTTTATTATT ATAGTAACTT TATTCTTTCT TCTCGATATA


 


6801
TCGAACTTGA ATCTTTTGTT TTCTAATTTA AATAATGTAA AAACGGAATG



AGCTTGAACT TAGAAAACAA AAGATTAAAT TTATTACATT TTTGCCTTAC


 


6851
TAAGTAGGCG AGGGGGCGGA TGTAGCCAAG TGGATCAAGG CAGTGGATTG



ATTCATCCGC TCCCCCGCCT ACATCGGTTC ACCTAGTTCC GTCACCTAAC


 


6901
TGAATCCACC ATGCGCGGGT TCAATTCCCG TCGTTCGCCC ATAATTACTC



ACTTAGGTGG TACGCGCCCA AGTTAAGGGC AGCAAGCGGG TATTAATGAG


 


6951
CTATTTTTTT TTTTTTTGTA AAAACGAAGA ATTTAATTCG ATTTTCTCTC



GATAAAAAAA AAAAAAACAT TTTTGCTTCT TAAATTAAGC TAAAAGAGAG


 


7001
CTATTTACTA CGGCGACGAA GAATCAAATT ATCACTATAT TTATTCCTTT



GATAAATGAT GCCGCTGCTT CTTAGTTTAA TAGTGATATA AATAAGGAAA


 


7051
TTCTACTTCT TCTTCCAAGT GCAGGATAAC CCCAAGGGGT TGTGGGTTTT



AAGATGAAGA AGAAGGTTCA CGTCCTATTG GGGTTCCCCA ACACCCAAAA


 


7101
TTTCTACCAA TTGGGGCTCT CCCTTCACCA CCCCCATGGG GATGGTCTAC



AAAGATGGTT AACCCCGAGA GGGAAGTGGT GGGGGTACCC CTACCAGATG


 


7151
AGGGTTCATA ACTACTCCTC TTACTACAGG ACGCTTACCT AGCCAACGCT



TCCCAAGTAT TGATGAGGAG AATGATGTCC TGCGAATGGA TCGGTTGCGA


 


7201
TAGATCCGGC TCTACCCAAA CTTTTCTGGT TCACCCCAAC ATTCCCCACT



ATCTAGGCCG AGATGGGTTT GAAAAGACCA AGTGGGGTTG TAAGGGGTGA


 


7251
TGTCCGACTG TTGCTGAGCA GTTTTTGGAT ATCAAACGGA CCTCCCCAGA



ACAGGCTGAC AACGACTCGT CAAAAACCTA TAGTTTGCCT GGAGGGGTCT


 


7301
AGGTAATTTT AATGTGGCCG ATTTCCCCTC TTTTGCAATC AGTTTCGCTA



TCCATTAAAA TTACACCGGC TAAAGGGGAG AAAACGTTAG TCAAAGCGAT


 


7351
CAGCACCCGC TGCTCTAGCT AATTGTCCAC CCTTTCCAAG TGTGATTTCT



GTCGTGGGCG ACGAGATCGA TTAACAGGTG GGAAAGGTTC ACACTAAAGA


 


7401
ATGTTATGTA TGGCCGTGCC TAAGGGCATA TCGGTTGAAG TAGATTCTTC



TACAATACAT ACCGGCACGG ATTCCCGTAT AGCCAACTTC ATCTAAGAAG


 


7451
TTTTGATCAA TCAAAACCCC TTCCCAAACT GTACAAGCTT GGCGTAATCA



AAAACTAGTT AGTTTTGGGG AAGGGTTTGA CATGTTCGAA CCGCATTAGT


 


7501
TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA



ACCAGTATCG ACAAAGGACA CACTTTAACA ATAGGCGAGT GTTAAGGTGT


 


7551
CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG



GTTGTATGCT CGGCCTTCGT ATTTCACATT TCGGACCCCA CGGATTACTC


 


7601
TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC TTTCCAGTCG



ACTCGATTGA GTGTAATTAA CGCAACGCGA GTGACGGGCG AAAGGTCAGC


 


7651
GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC GCGCGGGGAG



CCTTTGGACA GCACGGTCGA CGTAATTACT TAGCCGGTTG CGCGCCCCTC


 


7701
AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC



TCCGCCAAAC GCATAACCCG CGAGAAGGCG AAGGAGCGAG TGACTGAGCG


 


7751
TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG



ACGCGAGCCA GCAAGCCGAC GCCGCTCGCC ATAGTCGAGT GAGTTTCCGC


 


7801
GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG



CATTATGCCA ATAGGTGTCT TAGTCCCCTA TTGCGTCCTT TCTTGTACAC


 


7851
AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG



TCGTTTTCCG GTCGTTTTCC GGTCCTTGGC ATTTTTCCGG CGCAACGACC


 


7901
CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC



GCAAAAAGGT ATCCGAGGCG GGGGGACTGC TCGTAGTGTT TTTAGCTGCG


 


7951
TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT



AGTTCAGTCT CCACCGCTTT GGGCTGTCCT GATATTTCTA TGGTCCGCAA


 


8001
TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA



AGGGGGACCT TCGAGGGAGC ACGCGAGAGG ACAAGGCTGG GACGGCGAAT


 


8051
CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT



GGCCTATGGA CAGGCGGAAA GAGGGAAGCC CTTCGCACCG CGAAAGAGTA


 


8101
AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT



TCGAGTGCGA CATCCATAGA GTCAAGCCAC ATCCAGCAAG CGAGGTTCGA


 


8151
GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG



CCCGACACAC GTGCTTGGGG GGCAAGTCGG GCTGGCGACG CGGAATAGGC


 


8201
GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG



CATTGATAGC AGAACTCAGG TTGGGCCATT CTGTGCTGAA TAGCGGTGAC


 


8251
GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC



CGTCGTCGGT GACCATTGTC CTAATCGTCT CGCTCCATAC ATCCGCCACG


 


8301
TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG



ATGTCTCAAG AACTTCACCA CCGGATTGAT GCCGATGTGA TCTTCCTGTC


 


8351
TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT



ATAAACCATA GACGCGAGAC GACTTCGGTC AATGGAAGCC TTTTTCTCAA


 


8401
GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT



CCATCGAGAA CTAGGCCGTT TGTTTGGTGG CGACCATCGC CACCAAAAAA


 


8451
TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC



ACAAACGTTC GTCGTCTAAT GCGCGTCTTT TTTTCCTAGA GTTCTTCTAG


 


8501
CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT



GAAACTAGAA AAGATGCCCC AGACTGCGAG TCACCTTGCT TTTGAGTGCA


 


8551
TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT



ATTCCCTAAA ACCAGTACTC TAATAGTTTT TCCTAGAAGT GGATCTAGGA


 


8601
TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA



AAATTTAATT TTTACTTCAA AATTTAGTTA GATTTCATAT ATACTCATTT


 


8651
CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG



GAACCAGACT GTCAATGGTT ACGAATTAGT CACTCCGTGG ATAGAGTCGC


 


8701
ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT



TAGACAGATA AAGCAAGTAG GTATCAACGG ACTGAGGGGC AGCACATCTA


 


8751
AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC



TTGATGCTAT GCCCTCCCGA ATGGTAGACC GGGGTCACGA CGTTACTATG


 


8801
CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA



GCGCTCTGGG TGCGAGTGGC CGAGGTCTAA ATAGTCGTTA TTTGGTCGGT


 


8851
GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT



CGGCCTTCCC GGCTCGCGTC TTCACCAGGA CGTTGAAATA GGCGGAGGTA


 


8901
CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA



GGTCAGATAA TTAACAACGG CCCTTCGATC TCATTCATCA AGCGGTCAAT


 


8951
ATAGTTTGCG CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC



TATCAAACGC GTTGCAACAA CGGTAACGAT GTCCGTAGCA CCACAGTGCG


 


9001
TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG



AGCAGCAAAC CATACCGAAG TAAGTCGAGG CCAAGGGTTG CTAGTTCCGC


 


9051
AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC



TCAATGTACT AGGGGGTACA ACACGTTTTT TCGCCAATCG AGGAAGCCAG


 


9101
CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT



GAGGCTAGCA ACAGTCTTCA TTCAACCGGC GTCACAATAG TGAGTACCAA


 


9151
ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT



TACCGTCGTG ACGTATTAAG AGAATGACAG TGCGAGTGGC ATTCTACGAA


 


9201
TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC



AAGACACTGA CCACTCATGA GTTGGTTCAG TAAGACTCTT ATCACATACG


 


9251
GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA



CCGCTGGCTC AACGAGAACG GGCCGCAGTT ATGCCCTATT ATGGCGCGGT


 


9301
CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG



GTATCGTCTT GAAATTTTCA CGAGTAGTAA CCTTTTTCAA GAAGCCCCGC


 


9351
AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA



TTTTGAGAGT TCCTAGAATG GCGACAACTC TAGGTCAAGC TACATTGGGT


 


9401
CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT



GAGCACGTGG GTTGACTAGA AGTCGTAGAA AATGAAAGTG GTCGCAAAGA


 


9451
GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC



CCCACTCGTT TTTGTCCTTC CGTTTTACGG CGTTTTTTCC CTTATTCCCG


 


9501
GACACGGAAA TGTTGAATAC TCATACTCTT CCTTTTTCAA TATTAT



CTGTGCCTTT ACAACTTATG AGTATGAGAA GGAAAAAGTT ATAATA

## Claims

1. A fertile transplastomic plant comprising one or more plastids engineered to contain one or more expression cassettes for the plastid-encoded expression of genes encoding enzymes for the production of polyhydroxyalkanoate (PHA),
wherein the one or more expression cassettes comprise untranslated regions (UTRs) with a sequence length of about 55 nucleotides or fewer, or
wherein the one or more expression cassettes comprise a total plastidial DNA content of about 3% or less,
wherein the genes encoding enzymes for the production of PHA have codon usage similar to the plastome of the host plant and are inserted into an existing transcriptional unit of the host plastid, and are under the control of the transcriptional unit, and
wherein the fertile transplastomic plant produces greater than 10% polyhydroxyalkanoate per unit dry cell weight (dwt) in leaves and produces seeds that are able to germinate and produce viable transplastomic plants.

2. The transplastomic plant of claim 1 wherein the genes are modified or synthesized to improve codon usage for expression in the plastome of the host plant.

3. The transplastomic plant of claim 1 wherein the transgenic plant is engineered to express one or more genes selected from the group consisting of phaA, phaB, and phaC.

4. The transplastomic plant of claim 1 wherein the plant is a dicot or monocot.

5. The transplastomic plant of claim 1 wherein the plant is selected from the group consisting of B. napus, B. rappa, B. carinata and B. juncea; Camelina sativa, Crambe, jatropha, castor; Arabidopsis thaliana; maize; soybean; cottonseed; sunflower; palm; algae; coconut; safflower; peanut; Sinapis alba; sugarcane; silage corn; alfalfa; switchgrass; miscanthus; hemp; sorghum; and tobacco.

6. The transplastomic plant of claim 1, where some regions of the leaves of the plant produce greater than 10% dwt PHA.

7. The transplastomic plant of claim 1 wherein PHA levels can reach at least 20% dwt in some regions of leaf.

8. The transplastomic plant of claim 1 wherein the polyhydroxyalkanoate is poly(3-hydroxybutyrate) (P3HB).

9. A seed of the transplastomic plant of claim 1.

10. A biorefinery fuel or energy feedstock comprising plant material or plant parts of the transplastomic plant according to claim 1.

11. The feedstock of claim 10 wherein the feedstock comprises at least about 8% PHB throughout the whole plant.

12. A method for producing a fertile transplastomic plant that produces greater than 10% PHA per unit dwt in its leaves, the method comprising:
selecting a host plant; and
transfecting one or more plastids of the host plant with a vector containing one or more expression cassettes for the plastid-encoded expression of genes encoding enzymes for the production of polyhydroxyalkanoate (PHA), the expression cassettes comprising:
(a) genes encoding enzymes for the production of PHA, wherein the genes are selected for codon usage and GC content similar to the plastome of the host plant, are inserted into an existing transcriptional unit of the host plastid and are under the control of the transcriptional unit;
(b) untranslated regions (UTRs) with a sequence length of about 55 nucleotides or fewer; and
(c) a total plastidial DNA content of about 3% or less of the vector excluding a left flank and a right flank that together mediate integration of the vector into the one or more plastids;
wherein the fertile transplastomic plant produces seeds that are able to germinate and produce viable transplastomic plants.

13. The method of claim 12 wherein the polyhydroxyalkanoate comprises P3HB.

14. A method for producing a fertile transplastomic plant that produces PHA in its leaves, the method comprising:
selecting a host plant; and
transfecting one or more plastids of the host plant with a vector comprising:
(a) a left flank and a right flank that together mediate integration of the vector into the one or more plastids;
(b) genes between the left flank and right flank, wherein the genes encode enzymes for the production of PHA and have codon usage and GC content similar to the plastome of the host plant,
and
(c) one or more untranslated regions (UTRs) which allow a high level of expression of the genes, wherein sequence identity of the one or more UTRs with the host plastome is sufficiently low that, after the vector is integrated into the host plastid, the rate of recombination of the vector sequences with the host plastome is reduced relative to a corresponding host plastid not transfected with the UTRs;

thereby producing a fertile transplastomic plant that produces greater than 10% polyhydroxyalkanoate per unit dry cell weight (dwt) in its leaves and produces seeds that are able to germinate and produce viable transplastomic plants.

15. The method of claim 14 wherein the polyhydroxyalkanoate comprises P3HB.

16. A fertile transplastomic plant produced according to the method of claim 12.