US 5968805 A
The present invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a sequence of SEQ ID NO:1 where in, one or more amino acids are deleted, replaced or added, and the polypeptide having polyester synthase activity. A polyester synthase gene comprising DNA coding for the above polypeptide; a recombinant vector comprising the gene; and a transformant transformed with the recombinant vector is also provided.
1. An isolated polynucleotide encoding a polypeptide having a sequence as set forth in SEQ ID NO:1.
2. An isolated polynucleotide selected from the group consisting of:
a) SEQ ID NO:2;
b) SEQ ID NO:2, wherein T is U; and
c) nucleic acid sequences complementary to a) or b).
3. An isolated polynucleotide selected from the group consisting of:
a) SEQ ID NO:3;
b) SEQ ID NO:3, wherein T is U; and
c) nucleic acid sequences complementary to a) or b).
4. A vector containing a polynucleotide of claim 1, 2 or 3.
5. The vector of claim 4, wherein the vector is a viral vector.
6. The vector of claim 4, wherein the vector is a plasmid.
7. A host cell containing a vector of claim 4.
8. The host cell of claim 7, cultured under conditions which allow expression of the polynucleotide.
The present invention relates to polyester synthase, a gene coding for the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a process for producing polyester synthase by use of the transformant.
Polyesters (e.g. poly-3-hydroxyalkanoic acid) biosynthesized by microorganisms are biodegradable plastics with thermoplasticity ranging widely from rigid matter to viscoelastic rubber.
Poly-3-hydroxybutanoic acid (P(3HB)) is a typical polyester consisting of C4 monomer units, but it is a rigid and brittle polymeric material, so its application is limited. Accordingly, various polyesters such as P(3HB-co-3HV) having (P(3HB)) copolymerized with a C5 monomer unit (3HV) by adding propionic acid etc. to the medium have been prepared and examined to alter the physical properties of the polyester. On the other hand, polyesters consisting of at least C6 monomer units are soft polymeric materials having plasticity.
Polyester-synthesizing microorganisms are roughly divided into 2 groups, that is, those synthesizing polyesters with C3-5 monomer units and those synthesizing polyesters with C6-14 monomer units. The former microorganisms possess a polyester synthase using C3-5 monomer units as the substrate, while the latter microorganisms possess a polyester synthase using C6-14 monomer units as the substrate. Therefore, polyesters with different properties are synthesized by the respective microorganisms.
However, the respective polyesters from such known microorganisms are different in substrate specificity, so with one kind of enzyme given, polyesters (copolymers) having various monomer unit compositions adapted to the object of use are difficult to synthesize.
The object of the present invention is to provide a polyester synthase with monomer units having a wide range of carbon atoms as the substrate, a gene coding for the enzyme, a recombinant vector containing the gene, a transformant transformed with the vector, and a process for producing the polyester synthase by use of the transformant.
As a result of their eager research, the present inventors succeeded in cloning a polyester synthase gene from a microorganism belonging to the genus Pseudomonas isolated from soil, to arrive at the completion of the present invention.
That is, the present invention is a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a sequence where in said amino acid sequence, one or more amino acids are deleted, replaced or added, said polypeptide having polyester synthase activity.
Further, the present invention is a polyester synthase gene comprising DNA coding for said polypeptide. The DNA coding for the protein with polyester synthase activity includes e.g. that of SEQ ID NO:2.
Further, the present invention is a polyester synthase gene comprising the nucleotide sequence of SEQ ID NO:3.
Further, the present invention is a recombinant vector comprising the polyester synthase gene.
Further, the present invention is a transformant transformed with said recombinant vector.
Further, the present invention is a process for producing polyester synthase wherein said transformant is cultured in a medium and polyester synthase is recovered from the resulting culture.
Hereinafter, the present invention is described in detail.
(1) Cloning of the Polyester Synthase Gene
The polyester synthase gene of the present invention is separated from a microorganism belonging to the genus Pseudomonas.
First, genomic DNA is isolated from a strain having the polyester synthase gene. Such a strain includes e.g. Pseudomonas sp. Any known methods can be used for preparation of genomic DNA. For example, Pseudomonas sp. is cultured in a bouillon medium and then its genomic DNA is prepared by the hexadecyl trimethyl ammonium bromide method (Current Protocols in Molecular Biology, vol. 1, page 2.4.3., John Wiley & Sons Inc., 1994).
The DNA obtained in this manner is partially digested with a suitable restriction enzyme (e.g. Sau3AI, BamHI, BglII etc.). It is then ligated into a vector dephosphorylated by treatment with alkaline phosphatase after cleavage with a restriction enzyme (e.g. BamHI, BglII etc.) to prepare a library.
Phage or plasmid capable of autonomously replicating in host microorganisms is used as the vector. The phage vector includes e.g. EMBL3, M13, λgt11 etc., and the plasmid vector includes e.g. pBR322, pUC18, and pBluescript II (Stratagene). Vectors capable of autonomously replicating in 2 or more host cells such as E. coli and Bacillus brevis, as well as various shuttle vectors, can also be used. Such vectors are also cleaved with said restriction enzymes so that their fragment can be obtained.
Conventional DNA ligase is used to ligate the resulting DNA fragment into the vector fragment. The DNA fragment and the vector fragment are annealed and then ligated to produce a recombinant vector.
To introduce the recombinant vector into a host microorganism, any known methods can be used. For example, if the host microorganism is E. coli, the calcium chloride method (Lederberg, E. M. et al., J. Bacteriol. 119, 1072 (1974)) and the electroporation method (Current Protocols in Molecular Biology, vol. 1, page 1.8.4 (1994)) can be used. If phage DNA is used, the in vitro packaging method (Current Protocols in Molecular Biology, vol. 1, page 5.7.1 (1994)) etc. can be adopted. In the present invention, an in vitro packaging kit (Gigapack II, produced by Stratagene etc.) may be used.
To obtain a DNA fragment containing the polyester synthase gene derived from Pseudomonas sp., a probe is then prepared. The amino acid sequences of some polyester synthases have already been known (Peoples, O. P. and Sinskey, A. J., J. Biol. Chem., 264, 15293 (1989); Huisman, G. W. et al., J. Biol. Chem., 266, 2191 (1991); Pieper, U. et al., FEMS Microbiol. Lett., 96, 73 (1992); Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992), etc.) . Well-conserved regions are selected from these amino acid sequences, and nucleotide sequences coding for them are estimated to design oligonucleotides. Examples of such oligonucleotides include, but are not limited to, the sequence 5'-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3' (SEQ ID NO:4) reported by Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992).
Then, this synthetic oligonucleotide is labeled with a suitable reagent and used for colony hybridization of the above genomic DNA library (Current Protocols in Molecular Biology, vol. 1, page 6.0.3 (1994)).
The E. coli is screened by colony hybridization, and a plasmid is recovered from it using the alkaline method (Current Protocols in Molecular Biology, vol. 1, page 1.6.1 (1994)), whereby a DNA fragment containing the polyester synthase gene is obtained. The nucleotide sequence of this DNA fragment can be determined in e.g. an automatic nucleotide sequence analyzer such as 373A DNA sequencer (Applied Biosystems) using a known method such as the Sanger method (Molecular Cloning, vol. 2, page 13.3 (1989)).
After the nucleotide sequence was determined by the means described above, the gene of the present invention can be obtained by chemical synthesis or the PCR technique using genomic DNA as a template, or by hybridization using a DNA fragment having said nucleotide sequence as a probe.
(2) Preparation of Transformant
The transformant of the present invention is obtained by introducing the recombinant vector of the present invention into a host compatible with the expression vector used in constructing said recombinant vector.
The host is not particularly limited insofar as it can express the target gene. Examples are bacteria such as microorganisms belonging to the genus Alcaligenes, microorganisms belonging to the genus Bacillus, bacteria such as E. coli, yeasts such as the genera Saccharomyces, Candida etc., and animal cells such as COS cells, CHO cells etc.
If microorganisms belonging to the genus Alcaligenes or bacteria such as E. coli are used as the host, the recombinant DNA of the present invention is preferably constituted such that it contains a promoter, the DNA of the present invention, and a transcription termination sequence so as to be capable of autonomous replication in the host. The expression vector includes pLA2917 (ATCC 37355) containing replication origin RK2 and pJRD215 (ATCC 37533) containing replication origin RSF1010, which are replicated and maintained in a broad range of hosts.
The promoter may be any one if it can be expressed in the host. Examples are promoters derived from E. coli, phage etc., such as trp promoter, lac promoter, PL promoter, PR promoter and T7 promoter. The method of introducing the recombinant DNA into bacteria includes e.g. a method using calcium ions (Current Protocols in Molecular Biology, vol. 1, page 1.8.1 (1994)) and the electroporation method (Current Protocols in Molecular Biology, vol. 1, page 1.8.4 (1994)).
If yeast is used as the host, expression vectors such as YEp13, YCp50 etc. are used. The promoter includes e.g. gal 1 promoter, gal 10 promoter etc. To method of introducing the recombinant DNA into yeast includes e.g. the electroporation method (Methods. Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1333 (1978)), the lithium acetate method (J. Bacteriol., 153, 163-168 (1983)) etc.
If animal cells are used as the host, expression vectors such as pcDNAI, pcDNAI/Amp (produced by Invitrogene) etc. are used. The method of introducing the recombinant DNA into animal cells includes e.g. the electroporation method, potassium phosphate method etc.
(3) Production of Polyester Synthase
Production of the Polyester Synthase of the present invention is carried out by culturing the transformant of the present invention in a medium, forming and accumulating the polyester synthase of the present invention in the culture (the cultured microorganism or the culture supernatant) and recovering the polyester synthase from the culture.
A conventional method used for culturing the host is also used to culture the transformant of the present invention.
The medium for the transformant prepared from bacteria such as E. coli etc. as the host includes complete medium or synthetic medium, e.g. LB medium, M9 medium etc. The transformant is aerobically cultured at a temperature ranging from 25 to 37° C. for 12 to 48 hours so that the polyester synthase is accumulated in the microorganism and then recovered.
The carbon source is essential for the growth of the microorganism and includes e.g. carbohydrates such as glucose, fructose, sucrose, maltose etc.
The nitrogen source includes e.g. ammonia, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium phosphate etc., peptone, meat extract, yeast extract, corn steep liquor etc. The inorganic matter includes e.g. monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride etc.
Culture is carried out usually under aerobic conditions with shaking at 25 to 37° C. for more than 2 hours after expression is induced. During culture, antibiotics such as ampicillin, kanamycin, ampicillin, tetracycline etc. may be added to the culture.
To culture the microorganism transformed with the expression vector using an inducible promoter, its inducer can also be added to the medium. For example, isopropyl-β-D-thiogalactopyranoside (IPTG), indoleacrylic acid (IAA) etc. can be added to the medium.
To culture the transformant from animal cells as the host, use is made of a medium such as RPMI-1640 or DMEM which may be supplemented with fetal bovine serum. Culture is carried out usually in 5% CO2 at 30 to 37° C. for 1 to 7 days. During culture, antibiotics such as kanamycin, penicillin etc. may be added to the medium.
Purification of the polyester synthase can be performed by recovering the resulting culture by centrifugation (after disruption in the case of cells) and subjecting it to affinity chromatography, cation or anion exchange chromatography or gel filtration or to a suitable combination thereof.
Whether the resulting purified substance is the desired enzyme is confirmed by conventional methods such as SDS polyacrylamide gel electrophoresis, Western blotting etc.
Hereinafter, the present invention is described in more detail with reference to the Examples which however are not intended to limit the scope of the present invention.
(1) Cloning of the Polyester Synthase Gene from Pseudomonas sp.
First, a genomic DNA library of Pseudomonas sp. was prepared.
Pseudomonas sp. JCM 10015 was cultured overnight in 100 ml bouillon medium (1% meat extract, 1% peptone, 0.5% sodium chloride, pH 7.2) at 30° C. and then genomic DNA was obtained from the microorganism using the hexadecyl trimethyl ammonium bromide method (Current Protocols in Molecular Biology, vol. 1, page 2.4.3 (1994), John Wiley & Sons Inc.).
The resulting genomic DNA was partially digested with restriction enzyme Sau3AI. The vector plasmid used was cosmid vector pLA2917 (ATCC 37355). This plasmid was cleaved with restriction enzyme BglII and dephosphorylated (Molecular Cloning, vol. 1, page 5.7.2 (1989), Cold Spring Harbor Laboratory) and then ligated into the partially digested genomic DNA fragment by use of DNA ligase.
E. coli S17-1 was transformed with this ligated DNA fragment by the in vitro packaging method (Current Protocols in Molecular Biology, vol. 1, page 5.7.2 (1994)) whereby a genomic DNA library from Pseudomonas sp. was obtained.
To obtain a DNA fragment containing the polyester synthase gene from Pseudomonas sp., a probe was then prepared. An oligonucleotide consisting of the sequence 5'-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3' (SEQ ID NO:4) reported by Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992) was synthesized. This oligonucleotide was labeled with digoxigenin using a DIG DNA labeling kit (Boehringer Mannheim) and used as a probe.
Using the probe thus obtained, E. coli carrying a plasmid containing the polyester synthase gene was isolated by colony hybridization from the genomic DNA library from Pseudomonas sp.
When Alcaligenes eutrophus PHB-4 (DSM541) and Pseudomonas putida GPp104 (both of which are strains deficient in an ability to produce polyester) were transformed by the conjugation transfer method with the plasmid containing the polyester synthase gene, both the strains had a reverse ability to produce polyester and showed complementarity.
By recovering the plasmid from the E. coli, a DNA fragment containing the polyester synthase gene was obtained.
The nucleotide sequence of a PstI-Xbal fragment from this fragment was determined by the Sanger method.
As a result, the nucleotide sequence of the 1.8 kbp fragment shown in SEQ ID NO:3 was determined.
By further examining homology to this nucleotide sequence, the polyester synthase gene containing the nucleotide sequence (1680 bp) of SEQ ID NO:2 could be identified in this 1.8 kbp nucleotide sequence. The amino acid sequence encoded by SEQ ID NO:2 is shown in SEQ ID NO:1.
It should be understood that insofar as a protein containing the amino acid sequence of SEQ ID NO:1 or a sequence where in said amino acid sequence, one or more amino acids are deleted, replaced or added has polyester synthase activity, the gene (SEQ ID NO:2 or 3) containing DNA coding for said protein falls under the scope of the polyester synthase gene of the present invention.
Mutations such as deletion, replacement, addition etc. can be induced in the amino acid sequence or nucleotide sequence by the known site-direct mutagenesis method (e.g. Transfomer™ Site-Directed Mutagenesis Kit available from Toyobo).
(2) Preparation of E. coli transformant
The 1.8 kb PstI-Xbal fragment containing the polyester synthase gene was ligated into the XbaI, PstI site of plasmid vector pBluescript II KS+. The resulting recombinant vector was transformed by the calcium chloride method into Escherichia coli DH5α. The resulting transformant was designated Escherichia coli PX18. By extracting the plasmid from this transformant, the 1.8 kb PstI-Xbal fragment containing the polyester synthase gene can be easily obtained. Escherichia coli PX18 has been deposited as FERM BP-6297 with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan.
According to the present invention, there are provided a gene coding for polyester synthase, a recombinant vector containing the gene, and a transformant transformed with the vector. The gene of the present invention codes for a polyester synthase using monomers having a wide range of carbon atoms as the substrate, so it is useful in preparing copolymer polyesters having various physical properties.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 4 - - - - (2) INFORMATION FOR SEQ ID NO: 1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 559 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1: - - Met Ser Asn Lys Asn Ser Asp Asp Leu Asn Ar - #g Gln Ala Ser Glu Asn 1 5 - # 10 - # 15 - - Thr Leu Gly Leu Asn Pro Val Ile Gly Leu Ar - #g Gly Lys Asp Leu Leu 20 - # 25 - # 30 - - Thr Ser Ala Arg Met Val Leu Thr Gln Ala Il - #e Lys Gln Pro Ile His 35 - # 40 - # 45 - - Ser Val Lys His Val Ala His Phe Gly Ile Gl - #u Leu Lys Asn Val Met50 - # 55 - # 60 - - Phe Gly Lys Ser Lys Leu Gln Pro Glu Ser As - #p Asp Arg Arg Phe Asn 65 - #70 - #75 - #80 - - Asp Pro Ala Trp Ser Gln Asn Pro Leu Tyr Ly - #s Arg Tyr Leu Gln Thr 85 - # 90 - # 95 - - Tyr Leu Ala Trp Arg Lys Glu Leu His Asp Tr - #p Ile Gly Asn Ser Lys 100 - # 105 - # 110 - - Leu Ser Glu Gln Asp Ile Asn Arg Ala His Ph - #e Val Ile Thr Leu Met 115 - # 120 - # 125 - - Thr Glu Ala Met Ala Pro Thr Asn Ser Ala Al - #a Asn Pro Ala Ala Val130 - # 135 - # 140 - - Lys Arg Phe Phe Glu Thr Gly Gly Lys Ser Le - #u Leu Asp Gly Leu Thr 145 1 - #50 1 - #55 1 -#60 - - His Leu Ala Lys Asp Leu Val Asn Asn Gly Gl - #y Met Pro Ser GlnVal 165 - # 170 - # 175 - - Asp Met Gly Ala Phe Glu Val Gly Lys Ser Le - #u Gly Thr Thr Glu Gly 180 - # 185 - # 190 - - Ala Val Val Phe Arg Asn Asp Val Leu Glu Le - #u Ile Gln Tyr Arg Pro 195 - # 200 - # 205 - - Thr Thr Glu Gln Val His Glu Arg Pro Leu Le - #u Val Val Pro Pro Gln210 - # 215 - # 220 - - Ile Asn Lys Phe Tyr Val Phe Asp Leu Ser Pr - #o Asp Lys Ser Leu Ala 225 2 - #30 2 - #35 2 -#40 - - Arg Phe Cys Leu Ser Asn Asn Gln Gln Thr Ph - #e Ile Val Ser TrpArg 245 - # 250 - # 255 - - Asn Pro Thr Lys Ala Gln Arg Glu Trp Gly Le - #u Ser Thr Tyr Ile Asp 260 - # 265 - # 270 - - Ala Leu Lys Glu Ala Val Asp Val Val Ser Al - #a Ile Thr Gly Ser Lys 275 - # 280 - # 285 - - Asp Ile Asn Met Leu Gly Ala Cys Ser Gly Gl - #y Ile Thr Cys Thr Ala290 - # 295 - # 300 - - Leu Leu Gly His Tyr Ala Ala Leu Gly Glu Ly - #s Lys Val Asn Ala Leu 305 3 - #10 3 - #15 3 -#20 - - Thr Leu Leu Val Ser Val Leu Asp Thr Thr Le - #u Asp Ser Gln ValAla 325 - # 330 - # 335 - - Leu Phe Val Asp Glu Lys Thr Leu Glu Ala Al - #a Lys Arg His Ser Tyr 340 - # 345 - # 350 - - Gln Ala Gly Val Leu Glu Gly Arg Asp Met Al - #a Lys Val Phe Ala Trp 355 - # 360 - # 365 - - Met Arg Pro Asn Asp Leu Ile Trp Asn Tyr Tr - #p Val Asn Asn Tyr Leu370 - # 375 - # 380 - - Leu Gly Asn Glu Pro Pro Val Phe Asp Ile Le - #u Phe Trp Asn Asn Asp 385 3 - #90 3 - #95 4 -#00 - - Thr Thr Arg Leu Pro Ala Ala Phe His Gly As - #p Leu Ile Glu MetPhe 405 - # 410 - # 415 - - Lys Asn Asn Pro Leu Val Arg Ala Asn Ala Le - #u Glu Val Ser Gly Thr 420 - # 425 - # 430 - - Pro Ile Asp Leu Lys Gln Val Thr Ala Asp Il - #e Tyr Ser Leu Ala Gly 435 - # 440 - # 445 - - Thr Asn Asp His Ile Thr Pro Trp Lys Ser Cy - #s Tyr Lys Ser Ala Gln450 - # 455 - # 460 - - Leu Phe Gly Gly Lys Val Glu Phe Val Leu Se - #r Ser Ser Gly His Ile 465 4 - #70 4 - #75 4 -#80 - - Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Ly - #s Ser Arg Tyr MetThr 485 - # 490 - # 495 - - Ser Thr Asp Met Pro Ala Thr Ala Asn Glu Tr - #p Gln Glu Asn Ser Thr 500 - # 505 - # 510 - - Lys His Thr Asp Ser Trp Trp Leu His Trp Gl - #n Ala Trp Gln Ala Glu 515 - # 520 - # 525 - - Arg Ser Gly Lys Leu Lys Lys Ser Pro Thr Se - #r Leu Gly Asn Lys Ala530 - # 535 - # 540 - - Tyr Pro Ser Gly Glu Ala Ala Pro Gly Thr Ty - #r Val His Glu Arg 545 5 - #50 5 - #55 - - - - (2) INFORMATION FOR SEQ ID NO: 2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1680 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2: - - ATGAGTAACA AGAATAGCGA TGACTTGAAT CGTCAAGCCT CGGAAAACAC CT -#TGGGGCTT 60 - - AACCCTGTCA TCGGCCTGCG TGGAAAAGAT CTGCTGACTT CTGCCCGAAT GG -#TTTTAACC 120 - - CAAGCCATCA AACAACCCAT TCACAGCGTC AAGCACGTCG CGCATTTTGG CA -#TCGAGCTG 180 - - AAGAACGTGA TGTTTGGCAA ATCGAAGCTG CAACCGGAAA GCGATGACCG TC -#GTTTCAAC 240 - - GACCCCGCCT GGAGTCAGAA CCCACTCTAC AAACGTTATC TACAAACCTA CC -#TGGCGTGG 300 - - CGCAAGGAAC TCCACGACTG GATCGGCAAC AGCAAACTGT CCGAACAGGA CA -#TCAATCGC 360 - - GCTCACTTCG TGATCACCCT GATGACCGAA GCCATGGCCC CGACCAACAG TG -#CGGCCAAT 420 - - CCGGCGGCGG TCAAACGCTT CTTCGAAACC GGCGGTAAAA GCCTGCTCGA CG -#GCCTCACA 480 - - CATCTGGCCA AGGACCTGGT AAACAACGGC GGCATGCCGA GCCAGGTGGA CA -#TGGGCGCT 540 - - TTCGAAGTCG GCAAGAGTCT GGGGACGACT GAAGGTGCAG TGGTTTTCCG CA -#ACGACGTC 600 - - CTCGAATTGA TCCAGTACCG GCCGACCACC GAACAGGTGC ATGAGCGACC GC -#TGCTGGTG 660 - - GTCCCACCGC AGATCAACAA GTTTTATGTG TTTGACCTGA GCCCGGATAA AA -#GCCTGGCG 720 - - CGCTTCTGCC TGAGCAACAA CCAGCAAACC TTTATCGTCA GCTGGCGCAA CC -#CGACCAAG 780 - - GCCCAGCGTG AGTGGGGTCT GTCGACTTAC ATCGATGCGC TCAAAGAAGC CG -#TCGACGTA 840 - - GTTTCCGCCA TCACCGGCAG CAAAGACATC AACATGCTCG GCGCCTGCTC CG -#GTGGCATT 900 - - ACCTGCACCG CGCTGCTGGG TCACTACGCC GCTCTCGGCG AGAAGAAGGT CA -#ATGCCCTG 960 - - ACCCTTTTGG TCAGCGTGCT CGACACCACC CTCGACTCCC AGGTTGCACT GT -#TCGTCGAT 1020 - - GAGAAAACCC TGGAAGCTGC CAAGCGTCAC TCGTATCAGG CCGGCGTGCT GG -#AAGGCCGC 1080 - - GACATGGCCA AAGTCTTCGC CTGGATGCGC CCTAACGACC TGATCTGGAA CT -#ACTGGGTC 1140 - - AACAACTACC TGCTGGGTAA CGAGCCACCG GTCTTCGACA TTCTTTTCTG GA -#ACAACGAC 1200 - - ACCACCCGGT TGCCTGCTGC GTTCCACGGC GATCTGATCG AAATGTTCAA AA -#ATAACCCA 1260 - - CTGGTGCGCG CCAATGCACT CGAAGTGAGC GGCACGCCGA TCGACCTCAA AC -#AGGTCACT 1320 - - GCCGACATCT ACTCCCTGGC CGGCACCAAC GATCACATCA CGCCCTGGAA GT -#CTTGCTAC 1380 - - AAGTCGGCGC AACTGTTCGG TGGCAAGGTC GAATTCGTGC TGTCCAGCAG TG -#GGCATATC 1440 - - CAGAGCATTC TGAACCCGCC GGGCAATCCG AAATCACGTT ACATGACCAG CA -#CCGACATG 1500 - - CCAGCCACCG CCAACGAGTG GCAAGAAAAC TCAACCAAGC ACACCGACTC CT -#GGTGGCTG 1560 - - CACTGGCAGG CCTGGCAGGC CGAGCGCTCG GGCAAACTGA AAAAGTCCCC GA -#CCAGCCTG 1620 - - GGCAACAAGG CCTATCCGTC AGGAGAAGCC GCGCCGGGCA CGTATGTGCA TG -#AACGTTAA 1680 - - - - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1826 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3: - - CTGCAGTGCT CTCTGAACTA GAAAGCAACG TTGTGCAATT AACGGTCACC CG -#AGCAGTAG 60 - - TACCTGGCGG TTGCTGTGTG ACTACACAGC TGGTCCCGGT ACTCGTCTCA GG -#ACAATGGA 120 - - GCGTCGTAGA TGAGTAACAA GAATAGCGAT GACTTGAATC GTCAAGCCTC GG -#AAAACACC 180 - - TTGGGGCTTA ACCCTGTCAT CGGCCTGCGT GGAAAAGATC TGCTGACTTC TG -#CCCGAATG 240 - - GTTTTAACCC AAGCCATCAA ACAACCCATT CACAGCGTCA AGCACGTCGC GC -#ATTTTGGC 300 - - ATCGAGCTGA AGAACGTGAT GTTTGGCAAA TCGAAGCTGC AACCGGAAAG CG -#ATGACCGT 360 - - CGTTTCAACG ACCCCGCCTG GAGTCAGAAC CCACTCTACA AACGTTATCT AC -#AAACCTAC 420 - - CTGGCGTGGC GCAAGGAACT CCACGACTGG ATCGGCAACA GCAAACTGTC CG -#AACAGGAC 480 - - ATCAATCGCG CTCACTTCGT GATCACCCTG ATGACCGAAG CCATGGCCCC GA -#CCAACAGT 540 - - GCGGCCAATC CGGCGGCGGT CAAACGCTTC TTCGAAACCG GCGGTAAAAG CC -#TGCTCGAC 600 - - GGCCTCACAC ATCTGGCCAA GGACCTGGTA AACAACGGCG GCATGCCGAG CC -#AGGTGGAC 660 - - ATGGGCGCTT TCGAAGTCGG CAAGAGTCTG GGGACGACTG AAGGTGCAGT GG -#TTTTCCGC 720 - - AACGACGTCC TCGAATTGAT CCAGTACCGG CCGACCACCG AACAGGTGCA TG -#AGCGACCG 780 - - CTGCTGGTGG TCCCACCGCA GATCAACAAG TTTTATGTGT TTGACCTGAG CC -#CGGATAAA 840 - - AGCCTGGCGC GCTTCTGCCT GAGCAACAAC CAGCAAACCT TTATCGTCAG CT -#GGCGCAAC 900 - - CCGACCAAGG CCCAGCGTGA GTGGGGTCTG TCGACTTACA TCGATGCGCT CA -#AAGAAGCC 960 - - GTCGACGTAG TTTCCGCCAT CACCGGCAGC AAAGACATCA ACATGCTCGG CG -#CCTGCTCC 1020 - - GGTGGCATTA CCTGCACCGC GCTGCTGGGT CACTACGCCG CTCTCGGCGA GA -#AGAAGGTC 1080 - - AATGCCCTGA CCCTTTTGGT CAGCGTGCTC GACACCACCC TCGACTCCCA GG -#TTGCACTG 1140 - - TTCGTCGATG AGAAAACCCT GGAAGCTGCC AAGCGTCACT CGTATCAGGC CG -#GCGTGCTG 1200 - - GAAGGCCGCG ACATGGCCAA AGTCTTCGCC TGGATGCGCC CTAACGACCT GA -#TCTGGAAC 1260 - - TACTGGGTCA ACAACTACCT GCTGGGTAAC GAGCCACCGG TCTTCGACAT TC -#TTTTCTGG 1320 - - AACAACGACA CCACCCGGTT GCCTGCTGCG TTCCACGGCG ATCTGATCGA AA -#TGTTCAAA 1380 - - AATAACCCAC TGGTGCGCGC CAATGCACTC GAAGTGAGCG GCACGCCGAT CG -#ACCTCAAA 1440 - - CAGGTCACTG CCGACATCTA CTCCCTGGCC GGCACCAACG ATCACATCAC GC -#CCTGGAAG 1500 - - TCTTGCTACA AGTCGGCGCA ACTGTTCGGT GGCAAGGTCG AATTCGTGCT GT -#CCAGCAGT 1560 - - GGGCATATCC AGAGCATTCT GAACCCGCCG GGCAATCCGA AATCACGTTA CA -#TGACCAGC 1620 - - ACCGACATGC CAGCCACCGC CAACGAGTGG CAAGAAAACT CAACCAAGCA CA -#CCGACTCC 1680 - - TGGTGGCTGC ACTGGCAGGC CTGGCAGGCC GAGCGCTCGG GCAAACTGAA AA -#AGTCCCCG 1740 - - ACCAGCCTGG GCAACAAGGC CTATCCGTCA GGAGAAGCCG CGCCGGGCAC GT -#ATGTGCAT 1800 - - GAACGTTAAG TTGTAGGCAG TCTAGA - # - # 1826 - - - - (2) INFORMATION FOR SEQ ID NO: 4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: / - #desc = "synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4: - - CCSCAGATCA ACAAGTTYTA SGAC - # - # 24__________________________________________________________________________
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