CN100552018C - Myxobacteria specific plasmid and application thereof - Google Patents
Myxobacteria specific plasmid and application thereof Download PDFInfo
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- CN100552018C CN100552018C CNB2006100687847A CN200610068784A CN100552018C CN 100552018 C CN100552018 C CN 100552018C CN B2006100687847 A CNB2006100687847 A CN B2006100687847A CN 200610068784 A CN200610068784 A CN 200610068784A CN 100552018 C CN100552018 C CN 100552018C
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Abstract
The invention discloses a strain myxococcus fulvus (Myxococcus fulvus) 124B02 bacterial strain (CCTCC M206081).Also disclose a kind of plasmid pMX1 that derives from myxococcus fulvus (Myxococcus fulvus) 124B02 simultaneously, it has the nucleotide sequence of SEQ ID NO.1.Recombinant vectors or recombinant plasmid by plasmid pMX1 provides and makes up can transform or engage slime bacteria, especially myxococcus, are used for genetic analysis of slime bacteria resource and genetically engineered applied research.
Description
Technical field
The present invention relates to a strain myxococcus fulvus, a kind of proprietary plasmid that derives from described slime bacteria, contain segmental recombinant vectors of above-mentioned plasmid or recombinant plasmid, by microorganism and these application of microorganism of above-mentioned plasmid and derivative or recombinant vectors or recombinant plasmid transformed or joint, and the application of this plasmid and derivative, recombinant vectors and recombinant plasmid.
Background technology
Slime bacteria (myxobacteria) is the gram-negative unicellular rod-shaped bacterium of a class, the δ branch that belongs to the bacteroid door, mainly be present in various places soil, phytophagous animal muck, bark and the septic trees, having complicated many cells behavior and form generating process, is the high prokaryotic organism of a class.Can be divided into two suborders, four sections, 13 genus, i.e. Angiococcus, Myxococcus, Corallococcus, Anaeromyxobacter, Archangium, Cystobacter, Melittangium, Stigmatella, Haploangium, Chondromyces, Polyangium, Sorangium, Nannocystis according to its morphological feature.Though slime bacteria belongs to bacterium in phylogenetic systematics, the signal conduction mode between the cell that relates in its differentiation and development process is more near eukaryote.At the research of slime bacteria many cells form generation molecular regulation, will be all significant to the molecular regulation of being familiar with procaryotic cytodifferentiation and growing, understand the evolutionary process of life, Eukaryotic cell behavior and differentiation and development etc.Simultaneously slime bacteria can also be synthesized the secondary metabolite of many complexity, wherein much has antimycotic, antibacterium and anti-tumor activity, as bioactive moleculess such as soraphen, epothilones.At present, from slime bacteria, found the basic structure more than 100 kinds and the derivative of 600 number of chemical structures, accounted for 3.5% of microbe-derived sum, by the quantity of finding biologically active substance, slime bacteria only comes after actinomycetes and the genus bacillus, is the important medicine source bacterium of a class.Therefore slime bacteria has scientific research value and application and development prospect.
Though, in slime bacteria, set up a series of genetic operating systems successively by the research of decades, better be familiar with and understand this important resource bacterium, never find in the slime bacteria to exist can be outside karyomit(e) the plasmid DNA molecule of self-replicating.Therefore use genetic manipulation method commonly used at present (transduce, transform, engage) the external source fragment is imported slime bacteria, all need exogenous origin gene integrator be gone into karyomit(e) by specific site (transposon, homologous recombination arm), part as host genome is duplicated, and again its proterties or product is showed or expresses.Such operation will transform the process of expressing has undoubtedly increased this process of homologous recombination, and transformation efficiency is reduced.But since in slime bacteria, never find can be outside karyomit(e) the plasmid DNA molecule of self-replicating, therefore the expression of foreign gene in slime bacteria is a difficult problem always, and this has limited theoretical investigation and the genetically engineered exploitation of this class important microbe resource bacterium to a great extent.Relevant report has, the epothilone PKS/NRPS/PKS gene cluster electricity that is used for synthetic epothilones in sorangium cellulosum (Sorangium cellulosum) So ce90 source is transformed into yellow myxococcus (Myxococcusxanthus) DZ1, be integrated into chromosomal devRS district by the homologous recombination arm, come heterogenous expression epothilone natural product (Antimicrob.Agents Chemother.2002,46,2772-2778).
Though people also attempt the gene fragment with the slime bacteria source, import that some present genetic backgrounds are clearer, study in the bacterium that is easy to genetic manipulation or express, as intestinal bacteria (E.coli), self colour streptomycete (Streptomycescoelicolor).But the difference between bacterial classification often makes operation become very loaded down with trivial details, and adding as need to be the promotor of host cell identification, is transformed into the codon etc. that the host is easy to discern utilization, some mosaic genes of the pattern of wants.Because the characteristic of the high GC% content of slime bacteria, often make the mosaic gene instability, the heterologous host cell is sheared reorganization to its gene fragment, and the demolition purpose gene is eliminated the GC% difference in the gene.And the promoter sequence in the slime bacteria also has the characteristics of high GC%, often can not be by effectively identification and utilizing of heterologous host.
Therefore, for the such class important resource bacterium of slime bacteria, the genetic operating system of a cover plasmid method of being set up by himself naturally occurring plasmid DNA molecule and deutero-recombinant vectors or recombinant plasmid will be significant for genetic analysis and the applied research of this bacterium.
Summary of the invention
One of purpose of the present invention provides a strain myxococcus fulvus (Myxococcus fulvus) and mutant or varient.
Myxococcus fulvus of the present invention is myxococcus fulvus (Myxococcus fulvus) 124B02 CCTCC M206081.
Myxococcus fulvus of the present invention (Myxococcus fulvus) 124B02 is separated to from field, Changchun, Chinese Jilin, and Gram-negative is shaft-like, can grow in CTT, VY-2 etc. is usually used in cultivating the substratum of slime bacteria, can form sporophore.16s rDNA sequential analysis and myxococcus fulvus have 99% homology, and therefore deciding it is myxococcus fulvus.This bacterial strain is preserved in Chinese typical culture collection center (Wuhan University, Chinese Wuhan) on August 26th, 2006, and the preservation center is numbered: CCTCC M 206081.
Another object of the present invention provides a kind of isolating from above-mentioned myxococcus fulvus (Myxococcus fulvus) 124B02, but the plasmid DNA molecule of self-replicating outside karyomit(e), and it is built into the recombinant vectors or the recombinant plasmid that can be used for expression alien gene, be used for transforming or engaging into microorganism, slime bacteria particularly, thus better research and develop the slime bacteria resource.
The topology structure of plasmid DNA molecule can be for linear or annular.Use the total DNA extraction process and the alkaline lysis of extracting linear plasmid and circular plasmids to operate respectively, detect by pulsed field gel electrophoresis and the gel electrophoresis of plain agar sugar.
Slime bacteria Pseudomonas after testing comprises Myxococcus (Myxococcus), coral Coccus (Corallococcus), heap capsule Pseudomonas (Sorangium) etc.
Myxobacteria specific plasmid of the present invention is the plasmid pMX1 that derives from myxococcus fulvus (Myxococcus fulvus) 124B02, the double-stranded plasmid DNA molecule of a kind of annular, and it has the nucleotide sequence of SEQ ID NO.1, is made up of 18,634 Nucleotide.
Plasmid pMX1 of the present invention comprises restricted zymogram as shown in Figure 1.
The genes encoding of above-mentioned plasmid pMX1 is read frame, and (open reading frame orf) sees the following form.
Wherein, there are the essential functional zone of plasmid self-replicating in the 10953-13980 dna sequencing fragment of the SEQ ID NO.1 at pMX1.13 and pMX1.14 place.The dependency of these two genes encoding reading frames and plasmid DNA molecule self-replicating yet there are no report.
So every all or part of nucleotide sequence that comprises SEQ ID NO.1 and/or its complementary strand; and the plasmid DNA molecule or derivatives thereof of similar substantially with it nucleotide sequence, and the plasmid DNA molecule or derivatives thereof that comprises identical continuous 20 the base pair nucleotide segments of the partial sequence of 20 base pairs of successive in SEQ ID NO.1 and/or its complementary nucleotide sequence is protected by the present invention all.
Certainly,, contain the microorganism of recombinant vectors and conversion or the joint gained of plasmid pMX1, contain the microorganism of recombinant plasmid and conversion or the joint gained of pMX1, protected by the present invention by the microorganism of plasmid pMX1 conversion or joint gained.Wherein, described microorganism preferably is meant myxococcus or/and intestinal bacteria.
Detect through experiment, the plasmid DNA molecule electricity that comprises the 10953-13980 dna sequencing fragment in the described nucleotide sequence of SEQ ID NO.1 is transformed among yellow myxococcus (Myxococcus xanthus) DZ1, can be outside karyomit(e) self-replicating, and can extract by the method for alkaline lysis and to obtain the cyclic plasmid dna molecular.Therefore the 10953-13980 dna sequencing fragment among the SEQ ID NO.1 contains the essential functional zone of plasmid DNA molecule self-replicating in the slime bacteria.
Because plasmid pMX1 provides the necessary functional zone of plasmid self-replicating in slime bacteria, therefore, what it provided in assurance duplicates under the complete condition in essential functional zone, other function DNA fragments to wherein introducing external source just can constitute the recombinant vectors or the recombinant plasmid that can transform or engage into slime bacteria.Can guarantee that this duplicates under the complete situation in essential functional zone, be introduced in effective resistance selection gene in the various slime bacterias, just can constitute the pUC pUC of genetic manipulations such as being applicable to slime bacteria conversion or joint.Can also introduce the plasmid replication district of other bacteriums in addition on this basis, plasmid replication district as intestinal bacteria or streptomycete, and be suitable for the marker gene of in these bacteriums, screening, as resistance or lacZ selection markers gene, just can constitute the multiple shuttle vectors of operating in different strains and the slime bacteria that is applicable to, thereby make things convenient for the amplification of recombinant plasmid, and in slime bacteria, carry out genetic analysis and applied research.
Therefore proprietary plasmid and derivative thereof by the slime bacteria source of the present invention carry the recombinant vectors of the essential functional zone of this proprietary plasmid self-replicating or microorganism and these application of microorganism of recombinant plasmid and conversion or joint gained and protected by the present invention.
Myxobacteria specific plasmid of the present invention and derivative thereof; and carry the recombinant vectors of the essential functional zone of this proprietary plasmid self-replicating or recombinant plasmid at organic sphere, preferably the genetic analysis in slime bacteria field and genetically engineered applied research also should be subjected to protection of the present invention.
Description of drawings
A strain myxococcus fulvus provided by the invention (Myxococcus fulvus) 124B02, be preserved in Chinese representative microbial DSMZ on August 26th, 2006, the preservation address: Wuhan City, Hubei Province Wuhan University, postcode: 430072, its deposit number is CCTCC M 206081.
Fig. 1 is the restriction enzyme site collection of illustrative plates of plasmid pMX1 of the present invention.
Fig. 2 is a shuttle vectors pZJY7 structure collection of illustrative plates.
Embodiment
The separation and Culture of embodiment 1. myxococcus fulvuses (Myxococcus fulvus) 124B02 CCTCC M 206081
Preparation slime bacteria enrichment medium WCX[CaCl
20.15%, agar 1.5%, cycloheximide 0.5% (the sterilization back adds), KOH transfers to pH7.0, prepares with distilled water].On the WCX flat board that solidifies, smear coli somatic alive substrate as slime bacteria.The soil sample that to gather from field, Changchun, Chinese Jilin is sprinkling upon on the thalline equably.The sporophore of micrurgy picking myxococcus, purifying obtains pure bacterial strain 124B02.
Embodiment 2. extracts cyclic plasmid from slime bacteria
The slime bacteria that will detect is seeded in 3ml CTT[Casitone 1%, MgSO
48mM, TrisHCl (pH7.6) 10mM, potassium phosphate (pH7.6) 1mM, pH7.6] or VY-2 (Baker yeast 0.5%, VB
120.5mg/L, CaCl
20.1%, MgSO
47H
2O 0.05%, in liquid nutrient medium pH7.2), and in 30 ℃ of jolting 200rpm cultivations, growth 60-72h.Centrifugal collection thalline, add 500ul TE25S[sucrose 10.3%, TrisHCl (pH8.0) 25mM, EDTA (pH8.0) 25mM] and N,O-Diacetylmuramidase (final concentration 2mg/ml), the suspension thalline, 37 ℃ of temperature are bathed 1h, put upside down mixing frequently, add 250ul alkaline lysis liquid (NaOH 0.3M, SDS 2%), put upside down mixing 4-6 time, 55 ℃ of temperature are bathed 30min, add 250ul water-saturated phenol/chloroform/primary isoamyl alcohol (25: 24: 1) and put upside down mixing, the centrifuging and taking supernatant liquor adds the saturated phenol/chloroform of isopyknic Tris/primary isoamyl alcohol (25: 24: 1) extracting albumen, the centrifuging and taking supernatant, reuse the saturated phenol/chloroform of isopyknic Tris/primary isoamyl alcohol (25: 24: 1) extracting albumen, do not have tangible egg white layer until the interface.The centrifuging and taking supernatant liquor adds 3M NaAc and isopyknic isopropanol precipitating DNA0.5-1h of 1/10 volume, the centrifugal supernatant liquor of abandoning, 70% washing with alcohol precipitation, vacuum-drying.DNA is dissolved in 20ul TE[TrisHCl (pH8.0) 10mM, EDTA (pH8.0) 1mM], plain agar sugar detected through gel electrophoresis.
Embodiment 3. extracts the wire plasmid from slime bacteria
The slime bacteria that will detect is seeded in the liquid nutrient medium of 3ml CTT or VY-2, and cultivates in 30 ℃ of jolting 200rpm, growth 60-72h.Centrifugal collection thalline, add 500ul TE25S and N,O-Diacetylmuramidase (final concentration 2mg/ml), the suspension thalline, 37 ℃ of temperature are bathed 1h, put upside down mixing frequently, add SDS (final concentration 1%) and protein K (final concentration 50ug/ml), 37 ℃ of temperature are bathed 1h, add the saturated phenol/chloroform of isopyknic Tris/primary isoamyl alcohol (25: 24: 1) extracting albumen, the centrifuging and taking supernatant, reuse the saturated phenol/chloroform of isopyknic Tris/primary isoamyl alcohol (25: 24: 1) extracting albumen, do not have tangible egg white layer until the interface.The centrifuging and taking supernatant liquor adds 3M NaAc and isopyknic isopropanol precipitating DNA 0.5-1h of 1/10 volume, the centrifugal supernatant liquor of abandoning, 70% washing with alcohol precipitation, vacuum-drying.DNA is dissolved in 20ul TE, and pulsed field gel electrophoresis detects.
The subclone order-checking of embodiment 4.pMX1
Use the method for embodiment 2, from myxococcus fulvus (Myxococcus fulvus) 124B02, separate having obtained the double-stranded plasmid pMX1 of ring-type.A large amount of preparation thalline, extract plasmid, cutting glue reclaims, Xba I enzyme is cut pMX1, the endonuclease bamhi mixture is connected to the pSP72 carrier of using Xba I and Alkaline Phosphatase (CIAP) to handle, Transformed E .coli DH 5 α obtain subclone, and the subclone of selecting the different sizes of contained plasmid fragment checks order.
The evaluation of the essential functional zone of embodiment 5.pMX1 plasmid self-replicating
Make up plasmid pZJY1.Insert a kalamycin resistance gene (aminoglycoside-3 '-O-phosphotransferase gene) in the Hind of plasmid pSP72 III site, obtain pZJY1 from plasmid ColE I.Using different restriction enzymes to carry out enzyme the subclone that obtains among the embodiment 4 cuts, obtain the dna fragmentation of different zones among the SEQ ID NO.1, it is connected into pZJY1, and Transformed E .coli DH 5 α obtain containing the transformant of pMX1 different zones dna fragmentation, extract plasmid, electricity transforms yellow myxococcus (M.xanthus) DZ1, observes conversion results, the purifying transformant, method according to embodiment 2 is extracted plasmid, electrophoresis detection.The transformant of plasmid can be detected, the essential functional zone of pMX1 plasmid self-replicating must be contained in its plasmid.Find that through a series of tests the dna fragmentation that is positioned at the 10953-13980 dna sequence dna place of SEQ ID NO.1 contains the essential functional zone of pMX1 plasmid self-replicating.
The structure of embodiment 6. intestinal bacteria-myxococcus shuttle vectors pZJY7
Sal I site with the Sal I fragment at one section 10953-13980 dna sequence dna place that is positioned at SEQ ID NO.1 is inserted plasmid pZJY1 obtains shuttle vectors pZJY7 (see figure 2).This plasmid has the essential functional zone pMX1 ori (the 10953-13980 dna sequence dna of SEQ ID NO.1) of plasmid self-replicating of pMX1, can the mode with autonomously replicating plasmid exist in M.xanthus DZ1; The plasmid replication functional zone pSP72 ori that has pSP72 can increase by massive duplication in E.coli; Have kalamycin resistance gene (aminoglycoside-3 '-O-phosphotransferasegene), as the resistance selectable marker gene among the M.xanthus DZ1; (β-lactamase) is as the resistance selectable marker gene among the E.coli to have ampicillin resistance gene; Have the XbaI-BamH I-Kpn I-Sac I-EcoR I-Cla I-EcoR V multiple clone site that can be used for inserting foreign gene.Therefore pZJY7 is an effective intestinal bacteria-myxococcus shuttle vectors, but transformed into escherichia coli and myxococcus, and clonal expression foreign gene therein.
Sequence table
＜110〉Shandong University, Shanghai Inst. Of Life Science, Cas Plate Physiology Environmental Research Institute
＜120〉Myxobacteria specific plasmid and application thereof
<141>2006-8-22
<160>1
<210>1
<211>18634
<212>DNA
＜213〉myxococcus fulvus (Myxococcus fulvus)
＜221〉derive from the DNA of the plasmid pMX1 of myxococcus fulvus (Myxococcus fulvus) 124B02 CCTCC M 206081
<222>(1)…(18634)
<400>1
ctagagggcg?ctcccgtcga?gggggtgccc?tcttgtcgtt?ttggggtggg?cctcgagcgc 60
gggcgccagg?tggagctggt?ggagctgctc?gagcacctgg?tcggccagga?ggacctggtg 120
gaggccctcg?cgggccgggc?ctcgagcgcg?ggcgccaggt?ggagctggtg?gagctgctcg 180
agcacctggt?cggccaggag?gacctggtgg?aggccctcgc?gggccgggcc?tcgagcacgg 240
gcgcctggtg?gagctgctcg?agcacctggt?cggccaggag?gacctggtgg?aggtcctcgc 300
gggccgggcc?tcgagcgcgg?gcgccaggtg?gagctggtgg?agctgctcga?gcacctggtc 360
ggccaggagg?acctggtgga?ggtcctcgcg?ggccgggcct?cgagcacggg?cgcctggtgg 420
agctgctcga?gcacctggtc?ggccaggagg?acctggtgga?ggccctcgcg?ggccgggcct 480
cgagcacggg?cgcctggtgg?agctgctcga?gcacctggtc?ggccaggagg?acctggtgga 540
ggtcctcgcg?ggccgagcct?cgagcgcggg?cacctgcgcg?aaatgcgcgc?ggctgcgcga 600
aatgcccgag?accccctcgg?agcgaccatt?acgcctagct?ctcttttcgg?cgcggttttg 660
agaccttgca?tctcgttgat?gacggcggtg?cggacgtcag?tgcctcaacc?gctcccctcc 720
gaggacggga?ccgcgcgccc?atagaggccg?gggcgacgcc?gcgccgccgt?caccaacacc 780
acctttacgc?cgaggcgagg?cgatgcacga?cgagcttgga?ggacaggttg?cgcagcgtga 840
cgacgccccc?aagtcccggc?gtgcgaaacg?cgggcgcccc?gcgagcaacc?cggcgcgctg 900
gaagcgcacc?tatcgcgaga?tgctgttccg?cgctggcgac?agcacccgcg?aaatcgcggc 960
ggcgaatgca?tgggcgaggg?aacacggggt?cgagctgaat?ccgctggccg?tgctcgcgga?1020
ggaaggcgcg?aagtcgtttg?ctgcgcgggg?tggtgcgcct?gccgaggcgt?ccgcgcctgc?1080
cgaggcgtcc?gagagcgcct?gggatgccga?agccgcgagg?gctgaagctg?cggcgactgg?1140
tgcggatgct?gcccccgtcg?agggttccgc?gccttccagt?gcgccggccg?agggttccgc?1200
gccggctgag?ggcgccccgt?ccgccgagta?cgtgacgcca?gcggacccga?gcgcgttccc?1260
ggacatggct?ccgggtggcg?gtgcgcccga?ggcgaaagcc?gagccggcgg?ggccgcgctt?1320
catgggcgag?cgccccgacc?tggtcgaggg?atgcgcgcgc?ggtccgttgt?ttgtgctcgg 1380
ggcgttggcg?gaaatcacca?agggcagcct?tgttgacctg?acgcgccctg?tggagcggac 1440
gcttttcgcg?gggacgcccg?ttgagcgcaa?ggttcaggcg?gaccccgtaa?cgcgcctgtc 1500
ggagctggcg?ggcgttgcgc?tcgcgcgccg?agtcgaggcg?agcgcgaaag?tcatgggagg 1560
cgagggcggc?ggaaagccgt?ccgtggcgtg?ggagatggtt?ccgctcgcgc?tcgcgttcgc 1620
gggtgcggtc?gcagtcccgg?gtgcgggtct?cctgtcgggc?gcggcccgtg?ctgtcggcgg 1680
gtggttcaag?caaggcgcca?tcggcgcggc?tggtggcgtg?tcgaggctgt?ttgctcggcg 1740
gagtgcaggg?tgaggacggg?catctgcaag?gcgtgtcggc?aacctctgcc?cgatgaagtg 1800
cgaaaggtcg?agcggttcac?gcggccgtgg?atgccgcgcc?cctatgagaa?cgtgatgctg 1860
ttggggatga?caggaagcgg?gaagtccttc?ctgttcaaga?actggctcgc?gtacatgcag 1920
gccaacggtg?ccgcgtgctt?cgtgctcgac?gttggagacg?agtacagccg?caagggcaat 1980
gaccggggcg?gaacgctgcg?actcggaccg?ttgcggacgc?gcatgaccgt?gcctgaattt 2040
ttggcggcgg?tggagcgctc?aagcctgttc?gtgcgtcacg?cccgcgcgtc?ggtggccatt 2100
gtgccaacgg?tgcatgacgc?gggcgaggca?ctggccgagc?acatccggcc?tgtgctgcga 2160
atcctctacg?ggcgagggcg?gtgcgtaatc?gggtttgacg?agctgcaaaa?gtacggaccg 2220
agcctcacgg?cggagctgta?ccgagctgcc?ggtgagctgg?ggaaagacgg?ggtgatgccc 2280
ttcttccttt?cgcagtaccc?gggcggtgtc?ccggagattg?ttcgcaagca?ggttgggacc 2340
tgcgtcgcga?gcgaaatcgg?taaggcgacg?gaccggcgaa?tgctcgctgc?tgattttggc 2400
gacccgttcg?ccaacgcgct?cggcactccc?agggcgcccg?atgaccggac?cttccacgtc 2460
ggattttccc?gggcgcgatt?cgcgtccatc?ccaacgcggt?agcgaggtga?ggcacatggc 2520
cgacgagaag?aagcaggaga?acgcggagag?ctggggcaag?aacgccatga?ttttcggcgc 2580
gggcatggtg?acgggttcga?ccctgaccgt?cgtggcgggc?cacttctggc?cgaaggtgcc 2640
ggtgcccccc?gcgctcgcga?acgcgctcgg?ggcggacgcg?gcgaagaagt?agcgcgcgct 2700
caccatcgcg?cggcggtgag?cgtcgcgcac?cacgcacaac?acagacagga?caacgcatgc 2760
cgaacctcaa?caccacggtc?gcattcagga?agggtctcca?gcagttcgag?aaggacgtcg 2820
ggttccaggt?gggccagatg?gtggcccccg?ccgggaccgt?gacggacgcc?gtctgcatgg 2880
agttccagtg?cagcgtctcc?acgacgtcag?cggttggcgc?gctgacgatg?gtggagcggc 2940
tccaggtgct?cagcaagatt?cgcgcgtcct?tcgcgcagat?tcttggcgag?gacgccggcg 3000
ggctccgtct?ggaccccatc?cagggccagt?cgctcgacaa?catccgtctg?gatgccatcc 3060
gtctcctggg?gctcgacatg?gacgggctcg?aggacaacac?gacgggcctc?gcgaaggcgt 3120
tcgcggtggg?ctccaaccag?gtcacgttca?aggtgttcct?gccggtcggg?catctcgaga 3180
aggtggccga?gtcggcctac?ttcacgggcc?tgtcgccgga?acagctcctg?gactgcgagc 3240
tgacggtgac?gcgggacgtg?gaccccttcc?aggcggtcaa?gtccgcgctg?acgtcgacca 3300
tcaaggtcct?cttcgccccc?gggacgaaga?aggccgaggc?gcgccggatt?gggctcatgc 3360
cccactgccg?gtccatcacc?aacgcggagt?ccgacacgct?ggccacgccg?gaaggcttgg 3420
tgctggactt?cagccacatg?gcgccgatgc?aggacaagga?ggtcaaggcg?atgtccgtgc 3480
gcgtgggaaa?cgtgctcgtg?acggatgtcc?cgtcgacgcc?cgaacatgtg?tatgcggact 3540
tcctgcggaa?gttccccagc?atcagcgcgg?cggagaagac?gctcaccgcc?acgcgtacgc 3600
ccatcttcct?ggccgagccg?aacgtgatga?cccgcatgca?cgcgggcagg?gtggaagcca 3660
aggtgcagga?gcgcaccgcg?gaatgggacg?ggcgcgtgct?ctacgtgccg?ctccatgacc 3720
acgcccaggt?tttcgcgctg?gtgcgtacgt?acgcgaagcg?catcgagtcg?gggcgtcacc 3780
tgctcgcggt?gaatacggcc?atgtacgagg?gactcgacat?cgaggaccgt?ctgttgccct 3840
acgtggggat?gacggtcttc?ctcaacacgg?aggaaggctt?ctcggacttc?ccgggcatct 3900
actgcaagca?gggtggagag?ccctacgtca?cggtgcccga?gcaccgtctc?cgccaggcgg 3960
cggttcgggt?ggcggacgcg?atgcgctcga?cgaccacgca?ccccaacggg?aaccgcgacc 4020
tggtcaagtc?cgtggtcaac?gacgaggcgc?gctgggtgcc?cggcgcggtg?acgcacggtg 4080
atggtctccg?tgtctcgagc?aaggtccggc?aggacgtcca?ggcgctcatc?cgcgacgccg 4140
ccatcaagta?cgccgagacg?ctcggcaaga?cggacgcgga?gaaggcggcg?gcggcccggc 4200
tgctcggcag?cgcgttctag?ttcgtcgaga?tgttgggcgg?ggacgcgtcg?tgcgtccctg 4260
cccgcttcac?gcgtaatccc?tcccaaggtg?tctcgtgcct?ctcttcgtgt?ttggcgggct 4320
tgcgctcgcg?agcatcggtg?ttgctgcgcg?cattcgtgcg?acgtcccgtg?ctcgaggtgc 4380
ctcgagcgcg?ccgacgagca?caccgacaac?gacggccgag?actgccccgg?cgcaaaaggg 4440
ggaggtttcc?ggccagaagc?ccacgccggt?tgttgatgag?gtctccgctc?cccctccgcc 4500
ccctgcgcca?gctccgcccc?ctgcgccagc?tccggagcct?ccgccagctc?cagctccgca 4560
agcaccgccc?ccgccccctc?cgccccctcc?gccgatgccc?cctccgccgg?ctccgagcgg 4620
gacgggcgcc?accgtggcga?atggtggagc?tgctcttgct?gccgtgctcg?cggcgacgcc 4680
ggccgcgccg?ctggcgccgt?tcatcgcgat?tttcacgcgc?atcatgggcg?gctgggctga 4740
cgagaaagag?cggtcgcgcg?tcgcgcagag?gtacttgcgc?gatacgcaga?acatcgctct 4800
ccagcaggcc?cgttatgagg?ggtactgcgt?ggtctacaac?cagggcattg?cgctctggaa 4860
cgcgggccag?atttccgccg?ctcaggcgaa?gtttgcagcc?gccgaggcgg?ccggcgaaag 4920
caactggcga?aaggtggcgc?agcacatcga?cgacacggaa?gtggcgtcgt?accgggcatg 4980
gatggccaag?gcgccgtccg?acgcgtccgc?gtcgaagctg?gatgaccacc?tgtgggacgg 5040
atggttgctg?ggtgaggccg?gaatcacggg?ccagccgaac?acccgcgagt?tcaacacggg 5100
gactgcgggc?ggctgggtcc?aactccggcg?catgggtggc?gcggtgcggt?tcgctccgcg 5160
cccgggacat?gcggattacg?aggggacggt?gattccgccg?ctcacccccg?aggtgattgc 5220
gcggatggag?gctgcgcgcc?tcgagcgtga?gcggcggaag?ttcgtcgagg?agtcctcggg 5280
cctctcgcct?ggtggctacc?tgacgggcgt?gaaggccgac?aagcgcaacg?gcatgtatga 5340
ggaggatttc?ggccttgagg?agcctgtcat?ttacgagcct?ccgcgagcgc?ccgcgcctcc 5400
cccggctccc?gccccgcccc?ctccaccagc?tccaccagct?ccgcctgtgg?tggcgcctcc 5460
gccgaaagcc?cccgcgcctc?cgccaggagt?gccagcggac?cgcaagggtc?gtgttctcga 5520
gttcgagtga?ggaacggaca?tgtacgttgc?gattgttgga?gtgagcgcgt?tcaccatcgg 5580
ggccgcgctg?gcgctcgccg?cgtcgaatcg?ctgggcgcct?ggtgcccagt?cctcggcaac 5640
gccgccggcc?gggccggact?ggcgttccgc?cctgcctccc?cccgcgcctg?gtgccgctgg 5700
ccaggccgag?gccggcgcgg?cgaaacccgc?gcctggtgct?gtcgctgacc?aggccgtgag 5760
cggcgcggag?aagcccgcgc?ctggcgccac?ggagggcctc?tgggcgggtc?tccccggcag 5820
cgcggcgaaa?cccgcgcctg?gcgccacgga?gggcctctgg?gcgggtctcc?ctggcgaggc 5880
tgggggattc?gcgggcggag?cggcgaaacc?cgcgcctggc?gccactgaga?gcctctgggc 5940
ggggttgccg?gactcggatg?ggccggtgcc?ccctcccgct?ccccccgagt?cgggcctgtg 6000
ggcagggcat?ccggacgatg?gactgccgac?caaggcgcgg?cgcggcggtg?tgtacgagtc 6060
gccgccgacg?ctgccgtatt?caacccccac?catggaccgg?tgacacatgg?cgaaggacga 6120
cgagacgagt?agcaacacga?cggcgttggt?tgtgggcgga?gtcctcgcgc?tgggcgcaat 6180
tggagttggc?gcggcggtgc?tcttgcgcgg?tggcaagggt?gacgacgcga?cggccaagct 6240
caccgcgcag?cttgaggcgg?cggagagggc?cgcgaaggcg?gccgaggacg?cgcggagcgc 6300
ggatgcgctg?cgcgcggctc?aggccgaggt?ggagcgcgcc?aaggccgcgt?tggcggctca 6360
ggcccttgcg?gctcaggccg?aggcggagcg?cgcccgtgcc?gctgcggctg?ctgcggcggc 6420
cacgcctccc?gcgcagtacg?cgcccgcgcc?gcccgcgccg?gctgcccctg?ctgcgggtgg 6480
cggtggcctc?tttggtgcgc?tgcccggaat?cctcggtggc?ctggctggca?tcgcggaggc 6540
catcgggccg?agcctctggg?ggaaccccta?gccatggtcc?tcacgcgcga?gcaggaggaa 6600
accgtggtca?agctgcaagc?ggatgtccgt?tccctcctct?cgcgagttga?ggcggtggcg 6660
cgtccggtcg?acgccgcacg?agacctgttc?gggtggctgc?cggcgacatc?acttcccatc 6720
ccgggcttgg?ggctgctcga?ggcggtgcga?gacgcattcg?gcaacgacgt?gcagcaacag 6780
cacgtgggcg?tgttgcgcga?ggtggaaggg?cgggtgccga?tgtggattgg?ccccgatggc 6840
gcgaagtatc?gctggctcat?gcgtggctac?cgcgatgacg?gcaccgcgta?caggccggag 6900
ctgtggattg?acgagggcaa?cgcgattgcg?gatgcgctcg?cgactgcgct?cggagagacc 6960
cacgacaacg?ccacctggaa?gatttacgcg?gacacggtgg?acaagaccga?agaggagctg 7020
gcgtcagcgg?gccgcgcggt?tgtggaggcc?gccgtagaga?ttcccaaggc?ggtggtcggg 7080
ccctggtcgt?ggaaggtcaa?gctggcgctg?ggcgtgacgg?gcggaatcgc?cgccgttggg 7140
cttggcacca?tcgcctggca?ggccatgcgc?gcaacgcccc?tcgggcttgc?tgctcgaggc 7200
gctggcctgg?tgctgcggac?tggtgcgcag?ccgctcgagc?gcgcggctcg?agctgcgggg 7260
gctcgcgtgg?cgcaagcggt?ggttgaggcc?gaggagtcga?ggcgcacaac?gaagggaggc 7320
aaggcgaaat?gagttttctg?cttatcgctg?gtgggctggc?tgctgcgagc?gtggctggcg 7380
cggttgccgt?caagcggagg?ggagcccgcg?gaatcctctc?gctcgaggag?atgcgcgcgg 7440
tagcgccgaa?cctgcccgag?aacctgcgcg?tcgcgtatct?cccgttcctg?aacgaggcgc 7500
tcgaccgatt?cgacatccga?agctatgcgc?gggtgactgc?gcacctcggg?caaatcctgc 7560
acgagtgcaa?ccagttccga?agcctcgtgg?agttgagcga?cgcgaaggca?tacgaggggc 7620
gttcaaccct?tgggaacacg?aagccgggcg?acggcgagct?gttcaaggga?cggggcgcca 7680
tccagttgac?cgggaggcac?aactacgagc?gcgccgaggc?gttctttggc?gtgccgttca 7740
ctacgcagcc?cgagctggtg?gccacgccca?cgtgggcgtt?tctcacgggg?ggctggttct 7800
ggcggcacgg?ctcctcgagc?gacctgaaca?agctcgcgga?cgcgggtgat?ttcctcacca 7860
tcaccaagcg?aatcaacggc?gggacgaacg?gactcgcgca?ccgcgagagc?tactacgcca 7920
aggctcaggc?cgcgctcgcg?tcgtggaagc?aggtgacgac?atgactcccg?agacgagcca 7980
ggccctcgtg?ttgtgcgcgt?cagccgccgc?tggccagctt?gccgtgcacc?tggccaacag 8040
ccgccaaatc?cggcggaccc?tgcgccgcca?cgcccgacac?ctgcgcgcgc?tggatgcgcg 8100
ccagaagggc?ggtgacgcgc?aggtgcgcga?cgcgacggcg?aaggtggagg?ccctcgataa 8160
gcgcgtgacg?atgttggacg?gcaaggtcgt?gcggagctgg?cagcacatgc?gcggggccct 8220
cttggtggtc?gcccgcgctc?tgtccccgaa?gcagggcacc?taggagacat?caccatggcc 8280
atcatcccca?acgccgtgaa?gacgaagggc?gctggtgtct?tcgtcgctgc?cggcggacgt 8340
ggacgcgggt?tccgcgtcga?gcctgcgggc?gtgtacccgg?ttccgacttt?cccggtcctc 8400
gtgcgcatgc?acgtggacgc?caacgggacc?aagcgggact?tgttgacgtc?aacggccggc 8460
actcgctggc?cggaccacgg?tttcgtcaag?ctcgagctgg?aagccggcga?cgccatggag 8520
tggacggtta?ccgtctacga?gacgaattcg?gactgggacg?agaacccgcc?gacgcagctt 8580
gcgacggtgc?ggaacttgag?ccccggaagc?gtcccgactg?gaccggttgc?cctgtccgtc 8640
gtggcgatgc?gcccgacatt?ccttcccgct?ggcgtgatga?cgggtggcgt?gacggagctg 8700
cggagcacca?caatggccgt?tccgtgcttg?gtggagggac?cgaccggggc?ggcgtggccg 8760
ctcaaggccg?cgtcggatgg?tcggctcttg?gtgtccacgg?tgccgactcc?ctcggttgtc 8820
gcgctcggta?gcggcatcgt?cacgcgcaac?ccgggggctg?ggctcgcgga?ggaagtgctc 8880
ggcgcgccca?tcgacacctc?cgcatatcgc?ggaggggtgg?tgctgcggat?tggtcgcgtg 8940
gcgatgaatg?gcggcaccac?tccgacagtg?cggtgttcga?ttcggagcct?caaacagtcg 9000
atgttcagcg?gggttctgac?cgacttcgtc?gcggcgacct?acccgggagc?ggaagtgagc 9060
acgggtggcg?ctggtgagct?gacggtgggc?ctccagatgc?acagcacgac?gacggatgga 9120
gcgcggacca?tctcgggcta?tgtgatgcgc?gctcagccgg?tgctgacgtt?caccggcggg 9180
cccattgggt?acgaagtgcc?gtgggagctg?tggggcctgc?cgtagcgtcg?tcgcactgac 9240
ccgttccgca?acgaaatgag?gatgtgtccc?cgtggcgcgt?atgtgctcgc?cgcgctgggg 9300
ccttgctgtc?tcacgaccac?cacgacgcgg?cgtggttggt?cggtgtgtca?ctccccgccg 9360
cgcggaggct?gacgaaaatg?gcgaagggca?tcggtagtgc?ggtggtctgg?atcttcggtg 9420
gcgtgtgcgc?gctcttgggc?acgctctcca?cgttccttcc?ggcgggcgcg?ctccagcagg 9480
gcgcggacaa?gggcgctcag?ctctgcgagg?cgaacggctt?caccgcgtac?ccgcgcgggg 9540
atgacggcaa?gccgctcccc?aggcccgacc?aggccgaggg?cgaggccgag?ggccgctaca 9600
tgggcgtgat?gcccctgggg?cggagctggg?tgcccgtcct?ggtgcccgcc?ggtaagggcc 9660
ccagggcctc?gagcaccgtc?gaggacgtgg?gcccggccga?gtagccgcgc?cgaccaggtg 9720
ctcgagctgg?tggagctgcg?ccacgtcgac?caggtgctcg?agctggtgga?gctgcgccac 9780
gtcgaccagg?tgctcgagcc?tggccggcga?gggcctccac?cagctcggcc?tggttgagcc 9840
tccatggcgc?tgaccaggtg?ctcgagcggg?tccgccaggt?cggcctggcc?ccatctcagg 9900
tccgggatgg?ggctcccagc?tagttccgct?cgagtcgggg?attccggccc?tgagtcagag 9960
agcggccgag?ggggcgtaaa?tgatcgaggg?ggccggcccg?gcttgtgaat?agaagctgca?10020
ccttttcgtc?cttgtgcctg?cgcggctcaa?tggcggaaag?gctagtctgc?tcgaccgtgg?10080
agaccgcgcg?aatgacgaca?actgggacta?actgggacgg?acggaacctg?ggtgaccaca?10140
gggtgacgcg?ccgttattgg?cgagcagaga?aagacgctat?gagccagcga?ttcgcgcagg?10200
tcttcggaac?cgaacgccag?ggcgctgcgg?gcgtggccat?tgtgttggat?gaggccggcg?10260
gagaagtgca?actgacgcca?gggtggcgtg?tgacgctatc?cacgctgtcg?gccccgtccc?10320
gcgtaatggt?tgaggtcgaa?gcggctccgg?tgggcgtctc?ccccgcgagc?gtcgcgtcac?10380
tcgcggaagg?gatggatgcc?gcaacctctg?tcatggagtc?cctggtgggc?gaccccgtcg?10440
cgcgcgcaga?gttggcgcgc?tcggcagagg?cggccgagcg?atggcgcttg?gccatgcagt?10500
ggcagggcat?ggcggccgct?cttgctgcca?cgatgcccca?tcctgtttcg?gcgccccctc?10560
ggatgagcaa?ggggcgcgtt?ccggtgcttg?cggcgtgcgt?ggctacctct?gccgttgttt?10620
ccatcctcat?gggggcggtg?ctctcgcaag?agtcgcgcag?tgccgagcgg?ctcccgatgc?10680
cctcggttgt?ctctgtcgct?gatttcgcga?cgatgaccga?tgtggttgga?gccgggaccg 10740
gcgggggagg?cattgcgcat?gctgcgcttg?atgctgccga?cgaagacatt?tcggtcacgg 10800
tccggatgcc?gatgggcatc?tctcgcgaca?tgcccaagga?cccgggcccg?gtgactccag 10860
ggcaggccgt?ccccgacgag?gacgggaaat?gcaaagggtc?ggaggtcgcc?ttgcgcgggg 10920
cctgctggca?caagggcgac?ccgagcgaga?aggtcgacgg?gaagtgcccc?aaggacaagt 10980
gggagtggcg?cgggggctgc?tacgtccctg?tcctcgtggg?cgcgcgccag?gccgttcgcc 11040
cgtagagtga?gcagaaatcg?ttgagttttt?ggctggcctc?aagcaggctg?gcgagcaaat 11100
gttctgagag?ggccgttttg?tccggctccg?gccctcgttg?ggcacgtcct?gagtgttcgc 11160
agcgctctgg?gacgtgttat?agctgtggac?gcgttccatc?cgaggactcc?ttagccggac 11220
gcctcgcaga?acgcgcttag?aggggcccgt?tggcgcgtgc?tgcgaacacg?cgccggtggg 11280
cctctcgttt?ttggtggtcc?gtggacgact?ttcgcagagt?cgtggtcgcg?gttgacggta 11340
gtgacgttgg?tgagcgcgag?gcgctgaaag?cccgctggct?ccagtacgcg?ctagccgtga 11400
tgccgttcga?tgtccgggtc?gcggtggcct?accaggccgt?gcgggagggc?gaggatgcac 11460
tccgcgaggc?gggccggctg?cgcgagcagg?cgtgcgccga?cgcgaaagcc?ctggctgacc 11520
tccgtggtga?tgtcgcggag?ctgcgcgagc?agaacgcgga?gctacgcgag?cggaacgcgg 11580
agctgttccg?cacggcggtg?actgctcagc?gccggctccg?cgagctgcgg?cccggtgact 11640
ccgggttctc?gtttggccgg?gatggtgcac?cgcctgttcg?ccacgtcgcg?aacgaggagc 11700
ggtccgccct?ggtgcctgtc?gtcatggaga?ccccctggcc?ccccgcgccc?attgagggcg 11760
aggacggcca?ggcggagtga?agcgccgagc?gctgacatcg?cagccgacgc?ccgcgcgggc 11820
gctcgtgcac?ggagccggga?gcggagagcg?cccgactctg?cgcgagccgt?cgcgcgcgaa 11880
gagccaaccg?cggaatcggc?gctctgggct?ggatgtggag?ccgagtgccg?ctagcgcgtc 11940
gtcactcccc?gctgaccagg?tggctcagct?ccgcgcctca?accgcgcgac?tccgccgcga 12000
gcagtcgcag?cgccagcgcc?gcgtgaacgc?gggcaaggcc?cggcgcaatg?aggagcggaa 12060
gtgggccgcg?ctcgagctgg?cgacagggac?ggcgctcccc?ctggtcgata?cggtcggctt 12120
ccggccggcc?gaggtgagca?agcccgagac?gtacgtcacg?ctcgaggaca?cgcacgggct 12180
cgagggcctg?gcgcgcggcg?agcgcgcaga?gcaggagctg?gaggcgttcc?gcgcgagcca 12240
tccgtcgcgc?caggtcgcgc?ccccggccgc?gctggcgatc?gctgcgatcc?tctactcgac 12300
gcgcaaggcg?gagggtggtg?cggacgcgcg?ggactgtgcc?tctcttggat?ggcaagtccc 12360
ggtccgtgga?tggtgcgaaa?tgacgggcta?ccgcgagcgg?gccgtgcagg?ccgcgttcgc 12420
gtggctcgct?gacgaggggc?tcgtcagacg?attcggggac?tacgtcacgg?tcaagcacct 12480
cacccggctg?cgacctgctg?gcgagcgtct?ggatctctgg?ctcgacgcca?agggcaaagc 12540
acgccgctgg?gtgcaggtct?acagcgtgac?ttacctgacc?aacgctggcg?cggcctggct 12600
cgagcgcgcg?ggcttcgacc?tggccgagac?caggggccac?ggcgttgcgt?ggcgccgtag 12660
gggcttcctg?gcccgtgtgg?gccgtgccct?ggccggcctg?tggagaaccg?tgcggcggag 12720
gttgggcggt?ggcggctctg?ttgccgtagg?cgccggggct?acgtgcaccc?cttaccctgc 12780
gaagcaggta?gaagtctgtt?caaggcgtgg?tgacgactcc?cagcccactg?cctccctcga 12840
ggagctgata?ggggcggagt?ccccccccca?cgggggggac?gtcactggaa?gcgcatcagc 12900
caagcccgac?ccacgggccg?caaacgccga?gcacgctcgg?gtggagggcg?cgcccgctca 12960
aggcgggcac?gccagcatgg?ggagcgcgag?cgccaagctc?gaaggcagca?agccccctcc 13020
ctcggcactg?gggccggagt?gggagaagaa?gctgggcctc?accgccgaag?gcattgccgc 13080
tgggctggcc?tgggaggggg?gcggcctgct?cgcttacgcc?gaggtcgtgg?accggtgctg 13140
gaccaggacg?acgacgacga?cgcgggcggc?cccctcggac?gttccgagcg?gctcccgcaa 13200
catgttcggg?aggcgccgcg?tgtggcggtg?ccgtggctgt?ggtgccgagt?cccgctacaa 13260
gggcgccacc?gtgcgccacg?cgcaaggatg?taccgctgtg?ggggcctggc?acctggcgcg 13320
gggcccggcc?gtggagacct?ggtctcccgc?gatgacggcg?gagctggcct?ggcccgagga 13380
ctgggaggct?cacccggagc?tgcggccgag?gttgcggcgc?ctgtttgttc?cggtcgcggc 13440
cgagttgacg?cgccggattc?aagctcgagc?ggcggaggcg?cgccgcgccg?ccgaagctga 13500
agaggccgag?cgcagacatc?tggaagtctg?gatgtctaaa?catccagaat?gcggcggtgc 13560
gttccgcgcg?ccggtctatg?actcgtcggt?gtgcgtggcg?gatggccctc?cgaacgcgtg 13620
gggccgcgcg?ccgctttgct?actgtgatgc?gtgcagtcag?aagaggaggg?acatcgaatg 13680
aaaccctgta?gctgggaggc?tcgaggccgg?cttaatcccc?atcgcctagc?gcgcggcgtg 13740
agcgccttca?agccgccggt?cctctctccg?cacggtgcgg?gcgcggggtt?gtcgcccgag 13800
ctgccggaag?aggcccggga?ctcgacgcgg?tggcgggtgg?tcgtgcggtt?cgtgtcgctc 13860
aaggaggcgg?cgcgaaaggc?ccgggtggat?gagtgcacaa?tgcgtcggtg?gtgcactcgc 13920
ggttgggtga?tggccgagcg?cctggcctcc?ggacacggcc?cgtggattgt?tgcggtcgac 13980
atccgagggt?tgcccggtgg?acccgcctga?gaagcctgag?aagaagcgat?gagccgaggc 14040
aagggcgcga?gacgcaagcc?cgctcgaggg?aaggcaaaga?catccagggg?tctaaaagtc 14100
cagaagtcca?gacatctgga?cattcagaag?tctagacatc?tggatgtcca?gatggggttg 14160
tttgtgggtg?agccccgggc?gcggttgccg?gctgctctgt?atctgcgggt?ctcgagcgcc 14220
gagcagacgg?tcgaaaatca?gcgcgagccg?ctcttgcgcc?tggccgaggc?gcggggctgg 14280
gagccgacgc?tgtacgtgga?gacggtgagc?gggagcggca?agaagcgccg?gcccgtgttt 14340
gagcggatga?tgctcgacgc?gaaggcgggc?aaggtccggg?ctgtcgctgt?cactgcgttg 14400
gaccggctgg?ggcggtccct?cggtacggtc?gtggagacgc?tcaacatcct?gcacgcggct 14460
ggggcggtcg?tggtgtccat?tcgagagggt?atcgacacta?cgacgccggg?gcccgtgcag 14520
acgctcttgc?tggggctgtt?tgccgcgctc?ggccaggtgg?agctgcaact?gaaccgcgag 14580
cggacgcaag?cgggcctcga?gcgggctcgg?aagttcggca?cgaagtctgg?gcgccctatc 14640
ggtcgcccgc?gactggatgc?ggagctgctt?cgcaaggcgt?cgctgttggt?ggatggtggt 14700
gaaacggtgc?gccaggccgc?gctgactgcg?ggcgtctcga?agacgacgct?acagaactac 14760
cgacgcgagg?aactggcgaa?gcgggcggag?gccgagcgcc?aggcgcacga?ggacgtggcc 14820
cggatggcgc?gaggggtggc?ccgaaacccc?taggtgttcc?acggggcctg?gaggggccaa 14880
gttacggggg?gttgcgggct?ggtctgcgcc?tggccggaaa?ccggtcggtt?gttgtcccgg 14940
atgttaaagt?gtctagatgt?ctagatgtct?gaaatgcgat?gccgggcccg?acgctcgggg 15000
gccatacctc?ccaggtggta?tgtgtccgat?ggcgggaatc?gtgcttgcct?gacgctcaag 15060
cgaaggggag?gtgccatggc?agagaaggcg?gagcactcgt?cggaagagac?ggagcaggtg 15120
gggcaggaag?gccgggagta?tgtggccgtg?gagttcactg?gtgactccac?tcttacgcac 15180
aggttcacgg?cggctagcga?tgacgcggcg?aggtcgcatg?cgagcgcgcg?ctacatggac 15240
ggctacatcg?acaaggcgca?ctgctcgctt?tggaaggcga?aggattatga?ccaggcgcct 15300
ggccctggcc?tcgacgcgat?ttgcgtcgca?caccgccagc?tgggcgctac?ccgctggcgg 15360
gagacggagc?agcattggag?ggacagcaag?accgccgcgt?ttgcacctag?gacgtgggtg 15420
gcgaagcctc?cggggcccgt?cgatggcgcg?tagaatcggc?ttcccgtgca?cgggccatgg 15480
gccggtctgc?ctggccctgc?ctgacgtgcc?cattactgcg?tgctacgagc?gccagtctga 15540
cgtcgagtca?tcgccggacc?ttgcggcggt?tacgtgcatg?aggtgcaagg?cgttgctccc 15600
agaggagatg?ctgacgccgc?gcccccaccc?actgccccgt?gcgtactggt?gcgaggcgtg 15660
cgacacggac?tactgctctt?ctcaggtggc?gctccagcac?caggaccgga?cgcaccacat 15720
ggtggttgtg?gatgcggagc?tgcatgccga?ggaccccctc?cgctatccgt?tcctccctcc 15780
ggtcaacatg?gagccggggc?cgtcgtcgct?cgcgtacccg?aactagctgc?ggcggatgat 15840
gatgccaacg?ccgtgggggt?gcagaggtct?aggtgtctgg?aagtctggag?gtctggatgt 15900
cccgatgggg?gccagtgtga?tgtcgctggc?ttccaagtcg?ttgatgcggt?ctgccatgcg 15960
gccgctcacg?atgaccaggg?atgttgcgtc?ggtggggcgg?aaggcatccg?cgtccgccgg 16020
gattgactcc?gcgtcgagcc?agtagcgatg?gggcaacagg?cccccgcgcc?ggccgcatac 16080
cgcgcacggt?gttggccgac?ctggccgtgt?gcactcggga?gcgagccggc?cagcgggggc 16140
gagctgaatc?tctgcgatgc?gcattgtggg?ctgtcgtcgg?agctgtggcc?aggcgtgggt 16200
gaggttgagc?ccggccatgg?cgcgcaaagc?atcctctcgg?acaaggaggg?tggtgatgtc 16260
ctcgagcgtg?actggccccc?atttcccatg?ggcagtgcct?acgagggggc?cgaacgcggt 16320
tcccggctcg?agctgcgcgc?ctggtgggca?gtacgggcgc?accagctccc?ggaggcgcga 16380
gaattcgtcc?caggtctcgg?ggcgggcgtc?ctccagctcg?cgggccacgg?ggaggttgga 16440
gaggtctacg?gctgggtact?cgagtccgcc?gccccatgtt?tcgtggcatc?ccgggcagtc 16500
ctcgaggccc?gggagggacc?aacgtcgctc?ggctctaagg?gcgccgctcc?agcggtgctc 16560
agggctgctc?tgtacctcgt?agtaggtcat?ggagggggag?ggggaaggct?ccagctctgc 16620
cagtaggtca?tgggcaggcc?aaagaggttg?tagcgctgaa?tgagctgact?ggccttctcg 16680
aagtgcatcg?ccttggtagc?gtgctgctcg?gtgtccttta?tccactcgtt?ccattcagtg 16740
ttccacggac?cgcgccccgc?ttctcggtgg?agtcgcgcgt?gtacctctgc?gtccaacagc 16800
aaggtccact?cgtgcacgtt?gatgcccttg?ctcttgaagt?accgcgctcg?cgcctgcggg 16860
aagatgtgat?gccgctcgtg?gggtctgcgg?cgccactcct?cgaggcgttc?gcggaacgcg 16920
gcctcgctgg?gaagttcgtc?gggcgcgtac?cagtggaaga?ccatgaccgg?ttcgggcggg 16980
aggctctggg?cgcggccacg?gttgcgcgag?gggttgatgc?gaaccgatgg?cctggcggcg 17040
gatgttgcgc?cgcgtgtgcg?gaccacccgt?gggcctgggt?cgaccaggtc?ctcgagcgcg 17100
aagaggccgc?acacctcgtc?ggtgcacgcg?ggtacgagga?cgggggtgcc?gtcagagtgg 17160
cgcaggtcga?gcgggggaag?gggagtgggt?gcgctcgcac?atccggcgag?cgccaacagg 17220
agggctacgt?gggcgatggg?gcgcatcggg?tgagcgtaga?gggttccgcg?ggtcggttca 17280
cctggaagac?ttgaattgcg?gttttccact?acgtcgcccc?tcttgtgggt?ggggcgtcta 17340
ggtgttagga?agtctggaag?tctagatgtc?tagatgtttg?aatgtttgga?gtgggcgggc 17400
gtcacagggg?ggaacgcgat?gttggagggt?cggaaatgag?cgagtcgcaa?gcgttggttg 17460
agctgttggc?gaggattgag?gaagaggtca?gggcggcaaa?gaccacgatg?ccgacgcgca 17520
gtgacttcga?cgaggtcgag?gacgacgatt?actcagagtt?cgtcgagtgc?cttgggctcg 17580
tgcgcggaaa?cctgctcacg?ctcgagggaa?tggtcgcgca?ggcggtggag?ctggcgaaga 17640
aggcgggccg?atgattgtcg?cggtcgtgtc?ccagaagggg?ggggtctcga?agtcctccct 17700
tacgtgcgcc?atcgcgtggg?agctgcatgc?gcgcggctct?tccgtgctgg?tggtggatgc 17760
ggacccccag?gggaccgtcc?gccagtcggg?gcaggtgtcg?gcggatgagg?gccgggcgat 17820
gcccaccatc?gtcgccatgg?gggaaacgat?gttccgcccc?gaccagcttc?ctcggctggc 17880
gaggtcctat?gaccatgtca?tcgtggacac?gcccggccgt?agcgatgagg?tacagcgggc 17940
cgcgttgatg?gttgcggacc?tcgccctaat?cccgtgtggc?cagtctgcgc?cagatgggtg 18000
ggcgacggtg?cccacggtgg?agctggtgca?gcgcgctcag?cgcgcgcgcc?ctgacctcgc 18060
ggttgctctg?gtgctgacca?tgtgcctccc?gcgcaccgtg?gttggccggt?ctgcgcgtga 18120
tgtcgtggcg?gaggccggta?tccccgtcct?ctccgcctca?accacccatc?ggattgcgtg 18180
gcaagaggcc?ctcgcggcgg?gggtcggtgt?ggcgcagtac?gcgccccatg?acaaggcggc 18240
ggatgaggcg?cgggcggttg?ttgacgagct?gctggtgttg?accggcgaga?agcaggcgcg 18300
gaagcgccgc?acgacgaagc?ggaaggggag?ttgagatggc?tgccaagaag?aagaaaccga 18360
cgtactccat?gcgtgccccg?ccggccgagg?ttgaggcgtt?cgtgcagggc?gagcccgcga 18420
agaagatggg?gcgcactccg?aagagcgctc?cgagcgaccc?tcaacttgag?cggaagcaga 18480
agacgcttta?cctgacggtg?cagattcagc?gccggcttgc?tgtcgaggct?gctcggacgg 18540
gcaaggaaca?ctcccaaatt?gccgaggagt?tgttcgacaa?gtatctcccc?aagtagtgat 18600
gtctagaggt?ctggatgtct?agatgtctag?atgt 18660
Claims (9)
1. a strain myxococcus fulvus (Myxococcus fulvus) 124B02 CCTCC M 206081.
2. a plasmid pMX1 who derives from myxococcus fulvus (Myxococcus fulvus) 124B02 is characterized in that the dna molecular of described plasmid has whole nucleotide sequences of SEQ ID NO.1 and/or its complementary strand at least.
3. plasmid pMX1 as claimed in claim 2 is characterized in that the dna molecular of described plasmid has the nucleotide sequence of SEQ ID NO.1.
4. as the described plasmid pMX1 of one of claim 2～3, it is characterized in that the essential functional zone of described plasmid self-replicating are the dna sequence dnas of 10953-13980 in the described nucleotide sequence of SEQ ID NO.1.
5. contain right require 2 described plasmid pMX1 and can be in slime bacteria the recombinant vectors of self-replicating.
6. recombinant vectors as claimed in claim 5 is characterized in that, it contains the described plasmid DNA of claim 2, contain at least a can be in slime bacteria or other bacterial bodies can self-replicating dna sequence dna, and have a selective marker at least.
7. containing right requires 5 described recombinant vectorss to carry the recombinant plasmid that external source function DNA fragment constitutes.
8. by the described plasmid pMX1 of claim 2 or the described recombinant vectors of claim 5 or the described recombinant plasmid transformed of claim 7 or engage the microorganism of gained.
9. microorganism as claimed in claim 8 is characterized in that, described microorganism is myxococcus or intestinal bacteria.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316520A (en) * | 1999-12-01 | 2001-10-10 | 霍夫曼-拉罗奇有限公司 | Recombination producing of carotenoid especially astaxanthin and its utilized biological material |
| WO2002080846A3 (en) * | 2001-04-03 | 2003-12-04 | Kosan Biosciences Inc | Epothilone derivatives and methods for making and using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316520A (en) * | 1999-12-01 | 2001-10-10 | 霍夫曼-拉罗奇有限公司 | Recombination producing of carotenoid especially astaxanthin and its utilized biological material |
| WO2002080846A3 (en) * | 2001-04-03 | 2003-12-04 | Kosan Biosciences Inc | Epothilone derivatives and methods for making and using the same |
Non-Patent Citations (2)
| Title |
|---|
| 理化因子对橙色粘球菌色素形成的影响. 陈锡时等.沈阳农业大学学报，第30卷第2期. 1999 |
| 理化因子对橙色粘球菌色素形成的影响. 陈锡时等.沈阳农业大学学报，第30卷第2期. 1999 * |
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