CN1944631A - Myxobacteria specific plasmid and its use - Google Patents
Myxobacteria specific plasmid and its use Download PDFInfo
- Publication number
- CN1944631A CN1944631A CN 200610068784 CN200610068784A CN1944631A CN 1944631 A CN1944631 A CN 1944631A CN 200610068784 CN200610068784 CN 200610068784 CN 200610068784 A CN200610068784 A CN 200610068784A CN 1944631 A CN1944631 A CN 1944631A
- Authority
- CN
- China
- Prior art keywords
- plasmid
- pmx1
- myxococcus
- dna
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses one Myxococcus fulvus 124B02 strain in the preservation number of CCTCC M 206081, and one plasmid, pMX1, originated from Myxococcus fulvus 124B02 and with the nucleotide sequence as shown in SEQ ID No. 1. The recombinant carrier or recombinant plasmid via reconstituting pMX1 plasmid may convert or conjugating myxobacteria, especially myxococcus, and is used in the genetic analysis and genetic engineering research of myxobacteria resource.
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.
| Open reading frame | Position (bp) | Size (aa) | The E value | Similarity/function (biology) |
| pMX1.1 pMX1.2 pMX1.3 pMX1.4 pMX1.5 pMX1.6 | 803-1753 1750-2502 2516-2692 2757-4220 4664-5530 6107-6571 | 316 250 58 487 288 154 | 2×10 -8 2×10 -10 2×10 -7 | APEG precursor protein(Xenopus) Unknown Unknown Unknown Ca2+-binding protein(Magnetospirillum) Serine/threonine protein kinase(Streptomyces) |
| pMX1.7 pMX1.8 pMX1.9 pMX1.10 pMX1.11 pMX1.12 pMX1.13 pMX1.14 pMX1.15 pMX1.16 pMX1.17 pMX1.18 pMX1.19c pMX1.20c pMX1.21 pMX1.22 pMX1.23 | 6574-7332 7329-7964 7961-8263 8275-9225 9264-9704 10005-11045 11250-11780 11777-13681 13678-14010 14029-14853 14974-15453 15443-15826 16590-15823 17309-16587 17391-17654 17651-18334 18336-18596 | 252 211 100 316 146 346 176 634 110 274 159 127 255 240 87 227 86 | 9×10 -34 9×10 -6 2×10 -20 7×10 -42 2×10 -19 1×10 -31 | Unknown Lysozyme，putative(Pseudomonas) Unknown Unknown Unknown Protein kinase(Stigmatella) Unknown Unknown Unknown Invertase/recombinase(Pseudomonas) Unknown Unknown Hypothetical protein MXAN_6331(Myxococcus) Hypothetical protein MXAN_6330(Myxococcus) Unknown ParA(Pseudomonas) Unknown |
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 (10)
1. a strain myxococcus fulvus (Myxococcus fulvus) 124B02 CCTCC M 206081.
2. plasmid pMX1 who derives from myxococcus fulvus (Myxococcus fulvus) 124B02, it has the nucleotide sequence of SEQ IDNO.1.
3. plasmid pMX1 as claimed in claim 2 is characterized in that, described plasmid DNA molecule and derivative thereof contain all or part of nucleotide sequence of SEQ ID NO.1 and/or its complementary strand at least, and similar substantially with it nucleotide sequence.
4. plasmid pMX1 as claimed in claim 3, it is characterized in that described plasmid DNA molecule and derivative thereof contain identical continuous 20 the base pair nucleotide segments of partial sequence of 20 base pairs of successive in SEQ ID NO.1 and/or its complementary nucleotide sequence at least.
5. as the described plasmid pMX1 of one of claim 2～4, 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.
6. contain right require 2 described plasmid pMX1 and can be in slime bacteria the recombinant vectors of self-replicating.
7. recombinant vectors as claimed in claim 6, it is characterized in that, it contains the described plasmid DNA of claim 2 or its partial function dna fragmentation, contain at least a can be in slime bacteria and/or other bacterial bodies can self-replicating dna sequence dna, and have a selective marker at least.
8. containing right requires 6 described recombinant vectorss to carry the recombinant plasmid that external source function DNA fragment constitutes.
9. by the described plasmid pMX1 of claim 2 or the described recombinant vectors of claim 6 or the described recombinant plasmid transformed of claim 8 or engage the microorganism of gained.
10. microorganism as claimed in claim 9 is characterized in that, described microorganism is that myxococcus is or/and intestinal bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100687847A CN100552018C (en) | 2006-09-12 | 2006-09-12 | Myxobacteria specific plasmid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100687847A CN100552018C (en) | 2006-09-12 | 2006-09-12 | Myxobacteria specific plasmid and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1944631A true CN1944631A (en) | 2007-04-11 |
| CN100552018C CN100552018C (en) | 2009-10-21 |
Family
ID=38044286
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100687847A Expired - Fee Related CN100552018C (en) | 2006-09-12 | 2006-09-12 | Myxobacteria specific plasmid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100552018C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102132788A (en) * | 2011-03-22 | 2011-07-27 | 淮阴工学院 | Method for preparing and applying myxococcus fulvus probiotics for aquaculture |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2324625C (en) * | 1999-12-01 | 2011-09-27 | F. Hoffmann-La Roche Ag | Recombinant production of carotenoids, particularly astaxanthin |
| WO2002080846A2 (en) * | 2001-04-03 | 2002-10-17 | Kosan Biosciences, Inc. | Epothilone derivatives and methods for making and using the same |
-
2006
- 2006-09-12 CN CNB2006100687847A patent/CN100552018C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102132788A (en) * | 2011-03-22 | 2011-07-27 | 淮阴工学院 | Method for preparing and applying myxococcus fulvus probiotics for aquaculture |
| CN102132788B (en) * | 2011-03-22 | 2012-11-21 | 淮阴工学院 | Method for preparing and applying myxococcus fulvus probiotics for aquaculture |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100552018C (en) | 2009-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1171993C (en) | Novel genes coding for amino acid deacetylases with specificity for N-acetyl-L-phosphinothricin, their isolation and their use | |
| CN1197964C (en) | Directed evolution of microorganisms | |
| CN1513057A (en) | Host microorganisms | |
| CN1172003C (en) | Method for producing N-acetylneuraminic acid | |
| CN1283780C (en) | Method for constructing genetic engineering fungus of monascus with no citrinin | |
| CN1308457C (en) | Method for fermentatively producing s-adenosylmethionine | |
| CN1641019A (en) | Cephalosporin C acylases | |
| CN1262644C (en) | Nitrile hydratase and its coding gene and use | |
| CN1202244C (en) | Novel protein having aspartase activity and gene DNA coding for the same | |
| CN1181203C (en) | Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria | |
| CN1685057A (en) | Methods for producing biological substances in pigment-deficient mutants of bacillus cells | |
| CN1944631A (en) | Myxobacteria specific plasmid and its use | |
| CN1192107C (en) | Novel compound | |
| CN1237174C (en) | Temperature sensitive dtsR genes | |
| CN1527881A (en) | Novel enzymes and genes coding for the same derived from methylophilus methy lot rophus | |
| CN100347302C (en) | Nikkomycin biological synthesis regulating gene and its coded protein and engineering bacterium for expression | |
| CN101063140A (en) | Vancocin biological synthesis gene cluster | |
| CN1186447C (en) | Streptomyces avermitilis gene directing ratio of B2:B1 avermectins | |
| CN1186446C (en) | Streptomyces avermitilis gene directing ratio of B2:B1 avermectins | |
| CN1371423A (en) | Gene coding cyclic lipopeptide acrylase and expression of the same | |
| CN1177045C (en) | Trehalose synthase protein, gene, plasmides, microoganisms, and a process for producing trehalose | |
| CN1066470A (en) | The method and the preparation strain thereof that prepare the monocyte chemotactic factor polypeptide | |
| CN1763086A (en) | ANK protein for controlling fungus colony growth and pathogenicity and its coding gene and utilization | |
| CN1137806A (en) | Genes encoding branched-chain alpha-ketoacid dehydrogenase complex from streptomyces avermitilis | |
| CN1643151A (en) | Gene participating in acetic acid tolerance, acetic acid bacterium bred using the gene, and process for producing vinegar with the use of the acetic acid bacterium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20091021 Termination date: 20130912 |