CN108456703A - A kind of method of heterogenous expression Epothilones - Google Patents

Abstract

The invention discloses a kind of methods of heterogenous expression Epothilones, epothilone gene cluster is introduced in host strain, supplement Epothilones precursor route of synthesis simultaneously, in conjunction with the introducing of related tRNA genes and the insertion of promoter, the expression quantity of Epothilones can be greatly improved, about 4 orders of magnitude of output increased, can reach 8.5mg/L.In addition, the present invention also provides a kind of engineering strain of heterogenous expression Epothilones and the methods for producing Epothilones.

Description

A kind of method of heterogenous expression Epothilones

Technical field

The present invention relates to biological expression fields, and in particular to a kind of method and related gene work of heterogenous expression Epothilones Journey bacterial strain.

Background technology

Epothilones (epothilones) is generated by cellulose heap capsule bacterium Sorangium cellulosum fermentations, is A kind of similar taxol (paclitaxel) has the new type antineoplastic medicine of microtubule stabilization effect.There are 5 kinds angstroms to win now Mycin and derivative are used for clinic, and wherein Patupilone (epothilone B) and KOS-862 (Epothilone D) are fermentation productions Object, Ixabepilone and BMS-310705 are the chemical modification medicines of epothilone B, and ZK-EPO is the fully synthetic drug of chemistry. Ixabepilone is approved by the fda in the United States for breast cancer treatment of late stage in October, 2007.Currently, Epothilones mainly ferments Strain is to originate in strain cellulose heap capsule bacterium, and Natural strains mainly generate ebomycin A and B.

Short more capsule bacterium [Polyangium] the brachysporum DSM 7029 (=K481-B101=ATCC 53080) of spore It detached and obtains from soil from Greece in 1988,7029 bacterial strains of DSM can generate antimycotic and tumour drug and slide bacterium Element, as proteasome inhibitor, it is a kind of NRPS/PKS of heterozygosis (Nonribosomal Peptide Synthetases/polyketide synthases) class The natural products of type.The 7029 strain classification status DSM not yet determines, belongs to Burkholderia mesh by 16S rDNA analyses (Burkholderiales).Bacterium generation fungin heap capsule bacterium more original than Epothilones and pattern slime bacteria Myxococcus xanthus The Myxococcus xanthus speeds of growth are fast, two days visible single bacterium colonies.Studies have found that the bacterial strain can heterogenous expression angstrom it is rich mould Element.

By the prior art, original generation fungin heap capsule bacterium optimized through mutagenesis can reach about 100mg/L, but send out The ferment period is more than 20 days, easy to pollute, and is difficult to be improved space.And by other pattern bacterium such as Escherichia coli, or using not DSM 7029 before transformation, all can not high efficient expression Epothilones, yield can only achieve about 1ug/L.Therefore, exploitation one in need Plant the efficiently method of heterogenous expression Epothilones and the engineering strain for production.

Invention content

In order to overcome the problems, such as that Epothilones expression efficiency is low in the prior art, one aspect of the present invention provides one kind The method of heterogenous expression Epothilones.In a specific embodiment, this method introduces Epothilones gene in host strain Cluster, while supplementing Epothilones precursor route of synthesis.Wherein, epothilone gene cluster derives from cellulose heap capsule bacterium.

Further, host strain belongs to Burkholderia mesh (Burkholderiales).

Further, host strain is 7029 bacterial strains of Burkholderia mesh DSM.

Further, precursor route of synthesis is the route of synthesis of S- methylmalonyl CoAs.

Further, the route of synthesis for supplementing the S- methylmalonyl CoAs is, supplement PCC approach, MatB approach and It is one or more in mutase-isomery enzymatic pathway.

Preferably, the route of synthesis for supplementing the S- methylmalonyl CoAs is supplement PCC approach, MatB approach and change Position enzyme-isomery enzymatic pathway.

Further, PCC approach by add propionyl CoA carboxylase (propionyl-CoA carboxylase) come Supplement；MatB approach is by adding malonyl coenzyme A/methylmalonyl CoA synzyme (malonyl-CoA/ Methylmalonyl-CoA synthetase) it supplements；Mutase-isomery enzymatic pathway is by adding methylmalonyl CoA Isomerase (methylmalonyl-CoA epimerase) supplements.

Further, propionyl CoA carboxylase is the accA1/pccB of streptomyces coelicolor (S.coelicolor) A3 (2) Or pccA/pccB genes.

Further, methylmalonyl CoA isomerase is the epi of streptomyces coelicolor (S.coelicolor) A3 (2) Gene.

Further, malonyl coenzyme A/methylmalonyl CoA synzyme is streptomyces coelicolor (S.coelicolor) the matB genes of A3 (2).

Further, tRNA genes are also introduced in host strain.

Further, tRNA genes are Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti- It is one or more in CTC genes.

Further, Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC gene sources In orange-yellow myxobacter (Myxococcus xanthus) DK 1622.

Further, promoter sequence is added before one or more of epothilone gene cluster gene.

Preferably, in 6 genes of epoA, epoB, epoC, epoD, epoE and epoF in epothilone gene cluster Promoter sequence is added before one or more.

It is further preferred that 6 bases of epoA, epoB, epoC, epoD, epoE and epoF in epothilone gene cluster Promoter sequence is added before each because in.

Further, above-mentioned promoter is PKan.

Further, promoter sequence is added by the splicing again of gene.

Further, spliced using Bxb1 integrase splicings.

Another aspect of the present invention provides a kind of engineering strain of heterogenous expression Epothilones.It is specific at one In embodiment, epothilone gene cluster is introduced in engineering strain, while being supplemented with Epothilones precursor route of synthesis. Wherein, epothilone gene cluster derives from cellulose heap capsule bacterium.

Further, the basic bacterial strain of engineering strain is Burkholderia mesh (Burkholderiales) DSM 7029 Bacterial strain.

Further, the route of synthesis of Epothilones precursor S- methylmalonyl CoAs is supplemented in basic bacterial strain.

Further, the route of synthesis of S- methylmalonyl CoAs includes PCC approach, MatB approach and mutase-isomery Enzymatic pathway.

Further, in basic bacterial strain, by the accA1/ for adding streptomyces coelicolor (S.coelicolor) A3 (2) PccB supplements the PCC approach；Epi genes by adding streptomyces coelicolor (S.coelicolor) A3 (2) supplement displacement Enzyme-isomery enzymatic pathway；MatB genes by adding streptomyces coelicolor (S.coelicolor) A3 (2) supplement matB approach.

Further, tRNA genes are also added in basic bacterial strain.

Further, tRNA genes are Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti- CTC genes.

Further, it is added to promoter sequence before one or more genes of epothilone gene cluster.

Preferably, every in 6 genes of epoA, epoB, epoC, epoD, epoE and epoF in epothilone gene cluster Promoter sequence is all added before one.

Further, by way of splicing again, according to the sequence of epoA, epoB, epoC, epoD, epoE to epoF Splice successively to add the promoter sequence.

Further, promoter PKan.

Further, of the invention one preferably in specific implementation mode, the genetic engineering of heterogenous expression Epothilones Bacterial strain is the more capsule bacterium of short spore (Polyangium brachysporum) MMR11, deposit number CCTCC NO:M2017037, in On January 19th, 2017 is preserved in China typical culture collection administrative center, address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University.

Another aspect of the invention provides a kind of method producing Epothilones.In a specific embodiment, It provides one plant of bacterial strain as described above, ferments at 30 ± 2 DEG C in the fermentation medium.

Further, fermentation medium is CYMG fermentation mediums, and every liter of formula is junket peptone 8g, yeast extract 4g, Magnesium chloride hexahydrate 4.06g, 50% glycerine 10ml, micro- 1ml, sodium acetate 50mg, sodium propionate 100mg, methylmalonic acid 100mg, Cys2 .5mg, serine 5mg, XAD-16 macroporous absorbent resin weight in wet base 1%, adjustment pH 7.0-7.5.

Further, fermentation time is 3 days.

The method of Epothilones heterogenous expression in the present invention, by host strain gene group and epothilone gene cluster Analysis, to the Epothilones precursor S- methylmalonyl CoAs route of synthesis lacked in host strain and suitable Epothilones The Expression elements such as tRNA, the promoter of gene cluster are transformed, to improve the yield of Epothilones.Preferably implementing In mode, Epothilones high efficient expression in 7029 bacterial strains of improved DSM, about 4 orders of magnitude of output increased reach 8.5mg/L is 105 times of the Epothilones for the bacterial strain generation for containing only non-transformation gene cluster, meets industrial production Epothilones Needs.

Description of the drawings

Fig. 1 is that the supplement of precursor S- methylmalonyl CoA route of synthesis in the specific embodiment of the present invention is shown It is intended to.

Fig. 2 is that precursor S- methylmalonyl CoAs route of synthesis and tRNA add in the specific embodiment of the present invention Add rear Epothilones Yield mapping.

Fig. 3 is that the strain MMR1 bacterial strains angstrom containing epothilone gene cluster are rich mould in the specific embodiment of the present invention Plain gene cluster transcript profile peak figure (T is positive-sense strand, and F is antisense strand).

Fig. 4 is that the splicing again of Epothilones synthetic gene cluster and promoter add in the specific embodiment of the present invention Add schematic diagram.

Fig. 5 is the collection of illustrative plates of plasmid pST-BSD-epo in the specific embodiment of the present invention.

Fig. 6 is rich mould after splicing Epothilones biological synthesis gene cluster in the specific embodiment of the present invention again angstrom Plain Yield mapping.

Specific implementation mode

The present invention is further described below with reference to embodiment, it should be understood that the mesh of these embodiments only illustratively , it is not used in and limits the scope of the invention.

The analysis that the present invention passes through genome analysis and epothilone gene cluster to host strain, thus it is speculated that conventional host In bacterial strain the reason of heterogenous expression Epothilones low output, and host strain is transformed for these factors, it is rich to have reached raising angstrom The method of mycin heterogenous expression simultaneously provides related transformation bacterial strain.

7029 strain characteristics of 1.DSM are analyzed and epothilone gene cluster signature analysis

It is not sequenced before the genome of DSM 7029, the gene order-checking by DSM 7029 and analysis, discovery DSM The gene cluster G/C content of 7029 genome G/C content 67.51%, this and Epothilones 56kb is 69.5% close.

7029 genomes of DSM include multiple NRPSs and PKSs, and maximum CDS encodes Nonribosomal Peptide Synthetases (non-ribosomal peptide synthetase) (AAW51_3371), size 32,469bp.And it derives from The NRPS/PKS of the epothilone gene cluster encoding hybrid of Sorangium cellulosum, size 56kb, including 9 PKS Module, 1 NRPS module and 1 P450 oxidizing ferment.Complete epothilone gene cluster includes epoA (sequence such as SEQ ID Shown in No.1), epoB (sequence is as shown in SEQ ID No.2), epoC (sequence is as shown in SEQ ID No.3), (sequence is such as by epoD Shown in SEQ ID No.4), epoE (sequence is as shown in SEQ ID No.5), epoF (sequence is as shown in SEQ ID No.6) and 7 genes of epoK.Wherein, P450 oxidizing ferment (EpoK) can be catalyzed Ai BomeisuC D-shapeds into epoxy construction to Ai BomeisuA B is converted.Only in the case of 6 genes of epoA, epoB, epoC, epoD, epoE and epoF, then epothilones C and D are generated. Epothilone gene cluster from Sorangium cellulosum lacks the necessary promoter sequence expressed in DSM 7029 Row.

When producing Epothilones, the epothilone gene cluster from Sorangium cellulosum can be selected, It can also select the epothilone gene cluster from other strains.

TRNA is analyzed：

The codon preference of tRNA statistics and Epothilones to 7029 bacterial strains of DSM (uses tRNAscan- from the point of view of analyzing SE v1.23 analyses), 7029 bacterial strains of DSM lack 4 tRNA of main expression Epothilones：Arg anti-GCG、Arg Anti-TCG, Gln anti-CTG and Glu anti-CTC.Again by 7029 bacterial strains of DSM and Myxococcus xanthus, The tRNA of Sorangium cellulosum is compared (as shown in table 1), similarly finds that above-mentioned wherein three tRNA are lacked It is few.It is significant for the generation of Epothilones to supplement tRNAs.

TRNA in table 1,7029 strain gene group of M.xanthus DK1622, S.cellulosum So0157-2 and DSM The comparison of type and quantity

-：Missing

Precursor route of synthesis is analyzed：

Epothilone gene cluster is PKS/NRPS type gene clusters, and the synthesis precursor of ebomycin A includes 1 molecule acetate, 4 Molecule S- methylmalonyl CoAs, 4 molecule malonyl coenzyme As, the sources molecule SAM methyl carbon and a molecule cysteine. When epothilone B synthesizes, a molecule malonyl coenzyme A is substituted by methylmalonyl CoA.Wherein, S- methylmalonyl CoAs It is the important synthesis precursor of Epothilones.

S- methylmalonyl CoAs have following route of synthesis：

1) PCC approach：Propionate is by propionyl-CoA synthetase (propionyl-CoA synthetase, prpE, EC: 6.2.1.17 propionyl coenzyme A (propionyl-CoA)) is catalyzed and synthesized, then through propionyl CoA carboxylase (propionyl-CoA Carboxylase, pccA/pccB, EC:6.4.1.3) synthesis S- methylmalonyl CoAs ((2S)-methylmalonyl- CoA)；

2) MatB approach：Substrate methylmalonic acid passes through malonyl coenzyme A/methylmalonyl CoA synzyme (malonyl-CoA/methylmalonyl-CoA synthetase, matB, EC:6.2.1.-) synthesizing methyl malonyl coenzyme A (methylmalonyl-CoA)；

3) mutase-isomerase (mutase-epimerase) approach：Succinyl-coenzyme A (succinyl-CoA) passes through first Base malonyl coenzyme A mutase (methylmalonyl-CoA mutase, mcmA, EC:5.4.99.2 R- methyl-props two) are generated Acyl coenzyme A ((2R)-methylmalonyl-CoA), then by methylmalonyl CoA isomerase (methylmalonyl-CoA Epimerase, epi, 5.1.99.1) generate S- methylmalonyl CoAs ((2S)-methylmalonyl-CoA).

The annotation of 7029 strain gene groups of DSM is compared and is found, 7029 bacterial strains of DSM lack complete S- methylmalonyls Coacetylase route of synthesis does not have PCC approach and mutase-isomery enzymatic pathway.

Based on above-mentioned analysis, consider to start from supplement Epothilones precursor route of synthesis, addition tRNA appropriate and addition Sub- aspect is transformed, to improve the yield of Epothilones.

The present invention is further explained below with reference to specific embodiment.

If operating method in embodiment can be completed without specified otherwise by ordinary skill in the art means.

Restriction enzyme is purchased from Thermo Fisher Scientific；Ex Taq、GC buffer I/II、 PrimerSTAR^TMHS DNA polymerase, λ-EcoT14Marker are purchased from TakaRa- treasured bioengineering (Dalian) limited public affairs Department；DNA Ago-Gels QIAquick Gel Extraction Kit, bacterial genomes extraction agent box are purchased from Shanghai JaRa biology；PCR product recycling examination Agent box is purchased from raw work bioengineering；CloneExpress MultiS、CloneExpress II、Phanta Max Super- Fidelity DNA Polymerase only praise biology purchased from Nanjing promise；XAD-16 (macroporous absorbent resin) is purchased from Shanghai Mo Su sections Learn equipment；Epothilone B is purchased from Toronto Research Chemicals；Ebomycin A, epothilones C, Epothilone D Purchased from Dalian U.S. logical sequence；Hplc grade methanol, chromatographic grade acetonitrile are purchased from Germany MERCK；Ultimate XB-C18,5μm,4.6× 250mm is purchased from scientific and technological (Shanghai) limited liability company of the moon rising sun；Casein peptone is purchased from U.S. company BD；Yeast extract is purchased from OXIOD；Magnesium chloride, glycerine are purchased from raw work bioengineering.

Antibiotic：

Embodiment 1 adds precursor metabolic pathway pathway gene and tRNA to improve the yield of Epothilones

Such as Such analysis, 7029 bacterial strains of DSM lack complete S- methylmalonyl CoAs route of synthesis, do not have PCC Approach and mutase-isomery enzymatic pathway.Accordingly, it is considered to by supplementing the approach of S- methylmalonyl CoAs synthesis to improve angstrom The yield of rich mycin, the approach figure of supplement are as shown in Figure 1.The mode of operation be first build the plasmid containing epo gene clusters, and It is transferred in 7029 bacterial strains of DSM, then plasmid of the structure containing different supplemental passage genes and/tRNA genes, then is transferred to above-mentioned contain Have in the DSM7029 bacterial strains of epo gene clusters, obtains transformation bacterial strain.

Plasmid 1 (gene cluster containing epo) building process is as follows：The libraries cellulose heap capsule bacterium genome So0157-2 are through screening The plasmid Cosmid 10 and Fosmid3B11 arrived is (referring to Allopatric integrations selectively change host transcriptomes,leading to varied expression efficiencies of exotic genes In Myxococcus xanthus, Microb Cell Fact [J], 2015；14:105.), it is respectively provided with Epothilones biology The segments downstream of epoF is assigned at the 38.5kb segments of the preceding part of slave epoA to the epoD of synthetic gene cluster and the rear portion from epoC 34.4kb segments, the two segments have the covering in the regions 6.5kb.Full genome synthesizes 1 (SEQ ID of PCR targeting segments No.7) and segment 2 (SEQ ID No.8) (wherein, p15A replicons, resistant gene and att Post sections are located at fully synthetic piece In section 1 and segment 2), using PCR targeting technologies, reacts, obtain with plasmid Cosmid 10 and Fosmid3B11 respectively Plasmid pZLE21 (site containing attB0 and attP6) and pZLE22 (site containing attB6 and attP15).Finally, pZLE21, PZLE22 and pZLE19 (site containing attB15 and attP0) three integrate enzyme reaction (plasmid pZLE19 and integration using phi BT1 Enzyme reaction refers to Tandem assembly of the epothilone biosynthetic gene cluster by vitro site-specific recombination,Sci Rep.2011；1:141.doi:10.1038/srep00141), Plasmid pZL-epo is obtained, which includes whole Epothilones biological synthesis gene clusters.Wherein, the sites attB0 such as SEQ ID Shown in No.9, the sites attP6 are as shown in SEQ ID No.12, and the sites attB6 are as shown in SEQ ID No.11, and the sites attP15 are such as Shown in SEQ ID No.18

Plasmid pZL-epo containing epothilone gene cluster is transferred to GBred bacterial strains (being purchased from Gene Bridges), is prepared Electricity turns competence, using primer BSD-epo-F/R (as shown in SEQ ID No.71 and 72), with the plasmid pBSD containing transposase (refer to Nucleic Acids Res.2008Oct；36(17):e113.doi:10.1093/nar/gkn499 the plasmid can lead to Cross swivel base and be inserted into DSM7029 genomes) it is template, amplified production electricity is transferred in GBred/pZL-epo, passes through homologous recombination The plasmid pBSD-epo containing transposase and complete epothilone gene cluster is obtained, plasmid 1 is named as.

It is added to from streptomyces coelicolor (S.coelicolor according to the feed path of S- methylmalonyl CoAs A3 (2)) (be purchased from ATCC, number ATCC BAA-471) accA1/pccB or pccA/pccB genes, supplemented with PCC approach；Add The epi genes from streptomyces coelicolor (S.coelicolor A3 (2)) are added, polishing mutase-isomery enzymatic pathway； The matB genes from streptomyces coelicolor (S.coelicolor A3 (2)) are added to, other portion can be expressed by increasing The copy of malonyl-CoA/methylmalonyl-CoA synthetase.

Specifically, using streptomyces coelicolor genome as template, accA1-F/R is (as shown in SEQ ID No.21 and 22) Primer amplification accA1 genes (as shown in SEQ ID No.31), pccA-F/R (as shown in SEQ ID No.23 and 24) are to draw Object has expanded pccA genes (as shown in SEQ ID No.32), with pccB-F/R (as shown in SEQ ID No.25 and 26) for primer PccB genes have been expanded (as shown in SEQ ID No.33)；Expanded for primer with epi-F/R (as shown in SEQ ID No.27 and 28) Epi genes have been increased (as shown in SEQ ID No.34)；With MatB-F/R (as shown in SEQ ID No.29 and 30) for primer amplification MatB genes (as shown in SEQ ID No.35).

Using plasmid pBSD as template, BSD-F/R (as shown in SEQ ID No.19 and 20) is primer amplification carrier segments, profit It is connect with said gene with seamless link technology.

Such as Such analysis, 7029 bacterial strains of DSM lack 4 tRNA of main expression Epothilones：Arg anti-GCG、Arg Anti-TCG, Gln anti-CTG and Glu anti-CTC.Accordingly, it is considered to increasing this 4 tRNA to improve Epothilones Yield.

Full genome has synthesized four tRNA, Arg anti-GCG sequences such as SEQ ID for deriving from orange-yellow myxobacter Shown in No.36, Arg anti-TCG sequences are as shown in SEQ ID No.37, Gln anti-CTG sequences such as SEQ ID No.38 institutes Show, Glu anti-CTC sequences are as shown in SEQ ID No.39, the whole full genome synthesis tRNA sequences such as SEQ of 4 genes Shown in ID No.70, and it is that primer is cloned with tRNA-F/R (as shown in SEQ ID No.40 and 41).

Before each in gene in 5 above-mentioned precursor metabolic pathways of amplification and above-mentioned full genome synthesizes tRNA Whole gene is (when full genome synthesizes, according to Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti- The sequence of CTC synthesizes a whole segment) before plus after promoter PKan (shown in sequence such as SEQ ID No.42), then lead to Seamless link technology is crossed to be building up on the carrier pBSD containing transposase.Wherein, 7 plasmids are constructed, respectively：Plasmid 2 (pccA+pccB), plasmid 3 (pccA+pccB+tRNA), plasmid 4 (accA1+pccB), plasmid 5 (accA1+pccB+tRNA), matter 6 (accA1+pccB+epi+tRNA) of grain, plasmid 7 (accA1+pccB+matB+tRNA), 8 (accA1+pccB+epi+ of plasmid MatB+tRNA), checking influence of the different addition combinations to Epothilones yield.First the plasmid 1 that structure is completed is converted DSM7029 obtains the recombinant bacterium MMR1 containing epothilone gene cluster, then above-mentioned plasmid 2-8 is taken to convert MMR1 respectively, obtains each angstrom Rich mycin expression recombinant bacterium MMR2-8.Specific conversion process is as follows：Take 3 microlitres of plasmids to be transformed that DSM 7029 is added or is transferred to The MMR1 competent cells of epothilone gene cluster, 2mm is added after mixing and shocks by electricity cup, and 2500V carries out electrotransformation, activates 3 hours Tablet of the coating containing kalamycin (Kanamycin) or apramycin (Apramycin) afterwards.After 2 days on tablet picking Positive colony.

The CYMG fermentation mediums of 500ml are selected in the fermentation of Epothilones, are formulated as junket peptone (Casitone) (BD companies) 8g, yeast extract (OXOID companies) 4g, magnesium chloride hexahydrate 4.06g, 50% glycerine 10ml, micro- 1ml, sodium acetate 50mg/L, sodium propionate 100mg/L, methylmalonic acid 100mg/L, Cys2 .5mg/L, serine 5mg/L, XAD-16 macropore Resin weight in wet base 1% is adsorbed, 1L, adjustment pH 7.0-7.5,121 DEG C of moist heat sterilization 20min are added water to.Fermentation temperature is 30 DEG C, is shaken Bed rotating speed is 200rpm, is fermented 3 days.

Above-mentioned trace element is：Tetrahydrate manganese chloride 0.79g, white vitriol 0.15g, five water sulfuric acid are dissolved in 100mL water Copper 0.64g, ferrous sulfate heptahydrate 0.11g, it is spare as mother liquor.

After fermentation, resin is poured into 100 the polished standard screens and is cleaned for several times, addition 25ml methanol after drying, 30 DEG C It is parsed twice, each 12 hours every time.Methanol desorbed solution mixing twice is concentrated, by UHPLC-MS/MS to angstrom rich after filtering The yield of mycin A, B, C and D are quantified.

As shown in Fig. 2, original strain DSM 7029 does not have Epothilones yield.Epothilones base is added in DSM 7029 Because cluster (epo gene clusters) obtains recombinant bacterium MMR1 afterwards, the yield of epothilones C and D reach 61.27 μ g/L and 18.76 μ g/L. PccA/pccB/tRNAs approach is supplemented on the basis of this and obtains bacterial strain MMR3, and total output improves 10%；It supplements on this basis AccA1/pccB and accA1/pccB/tRNAs approach obtains bacterial strain MMR4 and MMR5, and total output doubles, respectively 100% With 130%, the yield of epothilones C and D reach 129.54 μ g/L and 59.35 μ g/L.Supplemented with accA1/pccB/tRNAs Epi genes are supplemented on the basis of approach respectively and MatB genes obtain bacterial strain MMR6 and MMR7, Epothilones yield continues substantially Increase, ebomycin A, B, C and D yield reach 65.57 μ g/L, 58.35 μ g/L, 508.30 μ g/L, 466.40 μ g/L and 19.35 μg/L、3.39μg/L、225.20μg/L、47.33μg/L.It is supplemented when accA1-pccB-tRNAs-epi-matB whole approach Obtain bacterial strain MMR8 afterwards, yield reaches highest, ebomycin A, B, C and D yield reach 45.85 μ g/L, 62.54 μ g/L, 399.12 μ g/L and 1101.03 μ g/L, total output reach 1.6mg/L, are only add epo gene cluster producing strains 20 times.

Embodiment 2epo gene clusters are spliced again improves the yield of Epothilones to add promoter

It is found after carrying out transcript profile sequencing to the strain MMR1 containing epothilone gene cluster, it is each in epothilone gene cluster Gene expression dose is all very low (Fig. 3), and low-level gene expression may be to restrict the another key factor of Epothilones yield.

In view of the foregoing, whole using Bxb1 to improve expression efficiency of the Epothilones synthetic gene cluster in DSM7029 Synthase splicing carries out such as Fig. 4 to 6 genes of epothilone gene cluster epoA, epoB, epoC, epoD, epoE and epoF Shown in splice again.In epothilone gene cluster before each gene, it is all added to promoter, is increased every in gene cluster The expression quantity of one gene.It attempts by adding promoter to improve the yield of Epothilones.

Specifically, iGEM, network address http (are derived from plasmid pSB1A3://parts.igem.org/Part:pSB1A3) For template, epo1A3-F/R (as shown in SEQ ID No.43 and 44) is that primer clones Amp sequences, with (the sources plasmid pSB3K5 In iGEM, network address http://parts.igem.org/Part:PSB3K5) it is template, epo3K5-F/R (such as SEQ ID No.45 Shown in 46) be that primer clones P15A sequences, two segments by seamless connection obtain plasmid p-vector (containing EcoRI, Tetra- restriction enzyme digestion sites of XbaI, SpeI and PciI).It is with the plasmid pZL-epo containing epothilone gene cluster Template, epoA-F/R (as shown in SEQ ID No.47 and 48) are that primer clones epoA genes, epoB-F/R (such as SEQ ID Shown in No.49 and 50) it is that primer clones epoB genes, epoC-F/R (as shown in SEQ ID No.51 and 52) clones for primer EpoC genes, epoF-F/R (as shown in SEQ ID No.57 and 58) is that primer clones epoF genes, with epo-vector-F/R (as shown in SEQ ID No.73 and 74) is primer, and p-vector is template amplification carrier segments, the above gene is passed through seamless The mode of connection clones to obtain pEpoA, pEpoB, pEpoC and pEpoF.

Plasmid pZL-epo containing epothilone gene cluster is transferred to GBred bacterial strains, electricity is prepared and turns competence, with centre Carrier p-vector is template, epoD-F/R (as shown in SEQ ID No.53 and 54) and epoE-F/R (such as SEQ ID No.55 Shown in 56) it is primer, the intermediate carrier linearized fragment containing homology arm is expanded, rear electricity goes to GBred/pZL-epo bacterial strains In, pass through homologous recombination, you can obtain the plasmid pEpoD and pEpoE containing epoD and epoE.

Contain plasmid pEpoA, the pEpoB of Epothilones gene using restriction enzyme EcoRI and XbaI enzyme cutting, PEpoC, pEpoD, pEpoE and pEpoF.Using pBSD as template, primer pKan-F/R is (as shown in SEQ ID No.59 and 60) Primer amplification promoter sequence, PKan promoters carry out double digestion using restriction enzyme EcoRI and SpeI, recycle digestion piece Section.Connect Epothilones gene and PKan promoter fragments after double digestion, you can obtain the Epothilones added with promoter Gene plasmid pPKan-EpoA, pPKan-EpoB, pPKan-EpoC, pPKan-EpoD, pPKan-EpoE and pPKan-EpoF.

Using restriction enzyme EcoRI and XbaI enzyme cutting plasmid pPKan-EpoB, using restriction enzyme EcoRI and SpeI digested plasmid pPKan-EpoA, pPKan-EpoA-PKan-EpoB is obtained after connection.Using restriction enzyme EcoRI and XbaI enzyme cutting plasmid pPKan-EpoF, using restriction enzyme EcoRI and SpeI digested plasmid pPKan-EpoE, after connection To pPKan-EpoE-PKan-EpoF.Using restriction enzyme EcoRI and XbaI enzyme cutting plasmid pPKan-EpoC, limitation is utilized Property restriction endonuclease EcoRI and SpeI digested plasmid pPKan-EpoA-PKan-EpoB, pPKan-EpoA-PKan- is obtained after connection EpoB-pPKan-EpoC.Utilize restriction enzyme EcoRI and XbaI enzyme cutting plasmid pPKan-EpoA-PKan-EpoB- PPKan-EpoC, pPKan-EpoD and pPKan-EpoE-PKan-EpoF are separately connected attB0 (as shown in SEQ ID No.9), AttB13 (as shown in SEQ ID No.15) and the site attB7 (as shown in SEQ ID No.13), obtain plasmid pattB0- PKan-EpoA-PKan-EpoB-pPKan-EpoC, pattB13-PKan-EpoD and pattB7-PKan-EpoE-PKan-EpoF. Using restriction enzyme SpeI and PciI digested plasmid pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC, PattB13-PKan-EpoD and pattB7-PKan-EpoE-PKan-EpoF is separately connected attP13 (such as SEQ ID No.16 institutes Show), attP7 (as shown in SEQ ID No.14) and the site attP15 (as shown in SEQ ID No.18) obtain pattB0- PKan-EpoA-PKan-EpoB-pPKan-EpoC-attP13, pattB13-PKan-EpoD-attP7 and pattB7-PKan- EpoE-PKan-EpoF-attP15。

Full genome synthesizes attP0-ccdB-attB15 segments (as shown in SEQ ID No.61), using composition sequence as template, CcdB-F/R (as shown in SEQ ID No.62 and 63) is primer amplification segment；Using pBSD as template, primer ccdB-vector- F/R (as shown in SEQ ID No.64 and 65) amplified fragments, the two are connected using seamless clone, obtain plasmid pBSD-ccdB.With PBSD-ccdB is template, and primer BSD-ccdB-F/R (as shown in SEQ ID No.66 and 67) is primer amplification segment；With plasmid PST-ccdB is template, and primer ST-F/R (as shown in SEQ ID No.68 and 69) amplified fragments, the two, which uses, to be seamlessly connected, and is obtained To plasmid pST-BSD.

Take 0.5 microlitre of vector plasmid pST-BSD, pattB0-PKan-EpoA-PKan-EpoB-pPKan-EpoC- 2 microlitres of attP13 plasmids, 2 microlitres of pattB13-PKan-EpoD-attP7 plasmids, pattB7-PKan-EpoE-PKan-EpoF- 1.5 microlitres of attP15 plasmids, 1 microlitre of Bxb1 integrases, 30 DEG C reaction 20 hours after, reaction system through high temperature and Proteinase K at It is converted after reason, correct clone is filtered out on antibiotic kanamycins tablet.Correct plasmid pST-BSD-epo collection of illustrative plates is sequenced As shown in Figure 5.

It takes 5 microlitres of electricity of pST-BSD-epo plasmids to go to 7029 bacterial strains of DSM, obtains containing the transformation angstrom added with promoter The recombinant bacterial strain MMR10 of rich mycin gene cluster.Continue electricity on the basis of the bacterial strain of transformation gene cluster to turn to contain accA1-pccB- The plasmid of tRNAs-epi-matB obtains the bacterial strain MMR11 of high yield Epothilones.

It is 30 DEG C that bacterial strain MMR1, MMR10, MMR8, MMR11, which are used 500mL CYMG fermentation mediums, fermentation temperature, is shaken Bed rotating speed is 200rpm, is fermented 3 days.

After fermentation, resin is concentrated in 100 the polished standard screens and is cleaned for several times, addition 25ml methanol after drying, 30 It DEG C is parsed twice, each 12 hours every time.Concentrate methanol desorbed solution mixing twice, after filtering by UHPLC-MS/MS to angstrom The yield of rich mycin C and D are quantified.

As shown in fig. 6,7029 zymotic fluids of DSM do not have Epothilones generation.The Ai Bo not spliced is added in DSM 7029 After mycin gene cluster, the epothilones C of bacterial strain MMR1 and the yield of D reach 61.27 μ g/L and 18.76 μ g/L.When spelling again The epothilone gene cluster of the addition promoter connect is transferred to the generation that Epothilones can be detected in DSM7029, epothilones C Yield with D is that 83.63 μ g/L and 25.38 μ g/L, total amount improve 36%.When tRNAs and accA1-pccB-epi-matB Gene is added containing in the epothilone gene cluster bacterial strain not being transformed, epothilones C and D yield reach 399.12 μ g/L and 1101.03μg/L.When tRNAs and the above precursor related gene add containing splice again and add promoter bacterial strain it Afterwards, yield is substantially improved, and the yield of epothilones C and D are 4721.47 μ g/L and 3812.25 μ g/L, and total output ratio contains only not The bacterial strain of transformation gene cluster improves 105 times.

In conclusion we in Burkholderiales DSM 7029 by adding synthesis S- methylmalonyl coenzyme The approach such as the PCC approach of A, MatB approach, mutase-isomery enzymatic pathway, it is necessary in conjunction with expression epothilone gene cluster TRNAs, promoter engineering make Epothilones high efficient expression in DSM 7029,3 days yield of shaking flask can reach 8.5mg/L, substantially Meet the needs of industrial production Epothilones.

Sequence table

<110>Fudan University

<120>A kind of method of heterogenous expression Epothilones

<160> 74

<170> PatentIn version 3.5

<210> 1

<211> 4266

<212> DNA

<213>Cellulose heap capsule bacterium（Sorangium cellulosum）

<400> 1

atggcggatc gtcccatcga gcgcgcagcc gaagatccga ttgcgatcgt cggagcgggt 60

tgccgtctgc ccggcggcgt gatcgatctg agcgggttct ggacgctcct cgagggctcg 120

cgcgacaccg tcgggcaagt ccccgcagaa cgctgggatg cagcagcgtg gtttgatccc 180

gacctcgatg ccccggggaa gacgcccgtt acgcgcgcat ctttcctgag cgacgtagcc 240

tgcttcgacg cccccttctt cggcatctcg cctcgcgaag cgctgcggat ggaccctgca 300

catcgactct tgctggaggt gtgctgggag gcgctggaga acgccgcgat cgctccatcg 360

gcgctcgtcg gtacggaaac gggagtgttc atcgggatcg gcccgtccga gtatgaggcc 420

gcgctgccgc gagcgacggc gtccgcagag atcgacgctc atggcgggct ggggacgatg 480

cccagcgtcg gagcgggccg aatctcgtat gtcctcgggc tgcgagggcc gtgtgtcgcg 540

gtggatacgg cctattcgtc ctcgctcgtg gccgttcatc tggcctgtca gagcttgcgc 600

tccggggaat gctccacggc cctggctggt ggggtatcgc tgatgttgtc gccgagcacc 660

ctcgtgtggc tctcgaagac ccgcgcgctg gccacggacg gtcgctgcaa ggcgttttcg 720

gcggaggccg atgggttcgg acgaggcgaa gggtgcgccg tcgtggtcct caagcggctc 780

agtggagccc gcgcggacgg cgatcggata ttggcggtga ttcgaggatc cgcgatcaat 840

cacgacggag cgagcagcgg tctgaccgtg ccgaacggga gctcccaaga aatcgtgctg 900

aaacgggccc tggcggacgc aggctgcgcc gcgtcttcgg tgggttatgt cgaggcacac 960

ggcacgggca cgacgcttgg tgaccccatc gaaatccaag cgctgaatgc ggtatacggc 1020

ctcgggcgag acgtcgccac gccgctgctg atcgggtcgg tgaagaccaa ccttggccat 1080

cctgagtatg cgtcggggat cactgggctg ctgaaggtcg tcttggccct tcagcacggg 1140

cagattcctg cgcacctcca cgcgcaggcg ctgaaccccc ggatctcatg gggtgatctt 1200

cggctgaccg tcacgcgcgc ccggacaccg tggccggact ggaatacgcc gcgacgggcg 1260

ggggtgagct cgttcggcat gagcgggacc aacgcgcatg tggtgctgga agaggcgccg 1320

gcggcgacgt gctcaccgcc ggcgccggag cggccggcag agctgctggt gctgtcggca 1380

aggaccgcgg cagccctgga tgcacacgcg gcgcggctgc gcgaccatct ggagacctac 1440

ccttcgcagt gtctgggcga tgtggcgttc agtctggcga cgacgcgcag cgcgatggag 1500

caccggctcg cggtggcggc gacgtcgagc gaggggctgc gggcagccct ggacgctgcg 1560

gcgcagggac agacgccgcc cggtgtggtg cgcggtatcg ccgattcctc acgcggcaag 1620

ctcgcctttc tcttcaccgg acagggggcg cagacgctgg gcatgggccg tgggctgtac 1680

gatgtatggc ccgcgttccg cgaggcgttc gacctgtgcg tgaggctgtt caaccaggag 1740

ctcgatcggc cgctccgcga ggtgatgtgg gccgaaccgg ccagcgtcga cgccgcgctg 1800

ctcgaccaga cagccttcac ccagccggcg ctgttcacct tcgagtatgc gctcgccgcg 1860

ctgtggcggt cgtggggcat agagccggag ttggtcgctg gccatagcat cggtgagctg 1920

gtggctgcct gcgtggcggg cgtgttctcg cttgaggacg cggtgttcct ggtggctgcg 1980

cgcgggcgcc tgatgcaggc gctgccggcc ggcggggcga tggtgtcgat cgcggcgccg 2040

gaggccgatg tggctgctgc ggtggcgccg cacgcagcgt cggtgtcgat cgccgcggtc 2100

aacggtccgg accaggtggt catcgcgggc gccgggcaac ccgtgcatgc gatcgcggcg 2160

gcgatggccg cgcgcggggc gcgaaccaag gcgctccacg tctcgcatgc gttccactca 2220

ccgctcatgg ccccgatgct ggaggcgttc gggcgtgtgg ccgagtcggt gagctaccgg 2280

cggccgtcga tcgtcctggt cagcaatctg agcgggaagg ctggcacaga cgaggtgagc 2340

tcgccgggct attgggtgcg ccacgcgcga gaggtggtgc gcttcgcgga tggagtgaag 2400

gcgctgcacg cggccggtgc gggcaccttc gtcgaggtcg gtccgaaatc gacgctgctc 2460

ggcctggtgc ctgcctgcct gccggacgcc cggccggcgc tgctcgcatc gtcgcgcgct 2520

gggcgtgacg agccagcgac cgtgctcgag gcgctcggcg ggctctgggc cgtcggtggc 2580

ctgttctcct gggccggcct cttcccctca ggggggcggc gggtgccgct gcccacgtac 2640

ccttggcagc gcgagcgcta ctggatcgac acgaaagccg acgacgcggc gcgtggcgac 2700

cgccgtgctc cgggagcggg tcacgacgag gtcgaggagg ggggcgcggt gcgcggcggc 2760

gaccggcgca gcgctcggct cgaccatcca ccgcccgaga gcggacgccg ggagaaggtc 2820

gaggccgccg gcgaccgtcc gttccggctc gagatcgatg agccaggcgt gcttgatcac 2880

ctggtgcttc gggtcacgga gcggcgcgcc cctggtctgg gcgaggtcga gatcgccgtc 2940

gacgcggcgg ggctcagctt caatgatgtc cagctcgcgc tgggcatggt gcccgacgac 3000

ctgccgggaa agcccaaccc tccgctgctg ctcggaggcg agtgcgccgg gcgcatcgtc 3060

gccgtgggcg agggcgtgaa cggccttgtg gtgggccaac cggtcatcgc cctttcggcg 3120

ggagcgtttg ctacccacgt caccacgtcg gctgcgctgg tgctgcctcg gcctcaggcg 3180

ctctcggcga ccgaggcggc cgccatgccc gtcgcgtacc tgacggcatg gtacgcgctc 3240

gacagaatag cccgccttca gccgggggag cgggtgctga tccacgcggc gaccggcggg 3300

gtcggtctcg ccgcggtgca gtgggcgcag cacgtcggag ccgaggtcca tgcgacggcc 3360

ggcacgcccg agaagcgcgc ccacctggag tcgctgggcg tgcggtatgt gagcgattcc 3420

cgctcggacc ggttcgtcgc cgacgtgcgc gcgtggacgg gcggcgaggg agtagacgtc 3480

gtgctcaact cgctttcggg cgagctggtc gacaagagtt tgaatctcct gcgatcgcac 3540

ggccggtttg tggagctcgg caagcgcgac tgttacgcgg ataaccagct cgggctgcgg 3600

ccgttcctgc gcaatctctc cttctcgctg gtggatctcc gggggatgat gctcgagcgg 3660

ccggcgcggg tccgtgcgct cttcgaggag ctcctcggcc tgatcgcggc aggcgtgttc 3720

acccctcccc ccatcgcgac gctcccgatc gctcgtgtcg ccgatgcgtt ccggagcatg 3780

gcgcaggcgc agcatcttgg gaagctcgta ctcacgctgg gtgacccgga cgtccagatc 3840

cgtattccga cccacgcagg cgccggcccg tccaccgggg atcgggacct gctcgacagg 3900

ctcgcgtcag ctgcgccggc cgcgcgcgcg gcggcgctgg aggcgttcct ccgtacgcag 3960

gtctcgcagg tgctgcgcac gcccgaaatc aaggtcggcg cggaggcgct gttcacccgc 4020

ctcggcatgg actcgctcat ggccgtggag ctgcgcaatc gtatcgaggc gagcctcaag 4080

ctgaagctgt cgacgacgtt cctgtccacg tcccccaata tcgccttgtt gacccaaaac 4140

ctgctggatg ctctcgccac agctctctcc ttggagcggg tggcggcgga gaacctacgg 4200

gcaggcgtgc aaagcgactt cgtctcatcg ggcgcagatc aagactggga aatcattgcc 4260

ctatga 4266

<210> 2

<211> 4233

<212> DNA

<213>Cellulose heap capsule bacterium（Sorangium cellulosum）

<400> 2

atgacgatca atcagcttct gaacgagctc gagcaccagg gtatcaagct ggcggccgat 60

ggggagcgcc tccagataca ggcccccaag aacgccctga acccgagcct gctcgctcga 120

atctccgagc acaaaagcac gatcctgacg atgctccgtc agagactccc cgcagagtcc 180

atcgtgcccg ccccagccga gcggcacgtt ccgtttcctc tcacagacat ccaaggatcc 240

tactggctgg gtcggacagg agcgtttacg gtccccagcg ggatccacgc ctatcgcgaa 300

tacgactgtg cggatctcga cgtggcgagg ctgagccgcg cctttcggaa agtcgtcgcg 360

cggcacgaca tgcttcgggc ccacacgctg cccgacatga tgcaggtgat cgagcctaaa 420

gtcgacgccg acatcgagat catcgatctg cgcgggctcg accggagcac gcgggaagcg 480

aggctcgtat cgttgcgaga tgcgatgtcg caccgcatct atgacaccga gcgccctccg 540

ctctatcacg tcgtcgccgt tcggctggac gagcggcaaa cccgtctcgt gctcagcatc 600

gatctcatta acgttgacct aggcagcctg tccatcatct tcaaggactg gctcagcttc 660

tacgaagatc ccgagacctc tctccctgtc ctggagctct cgtaccgcga ctatgtactc 720

gcgctggagt ctcgcaagaa gtctgaggcg catcaacgat cgatggatta ctggaagcgg 780

cgcatcgccg agctcccacc tccgccgatg ctcccgatga aggccgatcc atctaccctg 840

aaggagatcc gcttccggca cacggagcaa tggctgccgt cggactcctg gagtcgattg 900

aagcggcgtg tcggggagcg cgggctgacc ccgacgggcg tcatcctggc tgcattttcc 960

gaggtgatcg ggcgctggag cgcgagcccc cggtttacgc tcaacataac gctcttcaac 1020

cggctacccg tccatccgtg cgtgaacgat atcaccgggg acttcacgtc gatggttctc 1080

ctggacatcg acaccactcg cgacaagagc ttcgaacaac gcgctaagtg tattcaaaag 1140

cagctatggg aggcgatgga tcactgcgac gtgagcggta tcgaggtcca gcgagaggcc 1200

gcccgggtcc tggggatcca acgaggcgca ttgttccccg tagtgctcac gagcgcgctc 1260

aaccagcaag tcgtcggtgt cacctcgctg cagaggctcg gcactccggt gtacaccagc 1320

acgcagactc ctcagctgct gctggatcat cagctctacg agcacgatgg ggacctcgtc 1380

ctcgcgtggg acatcgtcga cggagtgttc ccgcccgacc ttctggacga catgctcgaa 1440

gcgtacgtcg ctcttctccg gcggctcact gaggaaccat ggggtgaaca gatgcgctgt 1500

tcgcttccgc ctgcccagct agaagcgcgg gcgagcgcaa acgagaccaa cgcgctgctg 1560

agcgagcata cgctgcacgg cctgttcgcg gcgcgggtcg agcagctgcc tatgcagctc 1620

gccgtggtgt cggcgcgcaa gacgctcacg tacgaagagc tttcgcgccg ttcgcggcga 1680

tttggcgcgc ggctgcgcga gcagggggca cgcccgaaca cattggtcgc ggtggtgatg 1740

gagaaaggct gggagcaggt tgtcgcggtt ctcgcggtgc tcgagtcagg cgcggcctac 1800

gtgccgatcg atgccgacct accggcggag cgtatccact acctcctcga tcatggtgag 1860

gtaaagctcg tgctgacgca gccatggctg gatggtaaac tgtcatggcc gccggggatc 1920

cagcggctgc tcgtgagcga ggccggcgtc gaaggcgacg gcgaccagct tccgatgatg 1980

cccattcaga caccttcgga tctcgcgtat gtcatctaca cctcgggatc cacagggttg 2040

cccaaggggg tgatgatcga tcatcggggt gccgtcaaca ccatcctgga catcaacgag 2100

cgcttcgaaa tagggcccgg agacagagtg ctggcgctct cctcgctgag cttcgatctc 2160

tcggtctatg atgtgttcgg gatcctggcg gcgggcggta cgatcgtggt gccggacgcg 2220

tccaagctgc gcgatccggc gcattgggca gagttgatcg aacgagagaa ggtgacggtg 2280

tggaactcgg tgccggcgct gatgcggatg ctcgtcgagc attccgaggg tcgccccgat 2340

tcgctcgcta ggtctctgcg gctttcgctg ctgagcggcg actggatccc ggtgggcctg 2400

cctggcgagc tccagaccat caggcccggc gtgtcggtga tcagcctggg cggggccacc 2460

gaagcgtcga tctggtccat cgggtaccca gtgatgaacg tcgatccatc gtgggcgagc 2520

atcccctacg gccgtccgct gcgcaaccag acgttccacg tgctcgatga ggcgctcgaa 2580

ccgcgcccgg tctgggttcc ggggcaactc tacattggcg gggtcggact ggcactgggc 2640

tactggcgcg atgaagagaa gacgcgcaag agcttcctcg tacaccccga gaccggggag 2700

cgcctttaca agaccggcga tctgggccgc tacctgcccg atggaaacat cgagttcatg 2760

gggcgggagg acaaccaaat caagcttcgc ggataccgcg ttgagctcgg ggaaatcgag 2820

gaaacgctca agtcgcatcc gaacgtacgc gacgcggtga ttgtgcccgt cgggaacgac 2880

gcggcgaaca agctccttct agcctatgtg gtcccggaag gcacacggag acgcgctgcc 2940

gagcaggacg cgagcctcaa gaccgagcgg atcgacgcga gagcacacgc cgccgaagcg 3000

gacggcttga gcgacggcga gagggtgcag ttcaagctcg ctcgacacgg actccggagg 3060

gatctggacg gaaagcccgt cgtcgatctg accgggctgg ttccgcggga ggcggggctg 3120

gacgtctacg cgcgtcgccg tagcgtccga acgttcctcg aggccccgat tccatttgtt 3180

gagttcggcc gattcctgag ctgcctgagc agcgtggagc ccgacggcgc ggcccttccc 3240

aaattccgtt atccatcggc tggcagcacg tacccggtgc agacctacgc gtacgccaaa 3300

tccggccgca tcgagggcgt ggacgagggc ttctattatt accacccgtt cgagcaccgt 3360

ttgctgaagg tctccgaaca cgggatcgag cgcggagcgc acgttccgca aaacttcgac 3420

gtgttcgatg aagcggcgtt cggtctcctg ttcgtgggca ggatcgatgc catcgagtcg 3480

ctgtatggat cgttgtcacg agagttctgc ctgctggagg ccggatatat ggcgcagctc 3540

ctgatggagc aggcgccttc ctgcaatatc ggcgtctgcc cggtgggtca attcaatttt 3600

gaacaggttc ggccggttct cgacctgcgg cgttcggacg tttacgtgca cggcatgctg 3660

ggtgggcggg tagacccgcg gcagttccag gtctgtacgc tcggtcagga ttcctcaccg 3720

aggcgcgtca cgacgcgcgg cgcccctcct ggccgcgatc agcacttcgc cgatatcctt 3780

cgcgacttct tgaggaccaa actacccgag tatatggtgc ctacagtctt cgtggagctc 3840

gatgcgttgc cgctgacgtc caacggcaag gtcgatcgta aggccctgcg cgagcggaag 3900

gatacctcat cgccgcggca ttcggagcac acggcgccac gggacgcctt ggaggagatc 3960

ctcgtcgcgg tcgtacggga ggtgctcggg ctggaggtgg tcgggctcca gcagagcttc 4020

gtcgatcttg gtgcgacatc gattcacatc gttcgcatga ggagcctgtt gcagaagagg 4080

ctggataggg agatcgccat caccgagttg ttccagtacc cgaacctcgg ctcgctggca 4140

tccggtttgc gccgagactc gaaagatcta gatcagcgga cgaacatgca ggaccgagtg 4200

gaggcccggc gcaagggcag gagacgtagc taa 4233

<210> 3

<211> 5499

<212> DNA

<213>Cellulose heap capsule bacterium（Sorangium cellulosum）

<400> 3

atggaagaac aagattcctc cgctatcgca gtcatcggca tgtcgggccg atttccgggg 60

gcgcgtaatc tggacgagtt ctggaggaac cttcgagacg gcacggaggc cgtgcagcgc 120

ttctccgagc aggagctcgc ggcgtccgga gtcgaccccg cgctggtgct ggacccgagc 180

tacgtccggg cgggcagcgt gctggaagat gtcgaccggt tcgacgctgc tttcttcggc 240

atcagcccgc gcgaggcaga gctcatggat ccgcagcacc gcatcttcat ggaatgcgcc 300

tgggaggcgc tggagaacgc cggatacgac ccgacggctt acgagggctc tatcggcgtg 360

tacgccggcg ccaacatgag ctcgtacttg acgtcgaacc tccacgagca cccagcgatg 420

atgcggtggc ccggctggtt tcagacgttg atcggcaacg acaaggatta cctcgcgacc 480

cacgtctcct acaggctgaa tctgagaggg ccgagcatct ccgttcaaac tgcctgctcc 540

acctcgctcg tggcggttca cttggcgtgc atgagcctcc tggaccgcga gtgcgacatg 600

gcgctggccg gcgggattac cgtccggatc ccccatcgag ccggctatgt atatgccgag 660

gggggcatct tctctcccga cggccattgc cgggccttcg acgccaaggc gaacggcacg 720

atcatgggca acggctgcgg ggttgtcctc ctgaagccgc tggaccgggc gctctccgat 780

ggtgatcccg tccgcgcggt catccttggg tctgccacaa acaacgacgg agcgaggaag 840

atcgggttca ctgcgcccag tgaggtgggc caggcgcaag cgatcatgga ggcgctggcg 900

ctggcagggg tcgaggctaa cccatcgatc gatttcgcga cccacgggac cggcacgctg 960

ctcggagacg ccatcgagac ggcggcgttg cggcgggtgt tcgatcgcga cgcttcggcc 1020

cggaggtctt gcgcgatcgg ctccgtgaag accggcatcg gacacctcga atcggcggct 1080

ggcatcgccg gtttcatcaa gacggtcttg gcgctggagc accggcagct gccgcccagc 1140

ctgaacttcg agtctcctaa cccatcgatc gatttcgcga gcagcccgtt ctacgtcaat 1200

acctctctta aggattggaa taccggctcg actccgcggc gggccggcgt cagctcgttc 1260

gggatcggcg gcaccaacgc ccatgtcgtg ctggaggaag cacccgcggc gaagcttcct 1320

gccgcggcgc cggcgcgctc tgccgagctc ttcgtcgtct cggccaagag cgcagcggcg 1380

ctggatgccg cggcggaacg gctacgagat catctgcagg cgcaccaggg gctctcgttg 1440

ggcgacgtcg ccttcagcct ggcgacgacg cgcagcccca tggagcaccg gctcgcgatg 1500

gcggcgccgt cgcgcgaggc gttgcgagag gggctcgacg cagcggcgcg aggccagacc 1560

ccgccgggcg ccgtgcgtgg ccgctgctcc ccaggcaacg tgccgaaggt ggtcttcgtc 1620

tttcccggcc agggctctca gtgggtcggt atgggccgtc agctcctggc tgaggaaccc 1680

gtcttccacg cggcgctctc ggcgtgcgac cgggccatcc aggccgaagc cggttggtcg 1740

ctgctcgccg agctcgccgc cgacgaaggg tcctcccagc tcgagcgcat cgacgtggtg 1800

cagccggtgc tgttcgcgct tgcggtggca cttgcggcgc tgtggcggtc gtggggtgtc 1860

gcgcccgacg tcgtgatcgg ccacagcatg ggcgaggtag ccgccgcgca tgtggccggg 1920

gcgctgtcgc tcgaggatgc ggtggcgatc atctgccggc gcagccggct gctccggcgc 1980

atcagcggtc agggcgagat ggcggtgacc gagctgtcgc tggccgaggc cgagacagcg 2040

ctccgaggct acgaggatcg ggtgagcgtg gccgtgagca acagcccgcg ctcgacggtg 2100

ctctcgggcg agccggcagc gatcggcgag gtgctgtcgt ccctgaacgc gaagggggtg 2160

ttctgccgtc gggtgaaggt ggatgtcgcc agccacagcc cgcaggtcga cccgctgcgc 2220

gaggacctct tggcagccct gggcgggctc cggccgcgtg cggctgcggt gccgatgcgc 2280

tcgacggtga cgggcgccat ggtagcgggc ccggagctcg gagcgaatta ctggatgaac 2340

aacctcaggc agcctgtgcg cttcgccgag gtagtccagg cgcagctcca aggcggccac 2400

ggtctgttcg tggagatgag cccgcatccg atcctaacga cttcggtcga ggagatgcgg 2460

cgcgcggtcc agcgggcggg cgcagcggtg ggctcgctgc ggcgggggca ggacgagcgc 2520

ccggcgatgc tggaggcgct gggcgcgctg tgggcgcagg gctaccctgt accctggggg 2580

cggctgtttc ccgcgggggg gcggcgggta ccgctgccga actatccctg gcagcgcgag 2640

cggtactgga tcgaagcgcc ggccaagagc gccgcgggcg atcgccgcgg cgtgcgtgcg 2700

ggcggtcacc cgctcctcgg tgaaatgcag accctgtcaa cccagacgag cacgcggctg 2760

tgggagacga cgctggatct caagcggctg ccgtggctcg gcgaccaccg ggtgcaggga 2820

gcggtcgtgt ttccgggcgc ggcgtacctg gagatggcga tttcgtcggg ggccgaggct 2880

ttgggcgatg gcccattgca gataactgac gtggtgctcg ccgaggcgct ggccttcgcg 2940

ggcgacgcgg cggtgttggt ccaggtggtg acgacggagc agccgtcggg acggctgcag 3000

ttccagatcg cgagccgggc gccgggcgct ggccacgcgt ccttccgggt ccacgctcgc 3060

ggcgcgttgc tccgagtgga gcgcaccgag gtcccggctg ggcttacgct ttccgctgtg 3120

cgcgcacggc tccaggccag catacccgcc gcggccacct acgcggagct gaccgagatg 3180

gggctgcagt acggccctgc ctttcagggg attgctgagc tatggcgggg tgaaggcgag 3240

gcgctgggac gggtacgcct gcccgacgcg gccggctcgg cagcggagta tcggttgcat 3300

cctgcgctgc tggacgcgtg cttccagatc gtcggcagcc tcttcgccgg cggtggcgag 3360

gcgacgccgt gggtgcccgt ggagttgggc tcgctgcggc tcttgcagcg gccttcgggg 3420

gagctgtggt gccatgcgcg cgtcgtgaac catgggcacc aaacccccga tcggcagggc 3480

gccgactttt gggtggtcga cagctcgggt gcagtggtcg ccgaagtctg cgggctcgtg 3540

gcgcagcggc ttccgggagc ggtgcgccgg cgcgaagaag acgattggtt cctggagctc 3600

gagtgggaac ccgcagcggt cggcacagcc aaggtcaacg cgggccggtg gctgctcctc 3660

ggcggcggcg gtgggctcgg cgccgcgttg cgctcgatgc tggaggccgg cggccatgcc 3720

gtcgtccatg cggcagagaa caacacgagc gctgccggcg tgcgcgcgct cctggcaaag 3780

gcctttggcg gccaggctcc gacggcggtg gtgcacctcg gcagcctcga tgggggtggc 3840

gagctcgacc cagggctcgg ggcgcaaggc gcattggacg cgccccggag cgccgacgtc 3900

agtcccgatg ccctcgatcc ggcgctggta cgtggctgcg acagcgtgct ctggaccgtg 3960

caggccctgg ccggcatggg ctttcgagac gccccgcgat tgtggcttct gacccgcggc 4020

gcacaggccg tcggcgccgg cgacgtctcc gtgacacagg caccgctgct ggggctgggc 4080

cgcgtcatcg ccatggagca cgcggatctg cgctgcgctc gggtcgacct cgatccggcc 4140

cggcccgatg gggagctcgg tgccctgctg gccgagctgc tggccgacga cgccgaagcg 4200

gaagtcgcgt tgcgcggtgg cgagcgatgc gtcgctcgga tcgtccgccg gcagcccgag 4260

acccggcccc gggggaggat cgagagctgc gttccgaccg acgtcaccat ccgcgcggac 4320

agcacctacc tcgtgaccgg cggtctgggt gggctcggtc tgagcgtggc cggatggctg 4380

gccgagcgcg gcgctggtca cctggtgctg gtgggccgct ccggcgcggc gagcgtggag 4440

caacgggcag ccgtcgcggc gctcgaggcc cgcggcgcgc gcgtcaccgt ggcgaaggca 4500

gatgtcgccg atcgggcgca gctcgagcgg atcctccgcg aggttaccac gtcggggatg 4560

ccgctgcggg gcgtcgtcca tgcggccggc atcttggacg acgggctgct gatgcagcag 4620

actcccgcgc ggtttcgtaa ggtgatggcg cccaaggtcc agggggcctt gcacctgcac 4680

gcgttgacgc gcgaagcgcc gctttccttc ttcgtgctgt acgcttcggg agtagggctc 4740

ttgggctcgc cgggccaggg caactacgcc gcggccaaca cgttcctcga cgctctggcg 4800

caccaccgga gggcgcaggg gctgccagcg ttgagcgtcg actggggcct gttcgcggag 4860

gtgggcatgg cggccgcgca ggaagatcgc ggcgcgcggc tggtctcccg cggaatgcgg 4920

agcctcaccc ccgacgaggg gctgtccgct ctggcacggc tgctcgaaag cggccgcgtg 4980

caggtggggg tgatgccggt gaacccgcgg ctgtgggtgg agctgtaccc cgcggcggcg 5040

tcttcgcgaa tgttgtcgcg cctggtgacg gcgcatcgcg cgagcgccgg cgggccagcc 5100

ggggacgggg acctgctccg ccgcctcgct gctgccgagc cgagcgcgcg gagcgcgctc 5160

ctggagccgc tcctccgtgc gcagatctcg caggtgctgc gcctccccga gggcaagatc 5220

gaggtggacg ccccgctcac gagcctgggc atgaactcgc tgatggggct cgagctgcgc 5280

aaccgcatcg aggccatgct gggcatcacc gtaccggcaa cgctgttgtg gacctatccc 5340

acggtggcgg cgctgagcgg gcatctggcg cgggaggcat gcgaagccgc tcctgtggag 5400

tcaccgcaca ccaccgccga ttctgctgtc gagatcgagg agatgtcgca ggacgatctg 5460

acgcagttga tcgcagctaa attcaaggcg cttacatga 5499

<210> 4

<211> 21774

<212> DNA

<213>Cellulose heap capsule bacterium（Sorangium cellulosum）

<400> 4

atgactactc gcggtcctac ggcacagcag aatccgctga aacaagcggc catcatcatt 60

cagcggctgg aggagcggct cgctgggctc gcacaggcgg agctggaacg gaccgagccg 120

atcgccatcg tcggtatcgg ctgccgcttc cctggcggcg cggacgctcc ggaagcgttt 180

tgggagctgc tcgacgcgga gcgcgacgcg gtccagccgc tcgacaggcg ctgggcgctg 240

gtaggtgtcg ctcccgtcga ggccgtgccg cactgggcgg ggctgctcac cgagccgata 300

gattgcttcg atgctgcgtt cttcggcatc tcgcctcggg aggcgcgatc gctcgacccg 360

cagcatcgtc tgttgctgga ggtcgcttgg gaggggctcg aggacgccgg tatctcgccc 420

cggtccatcg acgggagccg caccggtgtg ttcgtcggcg ctttcacggc ggactacgcg 480

cgcacggtcg ctcggctgcc gcgcgaggag cgagacgcgt acagcgccac cggcaacatg 540

ctcagcatcg ccgccggacg gctgtcgtac acgctggggc tgcagggacc ttgcctgacc 600

gtcgacacgg cgtgctcgtc atcgctggtg gcgattcacc tcgcctgccg cagcctgcgc 660

gcaggagaga gcgatctcgc gttggcggga ggggtcagcg cgctcctctc ccccgacatg 720

atggaagccg cggcgcgcac gcaagcgctg tcgcccgatg gtcgttgccg gaccttcgat 780

gcttcggcca acgggttcgt ccgtggcgag ggctgtggcc tggtcgtcct caaacggctc 840

tccgacgcgc aacgggatgg cgaccgcatc tgggcgctga tccggggctc ggccatcaac 900

catgatggcc ggtcgaccgg gttgaccgcg cccaacgtgc tggctcagga gacggtcttg 960

cgcgaggcgc tgcggagtgc ccacgtcgaa gctggggccg tcgattacgt cgagacccac 1020

ggcacaggga cctcgctggg cgatcccatc gaggtcgagg cgctgcgggc gacggtgggg 1080

ccggcgcgct ccgacggcac acgctgcgtg ctgggcgcgg tgaagaccaa catcggccat 1140

ctcgaggccg ccgcaggcgt agcgggtctg atcaaggcag cgctttcgct gacgcacgag 1200

cgcatcccgc gaaacctcaa cttccgcacg ctcaatccgc ggatccggct cgagggcagc 1260

gcgctcgcgt tggcgaccga gccggtgccg tggccgcgca cggaccggcc gcgcttcgcg 1320

ggggtgagct cgttcgggat gagcggaacg aacgcgcacg tggtgctgga agaggcgccg 1380

gcggtggagc tggggcctgc cgcgccggag cgctcggcgg agcttctggt gctgtcgggc 1440

aagagcgagg gggcgctcga cgcgcaggcg gcgcggctgc gcgagcacct ggacatgcac 1500

ccggagctcg ggctcgggga cgtggcgttc agcctggcga cgacgcgcag cgcgatgaac 1560

caccggctcg cggtggcggt gacgtcgcgc gaggggctgc tggcggcgct ctcggccgtg 1620

gcgcaggggc agacgccgcc gggggcggcg cgctgcatcg cgagctcgtc gcgcggcaag 1680

ctggcgttgc tgttcaccgg acagggcgcg cagacgccgg gcatgggccg ggggctttgc 1740

gcggcgtggc cagcgttccg ggaggcgttc gaccggtgcg tggcgctgtt cgaccgggag 1800

ctggaccgcc cgctgcgcga ggtgatgtgg gcggaggcgg ggagcgccga gtcgttgttg 1860

ctcgacgaga cggcgttcac ccagcccgcg ctcttcgcgg tggagtacgc gctgacggcg 1920

ctgtggcggt cgtggggcgt agagccggag ctcctggttg ggcatagcat cggggagctg 1980

gtggcggcgt gcgtggcggg ggtgttctcg ctggaagatg gggtgaggct cgtggcggcg 2040

cgcgggcggc tgatgcaggg gctctcggcg ggcggcgcga tggtgtcgct cggcgcgccg 2100

gaggcggagg tggcggcggc ggtggcgccg cacgcggcgc cggtgtcgat cgcggcggtc 2160

aatgggccgg agcaggtggt gatcgcgggc gtggagcgag cggtgcaggc gatcgcggcg 2220

gggttcgcgg cgcgcggcgt gcgcaccaag cggctgcatg tctcgcacgc gttccactcg 2280

ccgctgatgg aaccgatgct ggaggagttc gggcgggtgg cggcgtcggt gacgtaccgg 2340

cggccaagcg tttcgctggt gagcaacctg agcgggaagg tggtcacgga cgagctgagc 2400

gcgccgggct actgggtgcg gcacgtgcgg gaggcggtgc gcttcgcgga cggggtgaag 2460

gcgctgcacg aagccggcgc ggggacgttc gtcgaagtgg gcccgaagcc gacgctgctc 2520

gggctgttgc cagcctgcct gccggagacg gagccgacgc tgctggcgtc gttgcgcgcc 2580

gggcgcgagg aggctgcggg ggtgctcgag gcgctgggcg ggctgtgggc cggcggcggc 2640

tcggtcagct ggccgggcgt cttccccacg gctgggcggc gggtgccgct gccgacctat 2700

ccgtggcagc ggcagcggta ctggatcgag gcgccggccg aagggctcgg agccacggcc 2760

gccgatgcgc tggcgcagtg gttttaccgg gtggactggc ccgagatgcc tcgctcatcc 2820

gtggattcgc ggcgagcccg gtccggcggg tggctggtgc tggccgaccg gggtggagtc 2880

ggggaggcgg ccgcggcggc gctttcgtcg cagggatgtt cgtgcgccgt gctccatgcg 2940

ccggccgagg cctccgcggt cgccgagcag gtgacccagg ccctcggtgg ccgcaacgac 3000

tggcaggggg tgctgtacct ctggggtctg gacgccgtcg tggaggcggg ggcatcggcc 3060

gaagaggtcg gcaaagtcac ccatcttgcc acggcgccgg tgctcgcgct gattcaggcg 3120

ctgggcacgg ggccgcgctc accccggctc tggatcgtga cccgaggggc ctgcacggtg 3180

ggcggcgagc ctgacgctgc cccctgtcag gcggcgctgt ggggtatggg ccgggtcgcg 3240

gcgctggagc atcccggctc ctggggcggg ctcgtggacc tggatccgga ggagagcccg 3300

acggaggtcg aggccctggt ggccgagctg ctttcgccgg acgccgagga tcagctggca 3360

ttccgccagg ggcgccggcg cgcagcgcgg ctcgtggccg ccccaccgga gggaaacgca 3420

gcgccggtgt cgctgtctgc ggaggggagt tacttggtga cgggtgggct gggcgccctt 3480

ggcctcctcg ttgcgcggtg gttggtggag cgcggggcgg ggcacctcgt gctgatcagc 3540

cggcacggat tgcccgaccg cgaggaatgg ggccgagatc agccgccaga ggtgcgcgcg 3600

cgcattgcgg cgatcgaggc gctggaggcg cagggcgcgc gggtcaccgt ggcggcggtc 3660

gacgtggccg atgccgaagg catggcggcg ctcttggcgg ccgtcgagcc gccgctgcgg 3720

ggggtcgtgc acgccgcggg tctgctcgac gacgggctgc tggcccacca ggacgctggt 3780

cggctcgccc gggtgttgcg ccccaaggtg gagggggcat gggtgctgca cacccttacc 3840

cgcgagcagc cgctggacct cttcgtactg ttttcctcgg cgtcgggcgt cttcggctcg 3900

atcggccagg gcagctacgc ggcaggcaat gcctttttgg acgcgctggc ggacctccgc 3960

cgaacgcagg ggctcgccgc cctgagcatc gcctggggcc tgtgggcgga gggggggatg 4020

ggctcgcagg cgcagcgccg ggaacacgag gcatcgggaa tctgggcgat gccgacgcgt 4080

cgtgccctgg cggcgatgga atggctgctc ggtacgcgcg cgacgcagcg cgtggtcatc 4140

cagatggatt gggcccatgc gggagcggct ccgcgcgacg cgagccgagg ccgcttctgg 4200

gatcggctgg taactgccac gaaagcgacc tcctcctcgg ccgtgccagc tgtggagcgc 4260

tggcgtaacg cgtctgtcgt ggagacccgc tcggcgctct acgagcttgt gcgcggcgtg 4320

gtcgccgggg tgatgggctt taccgatcag ggcacgctcg acgtgcgacg aggcttcgcc 4380

gagcagggcc tcgactccct gatggccgtg gaaatccgca aacggcttca gggtgagctg 4440

ggtatgccgc tgtcggcgac gctggcgttc gaccatccga ccgtggagcg gctggtggaa 4500

tacttgctga gccaggcgct ggagctgcag gaccgcaccg acgtgcgaag cgctcggttg 4560

ccggcgacag aggacccgat cgccatcgtg ggtgccgcct gccgcttccc gggcggggtc 4620

gaggacctgg agtcctactg gcagctgttg accgagggcg tggtggtcag caccgaggtg 4680

ccggccgacc ggtggaatgg ggcagacggg cgcggccccg gctcgggaga ggctcagaga 4740

cagacctacg tgcccagggg tggctttctg cgcgaggtgg agacgttcga tgcggcgttc 4800

ttccacatct cgcctcggga ggcgatgagc ctggacccgc aacagcggct gctgctggaa 4860

gtgagctggg aggcgatcga gcgcgcgggc caggacccgt cggcgctgcg cgagagcccc 4920

acgggcgtgt tcgtgggcgc gggccccaac gaatatgccg agcgggtgca ggacctcgcc 4980

gatgaggcgg cggggctcta cagcggcacc ggcaacatgc tcagcgttgc ggcgggacgg 5040

ctgtcatttt tcctgggcct gcacgggccg accctggctg tggatacggc gtgctcctcg 5100

tcgctcgtgg cgctgcacct cggctgccag agcttgcgac ggggcgagtg cgaccaagcc 5160

ctggttggcg gcgtcaacat gctgctctcg ccgaagacct tcgcgctgct ctcacggatg 5220

cacgcgcttt cgcccggcgg gcggtgcaag acgttctcgg ccgacgcgga cggctacgcg 5280

cgggccgagg gctgcgccgt ggtggtgctc aagcggctct ccgacgcgca gcgcgaccgc 5340

gaacccatcg tggcggtgat ccggggtacg gcgatcaatc atgatggccc gagcagcggg 5400

ctgacagtgc ccagcggccc tgcccaggag gcgctgttac gccaggcgct ggcgcacgca 5460

ggggtggttc cggccgacgt cgatttcgtg gaatgccacg ggaccgggac ggcgctgggc 5520

gacccgatcg aggtgcgggc gctgagcgac gtgtacgggc aagcccgccc tgcggaccga 5580

ccgctgatcc tgggagccgc caaggccaac cttgggcaca tggagcccgc ggcgggcctg 5640

gccggcttgc tcaaggcggt gctcgcgctg gggcaagagc aaataccagc ccagccggag 5700

ctgggcgagc tcaacccgct cttgccgtgg gaggcgctgc cggtggcggt ggcccgcgca 5760

gcggtgccgt ggccgcgcac ggaccggccg cgcttcgcgg gggtgagctc gttcgggatg 5820

agcggaacga acgcgcacgt ggtgctggaa gaggcgccgg cggtggagct ggggcctgcc 5880

gcgccggagc gctcggcgga gcttctggtg ctgtcgggca agagcgaggg ggcgctcgac 5940

gcgcaggcgg cgcggctgcg cgagcacctg gacatgcacc cggagctcgg gctcggggac 6000

gtggcgttca gcctggcgac gacgcgcagc gcgatgaacc accggctcgc ggtggcggtg 6060

acgtcgcgcg aggggctgct ggcggcgctc tcggccgtgg cgcaggggca gacgccgccg 6120

ggggcggcgc gctgcatcgc gagctcgtcg cgcggcaagc tggcgttgct gttcaccgga 6180

cagggcgcgc agacgccggg catgggccgg gggctttgcg cggcgtggcc agcgttccgg 6240

gaggcgttcg accggtgcgt ggcgctgttc gaccgggagc tggaccgccc gctgcgcgag 6300

gtgatgtggg cggagccggg gagcgccgag tcgttgctgc tcgaccagac ggcgttcacc 6360

cagcccgcgc tcttcacggt ggagtacgcg ctgacggcgc tgtggcggtc gtggggcgta 6420

gagccggagc tggtggctgg gcatagcgcc ggggagctgg tggcggcgtg cgtggcgggg 6480

gtgttctcgc tggaagatgg ggtgaggctc gtggcggcgc gcgggcggct gatgcagggg 6540

ctctcggcgg gcggcgcgat ggtgtcgctc ggagcgccgg aggcggaggt ggccgcggcg 6600

gtggcgccgc acgcggcgtc ggtgtcgatc gcggcggtca atgggccgga gcaggtggtg 6660

atagcgggcg tggagcgagc ggtgcaggcg atcgcggcgg ggttcgcggc gcgcggcgtg 6720

cgcaccaagc ggctgcatgt ctcgcacgcg tcccactcgc cgctgatgga accgatgctg 6780

gaggagttcg ggcgggtggc ggcgtcggtg acgtaccggc ggccaagcgt ttcgctggtg 6840

agcaacctga gcgggaaggt ggtcacggac gagctgagcg cgccgggcta ctgggtgcgg 6900

cacgtgcggg aggcggtgcg cttcgcggac ggggtgaagg cgctgcacga agccggcgcg 6960

gggacgttcc tcgaagtggg cccgaagccg acgctgctcg ggctgttgcc agcctgcctg 7020

ccggagacgg agccgacgct gctggcgtcg ttgcgcgccg ggcgcgagga ggctgcgggg 7080

gtgctcgagg cgctgggcag gctgtgggcc ggcggcggct cggtcagctg gccgggcgtc 7140

ttccccacgg ctgggcggcg ggtgccgctg ccgacctatc cgtggcagcg gcagcggtac 7200

tggcccgaca tcgagcctga cagccgtcgc cacgcagccg cggatccgac ccaaggctgg 7260

ttctatcgcg tggactggcc ggagatacct cgcagcctcc agaaatcaga ggaggcgagc 7320

cgcgggagct ggctggtatt ggcggataag ggtggagtcg gcgaggcggt cgctgcagcg 7380

ctgtcgacac gtggacttcc atgcgtcgtg atccatgcgc cggcagagac atccgcgacc 7440

gccgagctgg tgaccgaggc tgccggcggt cgaagcgatt ggcaggtagt gctctacctg 7500

tggggtctgg acgccgtcgt cggtgcggag gcgtcgatcg atgagatcgg cgacgcgacc 7560

cgtcgtgcta ccgcgccggt gctcggcttg gctcggtttc tgagcaccgt gtcttgttcg 7620

ccccgactct gggtcgtgac ccggggggca tgcatcgttg gcgacgagcc tgcgatcgcc 7680

ccttgtcagg cggcgttatg gggcatgggc cgggtggcgg cgctcgagca tcccggggcc 7740

tggggcgggc tcgtggacct ggatccccga gcgagcccgc cccaagccag cccgatcgac 7800

ggcgagatgc tcgtcaccga gctattgtcg caggagaccg aggatcagct cgccttccgc 7860

catgggcgcc ggcacgcggc acggctggtg gctgccccgc cacaggggca agcggcaccg 7920

gtgtcgctgt ctgcggaggc gagctacctg gtgacgggag gcctcggtgg gctgggcctg 7980

atcgtggccc agtggctggt ggagctggga gcgcggcact tggtgctgac cagccggcgc 8040

gggttgcccg accggcaggc gtggcgcgag cagcagccgc ctgagatccg cgcgcggatc 8100

gcagcggtcg aggcgctgga ggcgcggggt gcacgggtga ccgtggcagc ggtggacgtg 8160

gccgacgtcg aaccgatgac agcgctggtt tcgtcggtcg agcccccgct gcgaggggtg 8220

gtgcacgccg ctggcgtcag cgtcatgcgt ccactggcgg agacggacga gaccctgctc 8280

gagtcggtgc tccgtcccaa ggtggccggg agctggctgc tgcaccggct gctgcacggc 8340

cggccgctcg acctgttcgt gctgttctcg tcgggcgcag cggtgtgggg tagccatagc 8400

cagggtgcgt acgcggcggc caacgctttc ctcgacgggc tcgcgcatct tcggcgttcg 8460

caatcgctgc ctgcgttgag cgtcgcgtgg ggtctgtggg ccgagggagg catggcggac 8520

gcggaggctc atgcacgtct gagcgacatc ggggttctgc ccatgtcgac gtcggcagcg 8580

ttgtcggcgc tccagcgcct ggtggagacc ggcgcggctc agcgcacggt gacccggatg 8640

gactgggcgc gcttcgcgcc ggtgtacacc gctcgagggc gtcgcaacct gctttcggcg 8700

ctggtcgcag ggcgcgacat catcgcgcct tcccctccgg cggcagcaac ccggaactgg 8760

cgtggcctgt ccgttgcgga agcccgcgtg gctctgcacg agatcgtcca tggggccgtc 8820

gctcgggtgc tgggcttcct cgacccgagc gcgctcgatc ctgggatggg gttcaatgag 8880

cagggcctcg actcgttgat ggcggtggag atccgcaacc tccttcaggc tgagctggac 8940

gtgcggcttt cgacgacgct ggcctttgat catccgacgg tacagcggct ggtggagcat 9000

ctgctcgtcg atgtactgaa gctggaggat cgcagcgaca cccagcatgt ttggtcgttg 9060

gcgtcagacg agcccatcgc catcgtggga gccgcctgcc gcttcccggg cggggtggag 9120

gacctggagt cctactggca gctgttggcc gagggcgtgg tggtcagcgc cgaggtgccg 9180

gccgaccggt gggatgcggc ggactggtac gaccctgatc cggagatccc aggccggact 9240

tacgtgacca aaggcgcctt cctgcgcgat ttgcagagat tggatgcgac cttcttccgc 9300

atctcgcctc gcgaggcgat gagcctcgac ccgcagcagc ggttgctcct ggaggtaagc 9360

tgggaggcgc tcgagagcgc gggtatcgct ccggatacgc tgcgagatag ccccaccggg 9420

gtgttcgtgg gtgcggggcc caatgagtac tacacgcagc ggctgcgagg cttcaccgac 9480

ggagcggcag ggctgtacgg cggcaccggg aacatgctca gcgttacggc tggacggctg 9540

tcgtttttcc tgggtctgca cggcccgacg ctggccatgg atacggcgtg ctcgtcatcc 9600

ctggtcgcgc tgcacctcgc ctgccagagc ctgcgactgg gcgagtgcga tcaagcgctg 9660

gttggcgggg tcaacgtgct gctcgcgccg gagaccttcg tgctgctctc acggatgcgc 9720

gcgctttcgc ccgacgggcg gtgcaagacg ttctcggccg acgcggacgg ctacgcgcgg 9780

ggcgaggggt gcgccgtggt ggtgctcaag cggctgcgcg atgcgcagcg cgccggcgac 9840

tccatcctgg cgctgatccg gggaagcgcg gtgaaccacg acggcccgag cagcgggctg 9900

accgtgccca acggaccggc ccagcaagca ttgctgcgcc aggcgctttc gcaagcaggc 9960

gtgtctccgg tcgacgttga ttttgtggag tgtcacggga cagggacggc gctgggcgac 10020

ccgatcgagg tgcaggcgct gagcgaggtg tatggtccag ggcgctccgg ggatcgaccg 10080

ctggtgctgg gggccgtcaa ggccaacgtc gcgcatctgg aggcggcatc cggcttggcc 10140

agcctgctca aggccgtgct tgcgctgcgg cacgagcaga tcccggccca gccggagctg 10200

ggcgagctca acccgcactt gccgtggaac acgctgccgg tggcggtgcc acgtaatgcg 10260

gtgccgtggg ggcgcggcgc acgcccgcgt cgggccggcg tgagcgcgtt cgggttgagc 10320

ggaaccaacg tgcatgtcgt gctggaggag gcaccggagg tggagccggc gcccgcggcg 10380

ccggcgcgac cggtggagct ggtcgtgcta tcggccaaga gcgcggcggc gctggacgcc 10440

gcggcggaac ggctctcggc gcacctgtcc gcgcacccgg agctgagcct cggcgacgtg 10500

gcgttcagcc tggcgacgac gcgcagcccg atggagcacc ggctcgccat cgcgacgacc 10560

tcgcgcgagg ccctgcgagg cgcgctggac gccgcggcgc agcaaaagac gccgcagggc 10620

gcggtgcgcg gcaaggccgt gtcctcacgc ggtaagctgg ctttcctgtt caccggacag 10680

ggcgcgcaaa tgccgggcat gggccgtggg ctgtacgaaa cgtggcctgc gttccgggag 10740

gcgttcgacc ggtgcgtggc gctcttcgat cgggagatcg accagcctct gcgcgaggtg 10800

atgtgggctg cgccgggcct cgctcaggcg gcgcggctcg atcagaccgc gtacgcgcag 10860

ccggctctct ttgcgctgga gtacgcgctg gctgccctgt ggcgttcgtg gggcgtggag 10920

ccgcacgtac tgctcggtca tagcatcggc gagctggtcg ccgcctgcgt ggcgggcgtg 10980

ttctcgctcg aagatgcggt gaggttggtg gccgcgcgcg ggcggctgat gcaggcgcta 11040

cccgccggcg gtgccatggt agccatcgca gcgtccgagg ccgaggtggc cgcctccgtg 11100

gcgccccacg ccgccacggt gtcgatcgcc gcggtcaacg gtcctgacgc cgtcgtgatc 11160

gccggcgccg aggtacaggt gctcgccctc ggcgcgacgt tcgcggcgcg tgggatacgc 11220

acgaagaggc tcgccgtctc ccatgcgttc cactcgccgc tcatggatcc gatgctggaa 11280

gacttccagc gggtcgctgc gacgatcgcg taccgcgcgc cggaccgccc ggtggtgtcg 11340

aatgtcaccg gccacgtcgc aggccccgag atcgccacgc ccgagtattg ggtccggcat 11400

gtgcgaagcg ccgtgcgctt cggcgacggg gcaaaggcgt tgcatgccgc gggtgccgcc 11460

acgttcgtcg agattggccc gaagccggtc ctgctcgggc tgttgccagc gtgcctcggg 11520

gaagcggacg cggtcctcgt gccgtcgcta cgcgcggacc gctcggaatg cgaggtggtc 11580

ctcgcggcgc tcggggcttg gtatgcctgg gggggtgcgc tcgactggaa gggcgtgttc 11640

cccgatggcg cgcgccgcgt ggctctgccc atgtatccat ggcagcgtga gcgccattgg 11700

atggacctca ccccgcgaag cgccgcgcct gcagggatcg caggtcgctg gccgctggct 11760

ggtgtcgggc tctgcatgcc cggcgctgtg ttgcaccacg tgctctcgat cggaccacgc 11820

catcagcctt tcctcggtga tcacctcgtg tttggcaagg tggtggtgcc cggcgccttt 11880

catgtcgcgg tgatcctcag catcgccgcc gagcgctggc ccgagcgggc gatcgagctg 11940

acaggcgtgg agttcctgaa ggccatcgcg atggagcccg accaggaggt cgagctccac 12000

gccgtgctca cccccgaagc cgccggggat ggctacctgt tcgagctggc gaccctggcg 12060

gcgccggaga ccgaacgccg atggacgacc cacgcccgcg gtcgggtgca gccgacagac 12120

ggcgcgcccg gcgcgttgcc gcgcctcgag gtgctggagg accgcgcgat ccagcccctc 12180

gacttcgccg gattcctcga caggttatcg gcggtgcgga tcggctgggg gccgctttgg 12240

cgatggctgc aggacgggcg cgtcggcgac gaggcctcgc ttgccaccct cgtgccgacc 12300

tatccgaacg cccacgacgt ggcgcccttg cacccgatcc tgctggacaa cggctttgcg 12360

gtgagcctgc tggcaacccg gagcgagccg gaggacgacg ggacgccccc gctgccgttc 12420

gccgtggaac gggtgcggtg gtggcgggcg ccggttggaa gggtgcggtg tggcggcgtg 12480

ccgcggtcgc aggcattcgg tgtctcgagc ttcgtgctgg tcgacgaaac tggcgaggtg 12540

gtcgctgagg tggagggatt tgtttgccgc cgggcgccgc gagaggtgtt cctgcggcag 12600

gagtcgggcg cgtcgactgc agccttgtac cgcctcgact ggcccgaagc gcccttgccc 12660

gatgcgcctg cggaacggat ggaggagagc tgggtcgtgg tggcagcacc tggctcggag 12720

atggccgtgg cgctcgcaac acggctcaac cgctgcgtcc tcgccgaacc ccaaggcctc 12780

gagacggccc tcgcgggggt gtctcccgca ggtgtgatct gcctctggga acctggagcc 12840

cacgaggaag ctccggcggc ggcgcagcgt gtggcgaccg agggcctctc ggtggtgcag 12900

gcgctcaggg atcgcgcggt gcgcctctgg tgggtgacca cgggcgcagt ggctgtcgag 12960

gccggcgagc gggtgcaggt cgccacagcg gcggtgtggg gcctgggccg gacagtgatg 13020

caggagcgcc cggagctcag ctgcactctg gtggatttgg agccggaggc cgatgcagcg 13080

cgttcagctg acgttctgtt gcgggagctc ggtcgcgctg acgacgagac ccaggtggtt 13140

ttccgttccg gaaagcgccg cgtagggcgg ctggtcaaag cgacaacccc cgaagggctc 13200

ttggtccctg acgcagaatc ctatcgactg gaggctgggc agaagggcac attggaccag 13260

ctccgcctcg cgccggcaca gcgccgggca cctggcccgg gcgaggtcga gatcaaggta 13320

accgcctcgg ggctcaactt ccggaccgtc ctcgctgtgc tgggaatgta tccgggcgac 13380

gctgggccga tgggcggaga ttgtgccggt atcgtcacgg cggtgggcca gggggtgcac 13440

cacctctcgg tcggcgatgc tgtcatgacg ctggggacgt tgcatcgatt cgtcacggtc 13500

gacgcgcggc tggtggtccg gcagcctgca gggctgactc ccgcgcaggc agctacggtg 13560

ccggtcgcgt tcctgacggc ctggctcgct ctgcacgacc tggggaatct gcagcgcggc 13620

gagcgggtgc tgatccatgc tgcggccggc ggtgtgggca tggccgcggt gcaaatcgcc 13680

cgatggatag gggccgaggt gttcgccacg gcgagcccgt ccaagtgggc agcgcttcag 13740

gccatgggcg tgccgcgcac gcacatcgcc agctcgcgga cgctggagtt tgctgagacg 13800

ttccggcagg tcaccggcgg ccggggcgtg gacgtggtgc tcaacgcgct ggccggcgag 13860

ttcgtggacg cgagcctgtc cctgctgtcg acgagcgggc ggttcctcga gatgggcaag 13920

accgacatac gggatcgagc cgcggtcgcg gcggcgcatc ccggtgttcg ctatcgggta 13980

ttcgacatcc tggagctcgc tccggatcga actcgagaga tcctcgagcg cgtggtcgag 14040

ggctttgctg cgggacatct gcgcgcattg ccggtgcatg cgttcgcgat caccaaggcc 14100

gaggcagcgt ttcggttcat ggcgcaagcg cggcatcagg gcaaggtcgt gctgctgccg 14160

gcgccctccg ccgcgccctt ggcgccgacg ggcaccgtac tgctgaccgg tgggctggga 14220

gcgttggggc tccacgtggc ccgctggctc gcccagcagg gcgtgccgca catggtgctc 14280

acaggtcggc ggggcctgga tacgccgggc gctgctaaag ccgtcgcgga gatcgaagcg 14340

ctcggcgctc gggtgacgat cgcggcgtcg gatgtcgccg atcggaatgc gctggaggct 14400

gtgctccagg ccattccggc ggagtggccg ttacagggcg tgatccatgc agccggagcg 14460

ctcgatgatg gtgtgcttga tgagcagacc accgaccgct tctcgcgggt gctggcaccg 14520

aaggtgactg gcgcctggaa tctgcatgag ctcacggcgg gcaacgatct cgctttcttc 14580

gtgctgttct cctccatgtc ggggctcttg ggctcggccg ggcagtccaa ctatgcggcg 14640

gccaacacct tcctcgacgc gctggccgcg catcggcggg ccgaaggcct ggcggcgcag 14700

agcctcgcgt ggggcccatg gtcggacgga ggcatggcag cggggctcag cgcggcgctg 14760

caggcgcggc tcgctcggca tgggatggga gcgctgtcgc ccgctcaggg caccgcgctg 14820

ctcgggcagg cgctggctcg gccggaaacg cagctcgggg cgatgtcgct cgacgtgcgt 14880

gcggcaagcc aagcttcggg agcggcagtg ccgcctgtgt ggcgcgcgct ggtgcgcgcg 14940

gaggcgcgcc atgcggcggc tggggcgcag ggggcattgg ccgcgcgcgt tggggcgctg 15000

cccgaggcgc gtcgcgccga cgaggtgcgc aaggtcgtgc aggccgagat cgcgcgcgtg 15060

ctttcatgga gcgccgcgag cgccgtgccc gtcgatcggc cgctgtcgga cttgggcctc 15120

gactcgctca cggcggtgga gctgcgcaac gtgctcggcc agcgggtggg tgcgacgctg 15180

ccggcgacgc tggcattcga tcacccgacg gtcgacgcgc tcacgcgctg gctgctcgat 15240

aaggtcctgg tcgtggccga gccgagcgta tcgcccgcaa agtcgtcgcc gcaggtcgcc 15300

ctcgacgagc ccattgcggt gatcggcatc ggctgccgtt tcccaggcgg cgtgaccgat 15360

ccggagtcgt tttggcggct gctcgaagag ggcagcgatg ccgtcgtcga ggtgccgcat 15420

gagcgatggg acatcgacgc gttctatgat ccggatccgg atgtgcgcgg caagatgaca 15480

acacgctttg gcggcttcct gtccgatatc gaccggttcg agccggcctt cttcggcatc 15540

tcgccgcgcg aagcgacgac gatggatccg cagcagcggc tgctcctgga gacgagctgg 15600

gaggcgttcg agcgcgccgg gattttgccc gagcggctga tgggcagcga taccggcgtg 15660

ttcgtggggc tcttctacca ggagtacgct gcgctcgccg gcggcatcga ggcgttcgat 15720

ggctatctag gcaccggcac cacggccagc gtcgcctcgg gcaggatctc ttatgtgctc 15780

gggctaaagg ggccgagcct gacggtggac accgcgtgct cctcgtcgct ggtcgcggtg 15840

cacctggcct gccaggcgct gcggcggggc gagtgttcgg tggcgctggc cggcggcgtg 15900

gcgctgatgc tcacgccggc gacgttcgtg gagttcagcc ggctgcgagg cctggctccc 15960

gacggacggt gcaagagctt ctcggccgca gccgacggcg tggggtggag cgaaggctgc 16020

gccatgctcc tgctcaaacc gcttcgcgat gcgcagcgcg atggggatcc gatcctggcg 16080

gtgatccgcg gcaccgcggt gaaccaggat gggcgcagca acgggctgac ggcgcccaac 16140

ggatcgtcgc agcaagaggt gatccgtcgt gccctggagc aggcggggct ggctccggcg 16200

gacgtcagct acgtcgagtg ccacggcacc ggcacgacgt tgggggaccc catcgaagtg 16260

caggccctgg gcgccgtgct ggcacagggg cgaccctcgg accggccgct cgtgatcggg 16320

tcggtgaagt ccaatatcgg acatacgcag gctgcggcgg gcgtggccgg tgtcatcaag 16380

gtggcgctgg cgctcgagcg cgggcttatc ccgaggagcc tgcatttcga cgcgcccaat 16440

ccgcacattc cgtggtcgga gctcgccgtg caggtggccg ccaaacccgt cgaatggacg 16500

agaaacggcg tgccgcgacg agccggggtg agctcgtttg gcgtcagcgg gaccaacgcg 16560

cacgtggtgc tggaggaggc gccagcggcg gcgttcgcgc ccgcggcggc gcgttcagcg 16620

gagcttttcg tgctgtcggc gaagagcgcc gcggcgctgg acgcgcaggc ggcgcggctt 16680

tcggcgcatg tcgttgcgca cccggagctc ggcctcggcg acctggcgtt cagcctggcg 16740

acgacccgca gcccgatgac gtaccggctc gcggtggcgg cgacctcgcg cgaggcgctg 16800

tctgcggcgc tcgacacagc ggcgcagggg caggcgccgc ccgcagcggc tcgcggccac 16860

gcttccacag gcagcgcccc aaaggtggtt ttcgtctttc ctggccaggg ctcccagtgg 16920

ctgggcatgg gccaaaagct cctctcggag gagcccgtct tccgcgacgc gctctcggcg 16980

tgtgaccgag cgattcaggc cgaagccggc tggtcgctgc tcgccgagct cgcggccgat 17040

gagaccacct cgcagctcgg ccgcatcgac gtggtgcagc cggcgctgtt cgcgatcgag 17100

gtcgcgctgt cggcgctgtg gcggtcgtgg ggcgtcgagc cggatgcagt ggtaggccac 17160

agcatgggcg aagtggcggc cgcgcacgtc gccggcgccc tgtcgctcga ggatgctgta 17220

gcgatcatct gccggcgcag cctgctgctg cggcggatca gcggccaagg cgagatggcg 17280

gtcgtcgagc tttccctggc cgaggccgag gcagcgctcc tgggctacga agaccggctc 17340

agcgtggcgg tgagcaacag cccgcgatcg acggtgctgg cgggcgagcc ggcagcgctc 17400

gcagaggtgc tggcgatcct tgccgcaaag ggggtgttct gccgtcgagt caaggtggac 17460

gtcgccagcc acagcccaca gatcgacccg ctgcgcgacg agctattggc agcattgggc 17520

gagctcgagc cgcgacaagc gaccgtgacg atgcgctcga cggtgacgag cacgatcgtg 17580

gcgggcccgg agctcgtggc gagctactgg gcggacaacg ttcgacagcc ggtgcgcttc 17640

gccgaagcgg tgcaatcgtt gatggaaggc ggtcatgggc tgttcgtgga gatgagcccg 17700

catccgatcc tgacgacatc ggtcgaggag atccgacggg cgacgaagcg ggagggagtc 17760

gcggtggggt cgttgcggcg tggacaggac gagcgcctgt ccatgttgga ggcgctggga 17820

gcgctctggg tacacggcca ggcggtgggc tgggagcggc tgttctccgc gggcggcgcg 17880

ggcctccgtc gcgtgccgct gccgacctat ccctggcagc gcgagcggta ctgggtcgat 17940

gcgccgaccg gcggcgcggc gagcggcagc cgctttgctc atgcgggcag tcacccgctc 18000

ctgggtgaaa tgcagaccct gtcgacccag aggagcacgc gcgtgtggga gacgacgctg 18060

gatctcaaac ggctgccgtg gctcggcgat caccgggtgc agggggcggt cgtgttcccg 18120

ggcgcggcgt acctggagat ggcgctttcg tccggggccg aggccttggg tgacggtccg 18180

ctccaggtca gcgatgtggt gctcgccgag gcgctggcct tcgcggatga tacgccggcg 18240

gcggtgcagg tcatggcgac cgaggagcga ccaggccgcc tgcaattcca cgttgcgagc 18300

cgggtgccgg gccacggcgg tgctgccttt cgaagccatg cccgcggggt gctgcgccag 18360

atcgagcgcg ccgaggtccc ggcgaggctg gatctggccg cgcttcgtgc ccggcttcag 18420

gccagcgcac ccgctgcggc tacctatgcg gcgctggccg agatggggct cgagtacggc 18480

ccagcgttcc aggggcttgt cgagctgtgg cggggggagg gcgaggcgct gggacgtgtg 18540

cggctccccg aggccgccgg ctccccagcc gcgtgccggc tccaccccgc gctcttggat 18600

gcgtgcttcc acgtgagcag cgccttcgct gaccgcggcg aggcgacgcc atgggtaccc 18660

gtggaaatcg gctcgctgcg gtggttccag cggccgtcgg gggagctgtg gtgtcatgcg 18720

cggagtgtga gccacggaaa gccaacaccc gaccggcgga gtaccgactt ctgggtggtc 18780

gacagcacgg gcgcgatcgt cgccgagatc tccgggctcg tggcgcagcg gctcgcggga 18840

ggtgtacgcc ggcgcgaaga agacgactgg ttcatggagc cggcttggga accgaccgcg 18900

gtccccggat ccgaggtcat ggcgggccgg tggctgctca tcggctcggg cggcgggctc 18960

ggcgctgcgc tcgactcggc gctgacggaa gctggccatt ccgtcgtcca cgcgacaggg 19020

cacggcacga gcgccgccgg gttgcaggcg ctcttgacgg cgtccttcga cggccaggcc 19080

ccgacgtcgg tggtgcacct cggcagcctc gatgagcgtg gcgggctcga cgcggacgcg 19140

cccttcgacg ccgatgcgct cgaggagtcg ctggtgcgcg gctgcgacag cgtgctctgg 19200

accgtgcagg ccgtggccgg ggcgggcttc cgagatcctc cgcggttgtg gctcgtgaca 19260

cgcggcgctc aggccatcgg cgccggcgac gtctctgtgg cgcaagcgcc gctcctgggg 19320

ctgggccgcg ttatcgcctt ggagcacgcc gagctgcgct gcgctcggat cgacctcgat 19380

ccagcgcggc gcgacggaga agtcgatgcg ctgcttgccg agctgttggc cgacgacgcc 19440

gaggaggaag tcgcgtttcg cggcggtgag cggcgcgtgg cccggctcgt ccgaaggccg 19500

cccgagaccg actgccgaga gaaaatcgag cccgcggaag gccggccgtt ccggctggag 19560

atcgatgggt ccggcgtgct cgacgacctg gtgctccgag ccacggagcg gcgccctcct 19620

ggcccgggcg aggtcgagat cgccgtcgag gcggcggggc tcaactttct cgacgtgatg 19680

agggccatgg ggatctaccc tgggcctggg gacggtccgg ttgcgctggg cgccgagtgc 19740

tccggccgaa ttgtcgcgat gggcgaaggt gtcgagagcc ttcgtatcgg ccaggacgtc 19800

gtggccgtcg cgcccttcag tttcggcacc cacgtcaccg tcgacgcccg gatggtcgca 19860

cctcgccccg cggcgctgac ggccgcgcag gcagccgcgc tgcccgtcgc attcatgacg 19920

gcctggtacg gtctcgtcca tctggggagg ctccgggccg gcgagcgcgt gctcatccac 19980

tcggcgacgg ggggcaccgg ccttgctgct gtgcagatcg cccgccacct cggcgcggag 20040

atatttgcga ccgctggtac accggagaaa cgggcgtggc tgcgcgagca ggggatcgcg 20100

cacgtgatgg actcgcgctc gctggacttc gccgagcaag tgctggccgc gacgaagggc 20160

gagggggtcg acgtcgtgtt gaactcgctg tctggcgccg cgatcgacgc gagcctttcg 20220

accctcgtgc cggacggccg cttcatcgag ctcggcaaga cggacatcta tgcagatcgc 20280

tcgctggggc tcgctcactt caggaagagc ctgtcctaca gcgccgtcga tcttgcgggt 20340

ttggccgtgc gtcggcccga gcgcgtcgca gcgctgctgg cggaggtggt ggacctgctc 20400

gcacggggag cgctgcagcc gcttccggta gagatcttcc ccctctcgcg ggccgcggac 20460

gcgttccgga aaatggcgca agcgcagcat ctcgggaagc tcgtgctcgc gctggaggac 20520

ccggacgtgc ggatccgcgt ttcgggcgaa tccggcgtcg ccatccgcgc ggacggcacc 20580

tacctcgtga ccggcggtct gggtgggctc ggtctgagcg tggctggatg gctggccgag 20640

cagggggctg ggcatctggt gctggtgggc cgctccggtg cggtgagcgc ggagcagcag 20700

acggctgtcg ccgcgctcga ggcgcacggc gcgcgtgtga cggtagcgag ggcagacgtc 20760

gccgatcggg cgcagatcga gcgcatcctc cgcgaggtta ccgcgtcggg gatgccgctc 20820

cgcggcgtcg ttcatgcggc cggtatcctg gacgacgggc tgctgatgca gcaaaccccc 20880

gcgcggttcc gcgcggtcat ggcgcccaag gtccgagggg ccttgcacct gcatgcgttg 20940

acacgcgaag cgccgctctc cttcttcgtg ctgtacgctt cgggagcagg gctcttgggc 21000

tcgccgggcc agggcaatta cgccgcggcc aacacgttcc tcgacgctct ggcgcaccac 21060

cggagggcgc aggggctgcc agcgttgagc gtcgactggg gcctgttcgc ggacgtgggt 21120

ctggccgccg ggcagcaaaa tcgcggcgcg cggctggtca cccgcgggac gcggagcctc 21180

acccccgacg aagggctgtg ggcgctcgag cgtctgctcg acggcgatcg cacccaggcc 21240

ggggtcatgc cgttcgacgt gcggcagtgg gtggagttct acccggcggc ggcatcttcg 21300

cggaggttgt cgcggctggt gacggcacgg cgcgtggctt ccggtcggct cgccggggat 21360

cgggacctgc tcgaacggct cgccaccgcc gaggcgggcg cgcgggcagg aatgctgcag 21420

gaggtcgtgc gcgcgcaggt ctcgcaggtg ctgcgcctcc ccgaaggcaa gctcgacgtg 21480

gatgcgccgc tcacgagcct gggaatggac tcgctgatgg ggctagagct gcgcaaccgc 21540

atcgaggccg tgctcggcat caccatgccg gcgaccctgc tgtggaccta ccccacggtg 21600

gcagcgctga gtgcgcatct ggctagccat gtcgtctcta cgggggatgg ggaatccgtg 21660

cgcccgcctg atacagggag cgtggctcca atgacccacg aagtcgcttc gctcgacgaa 21720

gacgggttgt tcgcgttgat tgatgagtca ctcgcgcgtg cgggaaagag gtga 21774

<210> 5

<211> 11433

<212> DNA

<213>Cellulose heap capsule bacterium（Sorangium cellulosum）

<400> 5

atgagtcact cgcgcgtgcg ggaaagaggt gattgcgtga cagaccgaga aggccagctc 60

ctggagcgct tgcgtgaggt tactctggcc cttcgcaaga cgctgaacga gcgcgatacc 120

ctggagctcg agaagaccga gccgatcgcc atcgtgggga tcggctgccg cttccccggc 180

ggagcgggca ctccggaggc gttctgggag ctgctcgacg acgggcgcga cgcgatccgg 240

ccgctcgagg agcgctgggc gctcgtaggt gtcgacccag gcgacgacgt accgcgctgg 300

gcggggctgc tcaccgaggc catcgacggc ttcgacgccg cgttcttcgg tatcgccccc 360

cgggaggcac ggtcgctcga cccgcagcat cgcctgctgc tggaggtcgc ctgggagggg 420

ttcgaagacg ccggcatccc gcccaggtcc ctcgtcggga gccgcaccgg cgtgttcgtc 480

ggcgtctgcg ccacggagta cctccacgcc gccgtcgcgc accagccgcg cgaagagcgg 540

gacgcgtaca gcaccaccgg caacatgctc agcatcgccg ccggacggct atcgtacacg 600

ctggggctgc agggaccttg cctgaccgtc gacaccgcgt gctcgtcatc gctggtggcc 660

attcacctcg cctgccgcag cctgcgcgct cgagagagcg atctcgcgct ggcgggaggg 720

gtcaacatgc tcctctcccc cgacacgatg cgagctctgg cgcgcaccca ggcgctgtcg 780

cccaatggcc gttgccagac cttcgacgcg tcggccaacg ggttcgtccg tggggagggc 840

tgcggtctga tcgtgctcaa gcgattgagc gacgcgcggc gggatgggga ccggatctgg 900

gcgctgatcc gaggatcggc catcaatcag gacggccggt cgacggggtt gacggcgccc 960

aacgtgctcg cccagggggc gctcttgcgc gaggcgctgc ggaacgccgg cgtcgaggcc 1020

gaggccatcg gttacatcga gacccacggg gcggcgacct cgctgggcga ccccatcgag 1080

atcgaagcgc tgcgcgccgt ggtggggccg gcgcgagccg acggagcgcg ctgcgtgctg 1140

ggcgcggtga agaccaacct cggccacctg gagggcgctg ccggcgtggc gggcctgatc 1200

aaggcgacgc tttcgctaca tcacgagcgc atcccgagga acctcaactt tcgtacgctc 1260

aatccgcgga tccggatcga ggggaccgcg ctcgagttgg cgaccgagcc ggtgccctgg 1320

ccgcggacgg gccggacgcg cttcgcggga gtgagctcgt tcgggatgag cgggaccaac 1380

gcgcatgtgg tgttggagga ggcgccggcg gtggagcctg aggccgcggc ccccgagcgc 1440

gctgcggagc tgttcgtcct gtcggcgaag agcgtggcgg cgctggatgc gcaggcagcc 1500

cggctgcggg atcacctgga gaagcatgtc gagcttggcc tcggcgatgt ggcgttcagc 1560

ctgacgacga cgcgcagcgc gatggagcac cggctggcgg tggccgcgag ctcgcgcgag 1620

gcgctgcgag gggcgctttc ggccgcagcg caggggcaca cgccgccggg agctgtgcgt 1680

gggcgggcct cgggcggcag cgcgccgaag gtggtcttcg tgtttcccgg ccagggctcg 1740

cagtgggtgg gcatgggccg aaagctcatg gccgaagagc cggtcttccg ggcggcgctg 1800

gagggttgcg accgggccat cgaggcggaa gcgggctggt cgctgctcgg ggagctctcc 1860

gccgacgagg ccgcctcgca gctcgggcgc atcgacgtgg ttcagccggt gctcttcgcc 1920

atggaagtag cgctttctgc gctgtggcgg tcgtggggag tggagccgga agcggtggtg 1980

ggccacagca tgggcgaggt tgcggcggcg cacgtggccg gcgcgctgtc gctcgaggac 2040

gctgtggcga tcatctgccg gcgcagccgg ctgctgcggc ggatcagcgg tcagggggag 2100

atggcgctgg tcgagctgtc gctggaggag gccgaggcgg cgctgcgtgg ccatgagggt 2160

cggctgagcg tggcggtgag caacagcccg cgctcgaccg tgctcgcagg cgagccggcg 2220

gcgctctcgg aggtgctggc ggcgctgacg gccaaggggg tgttctggcg gcaggtgaag 2280

gtggacgtcg ccagccatag cccgcaggtc gacccgctgc gcgaagagct ggtcgcggcg 2340

ctgggagcga tccggccgcg agcggctgcg gtgccgatgc gctcgacggt gacgggcggg 2400

gtgattgcgg gtccggagct cggagcgagc tactgggcgg gcaatcttcg gcagccggtg 2460

cgcttcgctg cggcggcgcg agcgctgctg gaaggtggcc ccacgctgtt catcgagatg 2520

agcccgcacc cgatcctggt gccgcccctg gacgagatcc agacggcggt cgagcaaggg 2580

ggcgctgcgg tgggctcgct gcggcgaggg caggacgagc gcgcgacgct gctggaggcg 2640

ctggggacgc tgtgggcgtc cggctatccg gtgagctggg ctcggctgtt ccccgcgggc 2700

ggcaggcggg ttccgctgcc gacctatccc tggcagcacg agcggtgctg gatcgaggtc 2760

gagcctgaag cccgccgcct cgccgcagcc gaccccacca aggactggtt ctaccggacg 2820

gactggcccg aggtgccccg cgccgccccg aaatcggaga cagctcatgg gagctggctg 2880

ctgttggccg acaggggtgg ggtcggtgag gcggtcgctg cagcgctgtc gacgcgcgga 2940

ctttcctgca ccgtgcttca tgcgtcggct gacgcctcca ccgtcgccga gcaggtatcc 3000

gaagctgcca gtcgccgaaa cgactggcag ggagtcctct acctgtgggg cctcgacgcc 3060

gtcgtcgatg ctggggcatc ggccgacgac gtcagcgagg ctacccgccg tgccaccgca 3120

cccgtccttg ggctggttcg attcctgagc gctgcgcccc atcctcctcg cttctgggtg 3180

gtgacccgcg gggcatgcac ggtgggcggc gagccagagg tctctctttg ccaagcggcg 3240

ttgtggggcc tcgcgcgcgt cgtggcgctg gagcatcccg ctgcctgggg tggcctcgtg 3300

gacctggatc ctcagaagag cccgacggag atcgagcccc tggtggccga gctgctttcg 3360

ccggacgccg aggatcagct ggcgttccgt agcggtcgcc ggcacgcagc acgccttgta 3420

gccgccccgc cggagggcga cgtcgcaccg atatcgctgt ccgcggaggg aagctacctg 3480

gtgacgggcg ggctgggcgg ccttggtctg ctcgtggctc ggtggctggt ggagcgggga 3540

gctcgacatc tggtgctcac cagccggcac gggctgccag agcgacaggc gtcgggcgga 3600

gagcagccgc cggaggcccg cgcgcgcatc gcagcggtcg aggggctgga agcgcagggc 3660

gcgcgggtga ccgtggcagc ggtggatgtc gccgaggccg atcccatgac ggcgctgctg 3720

gccgccatcg agcccccgtt gcgcggggtg gtgcacgccg ccggcgtctt ccccgtgcgt 3780

cccctggcgg agacggacga ggccctgctg gagtcggtgc tccgtcccaa ggtggccggg 3840

agctggctgc tgcaccggct gctgcgcgac cggcctctcg acctgttcgt gctgttctcg 3900

tcgggcgcgg cggtgtgggg tggcaaaggc caaggcgcat acgccgcggc caatgcgttc 3960

ctcgacgggc tcgcgcacca tcgccgcgcg cgctcgctgc cggcgttgag cctcgcctgg 4020

ggcttatggg ccgagggagg catggttgat gcaaaggctc atgcacgtct gagcgacatc 4080

ggggtcctgc ccatggccac ggggccggcc ttgtcggcgc tggagcgcct ggtgaagacc 4140

agcgctgtcc agcgttcggt cacacggatg gactggacgc gcttcgcgcc cgtctatgcc 4200

gcgcgagggc ggcgcaactt gctttcggct ctggtcgcgg aggacgagcg cactgcgtct 4260

ccccctgtgc cgacggcaaa ccgaatctgg cgcggcctgt ccgttgcgga gagccgctca 4320

gccctctacg agctcgttcg cggcatcgcc gcccgggtgc tgggcttcgc cgacccgggc 4380

gcgctcgacg tcggccgagg cttcgccgag caggggctcg actccctgat ggctctggag 4440

atccgtaacc gccttcagcg cgagctgggc gaacggctgt cggcgactct ggccttcgac 4500

cacccgacgg tggagcggct ggtggcgcat ctcctcaccg acgtgctgaa gctggaggac 4560

cggagcgaca cccggcacat ccggtcggtg gcggcggatg acgacatcgc catcgtcggt 4620

gccgcctgcc ggttcccagg tggggatgaa agcctggaga catactggcg gcatctggcc 4680

gagggcatgg tggtcagcgc cgaggtgcca gccgaccggt ggcgcgcggc ggactggtac 4740

gaccccgatc cggaggttcc gggccggacc tatgtggcca agggtgcctt cctccgcgat 4800

gtgcgcagct tggatgcggc gttcttctcc atttcccctc gtgaggcgat gagcctggac 4860

ccgcaacagc ggctgttgct ggaggtgagc tgggaggcga tcgagcgcgc tggccaggac 4920

ccgatggcgc tgcgcgagac cgccacgggc gtgttcgtgg gcatgatcgg gagcgagcac 4980

gccgagcggg tgcagggcct cgacgacgac gcggcgttgc tgtacggcac caccggcaac 5040

ctgctcagcg tcgccgctgg acggctgtcg ttcttcctgg gtctgcacgg cccgacgatg 5100

acggtggaca ccgcctgctc gtcgtcgctg gtggcgttgc acctcgcctg ccagagcctg 5160

cgattgggcg agtgcgacca ggccctggcc ggcgggtcca gcgtgctttt gtcgccgcgg 5220

tcattcgtcg cggcgtcgcg catgcgtttg ctttcgccag atgggcggtg caagacgttc 5280

tcggccgctg cagacggctt tgcgcgggcc gagggctgcg ccgtggtggt gctgaagcag 5340

ctccgtgacg cgcagcgcga ccgcgacccc atcctggcgg tggtcaggag cacggcgatc 5400

aaccacgatg gcccgagcag cgggctcacg gtgcccagcg gtcctgccca gcaggcgttg 5460

ctaggccagg cgctggcgca agcgggcgtg gcgccggccg aggtcgattt cgtggagtgc 5520

cacgggacgg ggacagcgct gggtgacccg atcgaggtgc aggcgctggg cgcggtgtat 5580

gggcggggcc gccccgcgga gcggccgctc tggctgggcg ctgtcaaggc caacctcggg 5640

cacctggagg ccgcggcggg cttggccggc gtgctcaagg tgctcttggc gctggagcac 5700

gagcagattc cggctcaacc ggagctcgac gagctcaacc cgcacatccc gtgggcagag 5760

ctgccagtgg ccgttgtccg cagggcggtc ccctggccgc gcggcgcgcg cccgcgtcgt 5820

gcaggcgtga gcgctttcgg cctgagcggg accaacgcgc atgtggtgtt ggaggaggcg 5880

ccgacggtgg agcctggggc cgcggccccc gagcgcgcag cggagctgtt cgtcctgtcg 5940

gcgaagagcg tggcggcgct ggatgcgcag gcagcccggc tgcgggatca cctggagaag 6000

catgtcgagc ttggcctcgg cgatgtggcg ttcagcctgg cgacgacgcg cagcgcgatg 6060

gagcaccggc tggcggtggc cgcgagctcg cgcgaggcgc tgcgaggggc gctttcggcc 6120

gcagcgcagg ggcacacgcc gccgggagct gtgcgtgggc gggcctcggg cggcagcgcg 6180

ccgaaggtgg tcttcgtgtt tcccggccag ggctcgcagt gggtgggcat gggccgaaag 6240

ctcatggccg aagagccggt cttccgggcg gcgctggagg gttgcgaccg ggccatcgag 6300

gcggaagcgg gctggtcgct gctcggggag ctctccgccg acgaggccgc ctcgcagctc 6360

gagcgcatcg acgtggttca gccggtgctc ttcgccatgg aagtagcgct ttctgcgctg 6420

tggcggtcgt ggggagtgga gccggaagcg gtggtgggcc acagcatggg cgaggttgcg 6480

gcggcgcacg tggccggcgc gctgtcgctc gaggacgctg tggcgatcat ctgccggcgc 6540

agccggctgc tgcggcggat cagcggccag ggggagatgg cgctggtcga gctgacgctg 6600

gaggaggccg aggcggcgct gcgtggccat gagggtcggc tgagcgtggc ggtgagcaac 6660

agcccgcgct cgaccgtgct cgcaggcgag ccggcggcgc tctcggaggt gctggcggcg 6720

ctgacggcca agggggtgtt ctggcggcag gtgaaggtgg acgtcgccag ccatagcccg 6780

caggtcgacc cgctgcgcga agagctggtc gcggcgctgg gagcgatccg gccgcgagcg 6840

gctgcggtgc cgatgcgctc gacggtgacg ggcggggtga ttgcgggtcc ggagctcgga 6900

gcgagctact gggcggacaa tcttcggcag ccggtgcgct tcgctgcggc ggcgcgagcg 6960

ctgctgggag gtggccccac gctgttcatc gagatgagcc cgcacccgat cctggtgccg 7020

cccctggacg agatccagac ggcggtcgag caagggggcg ctgcggtggg ctcgctgcgg 7080

cgagggcagg acgagcgcgc gacgctgctg gaggcgctgg ggacgctgtg ggcgtccggc 7140

tatccggtga gctgggctcg gctgttcccc gcgggcggca ggcgggttcc gctgccgacc 7200

tatccctggc agcacgagcg gtactggatc gaggacagcg tgcatggatc gaagccctcg 7260

ctgcggcttc ggcagcttcg caacggcgcc acggaccatc cgctgctcgg ggcttcattg 7320

ctcgtctcgg cgcgacccgg agctcacttg tgggagcaag cgctgagcga cgagaggctg 7380

tcctatcttt cggaacatag ggtccatggc gaagccgtgt tgccaagcgc ggcgtatgta 7440

gagatggcgc tcgccgccgg cgtagatctc tatggcacgg cgacgctggt gctggagcag 7500

ctggcgctcg agcgagccct cgccgtgcct tccgaaggcg gacgcatcgt gcaagtggcc 7560

ctcagcgaag aagggcccgg tcgggcctca ttccaggtat cgagtcgtga ggaggcaggt 7620

agaagctggg tgcggcacgc cacggggcac gtgtgtagcg accagagctc agcagtggga 7680

gcgttgaagg aagctccgtg ggagattcaa cagcgatgtc cgagcgtcct gtcgtcggag 7740

gcgctctatc cgctgctcaa cgagcacgcc ctcgactatg gtccctgctt ccagggtgtg 7800

gagcaggtgt ggctcggcac gggggaggtg ctcggccggg tacgcttgcc agaagacatg 7860

gcatcctcaa gtggcgccta tcggattcat cccgccttct tggatgcatg ttttcaagtg 7920

ctgaccgcgc tgctcaccac gccggaatcc atcgatattc ggaggcggct gacggatctc 7980

cacgaaccgg atctcccgcg gtccagggct ccggtgaatc aagcggtgag tgacacctgg 8040

ctgtgggacg ccgcgctgga cggtggacgg cgccagagcg cgagcgtgcc cgtcgagctg 8100

gtgctcggca gcttccatgc gaagtgggag gtcatggagc gcctcgcgca ggcgtacatc 8160

atcgacactc tccgcatatg ggacgtcttc tgcgctgctg gagagcgtca cacgatagac 8220

gagttgctcg tcaggcttca aatctctgtc ggctacagga aggtcatcaa gcgatggatg 8280

gatcaccttg tcgcgatcgg cgtcctcgta ggggacggag agcattttgt gagctctcag 8340

ccgctgccgg agcctgattt ggcggcggtg ctcgaggagg ccgggagggt gttcgccgac 8400

ctcccagtcc tacttgagtg gtgcaagttt gccggggaac ggctcgcgga cgtattgacc 8460

ggtaagacgc tcgcgctcga gatcctcttc cctggtggct cgttcgatat ggcggagcga 8520

atctatcaag attcgcccat cgcccgttac tcgaacggca tcgtgcgcgg tgtcgtcgag 8580

tcggcggcgc gggtggtagc accgtcggga atgttcagca tcttggagat cggagcaggg 8640

acgggcgcga ccaccgccac cgtcctcccg gtgttgctgc ctgaccggac agagtaccat 8700

tttaccgatg tttctccgct cttccttgct cgtgcggagc aaaaatttcg agatcatcca 8760

ttcctgaagt atggcattct ggatatcgac caggagccag ctggccaggg atacgcacat 8820

cagaagttcg acgtcatcgt cgcggccaac gtcatccatg cgacccgcga tataagagcc 8880

acggcgaagc gtctcctgtc gttgctcgcg cccggaggcc ttctggtgct ggtcgagggc 8940

acagggcatc cgatctggtt cgatatcacc acgggattga ttgaggggtg gcagaagtac 9000

gaagatgatc ttcgtaccga ccatccgctc ctgcctgctc ggacctggtg tgacctcctg 9060

cgccgggtag gctttgcgga cgccgtgagt ctgccaggcg acggatctcc ggcggggatc 9120

ctcggacagc acgtgatcct ctcgcgcgcg ccgggcatag caggagccgc ctgtgacagt 9180

tccggtgagt cggcgaccga atcgccggcc gcgcgtgcag tacggcagga atgggccgat 9240

ggctccgctg acgtcgtcca tcggatggcg ttggagagaa tgtacttcca ccgccggccg 9300

ggccggcagg tctgggtcca cggtcgattg cgtaccggtg gagacgcgtt cacgaaggcg 9360

ctcgctggag atctgctcct gttcgacgac accgggcagg tcgtggcaga ggttcagggg 9420

cttcgcctgc cgcagctcga ggcttctgct ttcgcgccgc gggacccgcg ggaagagtgg 9480

ttgtacgcgt tggaatggca gcgcaaagac cctataccag aggctccggc agccgcgtct 9540

tcttcctccg cgggggcttg gctcgtgctg atggaccagg gcgggacagg cgctgcgctc 9600

gtatcgctgc tggaagggcg aggcgaggcg tgcgtgcgcg tcatcgtggg tacggaatac 9660

gcctgcctcg cgccggggct gtatcaagtc gatccggcgc agtcagatgg ctttcatacc 9720

ctgctccgcg atgcattcgg cgaggaccgg atttgtcgcg cggtagtgca tatgtggagc 9780

cttgatgcga cggcagcagg ggagaggacg acaggggagt cgcttcaggc cgatcaactc 9840

ctggggagcc tgagcgcgct ttctctggtg caggcgctgg tgcgccggag gtggcgcaac 9900

atgccgcgac tttggctctt gacccgcgcc gtgcatgcgg tgggcgcgga ggacgcagcg 9960

gcctcggtgg cgcaggcgcc ggtgtggggc ctcggtcgga cgctcgcgct cgagcatcca 10020

gagctgcggt gcacgctcgt ggacgtgaac ccggcgccgt ctccagagga cgcagctgca 10080

ctggcggtgg agctcggggc gagcgacaga gaggaccaga tcgcattgcg ctcggatggc 10140

cgctacgtgg cgcgcctcgt gcggagctcc ttttccggca agcctgctac ggatcgcggc 10200

atccgggcgg acggcagtta tgtgatcacc gatggcatgg ggagagtggg gctctcggtt 10260

gcgcaatgga tggtgatgca gggggcccgc catgtggtgc tcgtggatcg cggcggcgct 10320

tccgaggcct cccgggatgc cctccggtcc atggccgagg ctggcgcgga ggtgcagatc 10380

gtggaggccg acgtggctcg gcgcgacgat gtcgctcggc tcctctcgaa gatcgaaccg 10440

tcgatgccgc cgcttcgggg gatcgtgtac gtggacggga ccttccaggt cgactcctcg 10500

atgctggagc tggatgccca tcgcttcaag gagtggatgt atcccaaggt gctcggagcg 10560

tggaacctgc acgcgctgac cagggataga tcgctggact tcttcgtcct gtactcctcg 10620

ggcacctcgc ttctgggctt gcccggacag gggagccgcg ccgccggtga cgccttcttg 10680

gacgccatcg cgcatcaccg gtgtaggctg ggccttacag cgatgagcat caactgggga 10740

ttgctcttcg aagcatcatc gccggcgacc ccgaacgacg gcggagcacg gctcgaatac 10800

cgggggatgg aaggtctcac gctggagcag ggagcggcgg cgctcgggcg cttgctcgca 10860

caacccaggg cgcaggtagg ggtgatgcgg ctgaatctgc gccagtggct ggatttctat 10920

cccaatgcgg cccgattggc gctgtgggcg gagttgatga aggagcgtga ccgcgccgac 10980

cgaggcgcgt cgaacgcatc gaacctgcgc gaggcgctgc agagcggcag gcccgaagat 11040

cgtcagttga ttctggagaa gcacttgagc gagctgttgg ggcgggggct gcgccttccg 11100

ccggcgagga tcgagcggca cgtgccgttc agcaatctcg gcatggactc gctgataggc 11160

ctggagctcc gcaaccgcat cgaggccgcg ctcggcatca ccgtgccggc gaccctgcta 11220

tggacctacc ctaccgtagc agctctgagc gggaacttgc tagacattct gtttccgaac 11280

gccggcgcga cccacgctcc ggccaccgag cgggagaaga gcttcgagaa cgatgccgca 11340

gatctcgagg ctctgctggg tatgacggac gagcagaagg acgcgttgct cgccgaaaag 11400

ctggcgcagc tcgcgcagat cgttggtgag taa 11433

<210> 6

<211> 7320

<212> DNA

<213>Cellulose heap capsule bacterium（Sorangium cellulosum）

<400> 6

atggcgacca cgaatgccgg gaagcttgag catgcccttc tgctcatgga caagcttgcg 60

aaaaagaacg cgtctttgga gcaagagcgg accgagccga tcgccatcat aggcattggc 120

tgccgcttcc ccggcggagc ggacactccg gaggcattct gggagctgct cgactcaggc 180

cgagacgcgg tccagccgct cgaccggcgc tgggcgctgg tcggggtcca tcccagtgag 240

gaggtgccgc gctgggccgg actgctcacc gaggcggtgg acggcttcga cgccgcgttc 300

tttggcacct cgcctcggga ggcgcggtcg ctcgatcctc agcaacgtct gctgctggag 360

gtcacctggg aagggctcga ggacgccggc atcgcacccc ggtccctcga cggcagccgc 420

accggggtat tcctgggcgc atgcagcagc gactactcgc ataccgttgc gcaacagcgg 480

cgcgaggagc aggacgcgta cgacatcacc ggcaatacgc tcagcgtcgc cgccggacgg 540

ttgtcttata cgctagggct gcagggaccc tgcctgaccg tcgacacggc ctgctcgtcg 600

tcgctcgtgg ccatccacct tgcctgccgc agcctgcgcg ctcgcgagag cgatctcgcg 660

ctggcgggag gcgtcaacat gctcctttcg tccaaagacg tgataatgct ggggcgcatc 720

caggcgctgt cgcccgatgg ccactgccgg acattcgacg cctcggccaa cgggttcgtc 780

cgtggggagg gctgcggtat ggtcgtgctc aaacggctct ccgacgccca gcgacatggc 840

gatcggatct gggctctgat ccggggttcg gccatgaatc aggatggccg gtcgacaggg 900

ttgatggcac ccaatgtgct cgctcaggag gcgctcttgc gcgaggcgct gcagagcgct 960

cgcgtcgacg ccggggccat tggttatgtc gagacccacg gaacggggac ctcgctcggc 1020

gacccgatcg aggtcgatgc gctgcgcgcc gtgatggggc cggcgcgggc cgatgggagc 1080

cgctgcgtgc tgggcgcagt gaagaccaac ctcggccacc tggagggcgc tgcaggcgtg 1140

gcgggtttga tcaaggcggc gctggctctg caccacgaac tgatcccgcg aaacctccat 1200

ttccacacgc tcaatccgcg gatccggatc gaggggaccg cgctcgcgct ggcgacggag 1260

ccggtgccgt ggccgcgggc gggccgaccg cgcttcgcgg gggtgagcgc gttcggcctc 1320

agcggcacca acgtccatgt cgtgctggag gaggcgccgg ccacggtgct cgcaccggcg 1380

acgccggggc gctcagcaga gcttttggtg ctgtcggcga agagcgccgc cgcgctggac 1440

gcacaggcgg cgcggctctc agcgcacatc gccgcgtacc cggagcaggg cctcggagac 1500

gtcgcgttca gcctggtagc gacgcgtagc ccgatggagc accggctcgc ggtggcggcg 1560

acctcgcgcg aggcgctgcg aagcgcgctg gaggttgcgg cgcaggggca gaccccggca 1620

ggcgcggcgc gcggcagggc cgcttcctcg cccggcaagc tcgccttcct gttcgccggg 1680

cagggcgcgc aggtgccggg catgggccgt gggttgtggg aggcgtggcc ggcgttccgc 1740

gagaccttcg accggtgcgt cacgctcttc gaccgggagc tccatcagcc gctctgcgag 1800

gtgatgtggg ccgagccggg cagcagcagg tcgtcgttgc tggaccagac ggcgttcacc 1860

cagccggcgc tctttgcgct ggagtacgcg ctggccgcgc tcttccggtc gtggggcgtg 1920

gagccggagc tcgtcgctgg ccatagcctc ggcgagctgg tggccgcctg cgtggcgggt 1980

gtgttctccc tcgaggacgc cgtgcgcttg gtggttgcgc gcggccggtt gatgcaggcg 2040

ctgccggccg gcggtgcgat ggtatcgatc gccgcgccgg aggccgacgt ggctgccgcg 2100

gtggcgccgc acgcagcgtc ggtgtcgatc gcggcagtca atgggccgga gcaggtggtg 2160

atcgcgggcg ccgagaaatt cgtgcagcag atcgcggcgg cgttcgcggc gcggggggcg 2220

cgaaccaaac cgctgcatgt ctcgcacgcg ttccactcgc cgctcatgga tccgatgctg 2280

gaggcgctcc ggcgggtggc ggagtcggtg acgtatcggc ggccttcgat ggcgctggtg 2340

agcaacctga gcgggaagcc ctgcacggat gaggtgtgcg cgccgggtta ctgggtgcgt 2400

cacgcgcgag aggcggtgcg cttcgcggac ggcgtgaagg cgctgcacgc ggccggtgcg 2460

ggcatcttcg tcgaggtggg cccgaagccg gcgctgctcg gccttttgcc ggcctgcctg 2520

ccggatgcca ggccggtgct gctgccagcg tcgcgcgccg ggcgtgacga ggctgcgagc 2580

gcgctggagg cgctgggtgg gttctgggtc gtcggtggat cggtcacctg gtcgggagtc 2640

ttcccttcgg gcggacggcg ggtaccgctg ccaacctatc cctggcagcg cgagcgttac 2700

tggatcgaag cgccggtcga tggtgaggcg gacggcatcg gccgtgctca ggcggggggc 2760

cacccccttc tgggtgaagt cttttccgtg tcgacccatg ccgatctgcg cctgtgggag 2820

acgacgctgg accgaaagcg gctgccgtgg ctcggcgagc accgggcgca gggggaggtc 2880

gtgtttcctc gcgccgggta cctggagatg gcgctgtcgt cgggggccga gatcttgggc 2940

gatggaccga tccaggtcac ggatgtggtg ctcatcgaga cgctgacctt cgcgggcgat 3000

acggcggtac cggtccaggt ggtgacgacc gaggagcgac cgggacggct acggttccag 3060

atagcgagtc gggggccggg tgaacgtcgc gcgtccttcc ggatccacgc ccgcggcgtg 3120

ctgcgccggg tcgggcgcgc cgagaccccg gcgaggttgg acctcgccgc cctgcgcgcc 3180

cggcttcatg ccgccgtgcc cgctgcggct acctatgggg cgctcgccga gatggggctt 3240

cgatacggcc cggcgttgcg ggggctcgcc gagctgtggc ggggtgaggg cgaggcgctg 3300

ggcagggtga gactgcctga ggccgccggc tccgccacag cctaccagct gcatccggtg 3360

ctgctggacg cgtgcgtcca aatgattgtt ggcgcgttcg ccgatcgcga tgaggcggcg 3420

ccgtgggcgc cggtggaggt gggctcggtg cggctgttcc agcggtctcc tggggagcta 3480

tggtgccatg cgcgcgtcgt gagcgatggt caacaggccc ccagccggtg gagcgccgac 3540

tttgagttga tggacggtac gggcgcggtg gtcgccgagg tctccgggct ggtggtggag 3600

cggcttgcga gcggtgtacg ccggcgcgaa gcagacgact ggttcctgga gctggattgg 3660

gagcccgcgg cgctcggtgg gcccaagatc acagccggcc ggtggctgct gctcggcgag 3720

ggtggcgggc tcgggctctc gttgtgctca gcgctgaagg ccgccggcca tgtcgtcgtg 3780

cacgccacgg gggacgacac gagcgccgca ggaatgcgcg cgctcctggc caacgcgttc 3840

gacggccagg ccccgacggc cgtggtgcac ctcagcagcc tcgacggggg cggccagctc 3900

gacccggggc tcggggcgca gggcgcgctc gacgcgcccc ggagcccaga tgtcgatgcc 3960

gatgccctcg agtcggcgct gatgcgtggc tgcgacagcg tgctctccct ggtgcaagcc 4020

ctggtcggca tggacctccg aaatgcgccg cggctgtggc tcttgacccg cggggctcag 4080

gcggccgccg ccggcgacgt ctccgtggtg caagcgccgc tgttggggct gggccgcacc 4140

atcgccttgg agcacgccga gctgcgctgt atcagcgtcg acctcgatcc agcccagcct 4200

gaaggggaag ccgatgcttt gctggccgag ctacttgcag atgatgccga ggaggaggtc 4260

gcgctgcgcg gtggcgagcg gtttgttgcg cggctcgtcc accggctgcc cgacgctcag 4320

cgccgggaga aggtcgagcc cgccggtgac aggccgttcc ggctagagat cgatgaaccc 4380

ggcgcgctgg accaactggt gctccgggcc acggggcggc gcgctcctgg tccgggcgag 4440

gtcgagatcg ccgtcgaagc ggcggggctc gactcgatcg acatccagct ggcgttgggc 4500

gttgctccca atgacctgcc tggagaagaa atcgagccgt cggtgctcgg acgcgagtgc 4560

gccgggcgca tcgtcgctgt gggcgagggc gtgaacggcc ttgtggtggg ccagccggtg 4620

atcgcccttg cggcgggagt atttgctacc catgtcacca cctcggccac gctggtgttg 4680

cctcggcctc tggggctctc ggcgaccgag gcggccgcga tgcccctcgc gtatttgacg 4740

gcctggtacg ccctcgacaa ggtcgcccac ctgcaggcgg gggagcgggt gctgatccat 4800

gcggaggccg gtggtgtcgg cctctgcgcg gtgcgatggg cgcagcgcgt gggcgccgag 4860

gtgtatgcga ccgccgacac gcccgagaaa cgtgcctacc tggcgtcgct gggcgtgcgg 4920

tacgtgagcg attcccgctc gggccggttc gccgcagacg tgcatgcatg gacggacggc 4980

gagggtgtgg acgtcgtgct cgactcgctt tcgggcgagc acatcgacaa gagcctcatg 5040

gtcctgcgcg cctgtggtcg ccttgtgaag ctgggcaggc gcgacgactg ccccgacacg 5100

cagcctgggc tgccgccgct cctacggaat ttttccttct cgcaggtgga cttgcgggga 5160

atgatgctcg atcaaccggc gaggatccgt gcgctcctcg acgagctgtt cgggttggtc 5220

gcagccgatc ccatcagccc actggggtgg gggttgcgcg ttggcggatc cctcacgcca 5280

ccgccggtcg agaccttccc gatctctcgc gcagccgagg cattccggag gatggcgcaa 5340

agacagcatc tcgggaagct cgtgctcacg ctggacgacc cggaggtgcg gatccgcgct 5400

ccggccgaat ccagcgtcgc cgtccgcgcg gacggcacct accttgtgac cggcggtctg 5460

ggtgggctcg gtctgcgcgt ggccggatgg ctggccgagc ggggcgcggg gcaactggtg 5520

ttggtgggcc gctccggtgc ggcgagcgca gagcagcgag ccgccgtggc ggcgctggag 5580

gcccacggcg cgcgcgtcac ggtggcgaaa gcggacgtcg ccgatcggtc acagatcgag 5640

cgggtcctcc gcgaggttac cgcgtcgggg atgccgctgc ggggtgtcgt gcatgcggca 5700

ggtcttgtgg atgacgggct gctgatgcag cagactccgg cgcggttccg cacggtgatg 5760

ggacctaagg tccagggagc cttgcacttg cacacgctga cacgcgatgc gcctctttcc 5820

ttcttcgtgc tgtacgcttc tgcagctggg ctgttcggct cgccaggcca gggcaactat 5880

gccgcagcca acgctttcct cgacgccctt tcgcatcacc gaagggcgca gggcctgccg 5940

gcgctgagca tcgactgggg catgttcacg gaggtgggga tggccgttgc gcaagcaaac 6000

cgtggcgcgc ggctgatctc tcgcgggatg cggggcatca cccccgatga ggggctgtcc 6060

gctctggcgc gcttgctcga gggtgatcgc gtgcagacgg gggtgatacc gatcactccg 6120

cgccagtggg tggagttcta cccggcaacg gcggcctcac ggaggttgtc gcggctggtg 6180

accacgcagc gcgcggtcgc tgatcggacc gccggggatc gggacctgct cgaacagctt 6240

gcctcggctg agccgagcgc gcgggcgggg ctgctgcagg acgtcgtgcg cgtgcaggtc 6300

tcgcatgtgc tgcgtctccc tgaaggcaag atcgaggtgg atgccccgct ctcgagcatg 6360

ggcatggact cgctgatgag cctggagctg cgcaaccgca tcgaggctgc gctgggcgtc 6420

gccgcgcctg cagccttggg gtggacgtac ccaacggtag cagcgataac gcgctggctg 6480

ctcgacgacg ccctcgtcgt ccggcttggc ggcgggtcgg acacggacga atcgacggca 6540

agcgccggtt cgttcgtcca cgtcctccgc tttcgtcctg tcgttaagcc gcgggctcgt 6600

ctcttctgct ttcacggttc tggcggctcg cccgagggct tccgttcctg gtcggagaag 6660

cctgagtgga gcgatctgga aatcgtggcc atgtggcacg atcgcagcct cgcctccgag 6720

gacacgcctg gtaagaagta cgtccaagag gcggcctcgc tgattcagca ctatgcagac 6780

gcaccgtttg cgttagtagg gttcagcctg ggtgtccggt tcgtcatggg gacagccgtg 6840

gagctcgcca gtcgttccgg cgcaccggct ccgctggccg ttttcacgtt gggcggcagc 6900

ttgatctctt cttcagagat cgccccggag atggagaccg acataatagc caagctcttc 6960

ttccgaaatg ccgcgggttt cgtgcgatcc acccaacaag ttcaggccga tgctcgcgca 7020

gacaaggtca tcacagacac gatgatggct ccggcccccg gggattcgaa ggagccgccc 7080

gtgaagatcg cggtccctat cgtcgccatc gccggctcgg acgatgtgat cgtgcctcca 7140

agcgacgttc aggatctaca atctcgcacc acggagcgct tctatatgca tctccttccc 7200

ggagatcacg agtttctcgt cgatcgaagt cgcgagatca tgcacatcgt cgactcgcat 7260

ctcaatccgc tgctcgccgc gaagacgacg tcgtcaggcc cggcgttcga ggcaaaatga 7320

<210> 7

<211> 2179

<212> DNA

<213>Artificial sequence

<220>

<223>PCR targetin segments 1

<400> 7

ccctgctgtg gacctacccc acggtggcag cgctgagtgc gcatctggct agcttggctg 60

agccattcga gtgctgggtt gttgtctctg gacactgatc catgggaaac tactcagcac 120

catctctagt cgacctgcag gcatgcaagc ttcgattggc taggtctagc ggagtgtata 180

ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa 240

aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc 300

tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg 360

gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc 420

aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa 480

tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc 540

cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc 600

gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg 660

actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc 720

ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat 780

ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt 840

ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc 900

ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga 960

ccaaaacgat ctcaagaaga tcatcttatt aatcagataa aatatttcta gagtcgacct 1020

gcagcggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 1080

ttatcaaaaa ggatcttcac ctagatcctt ttcgaccgaa taaatacctg tgacggaaga 1140

tcacttcgca gaataaataa atcctggtgt ccctgttgat accgggaagc cctgggccaa 1200

cttttggcga aaatgagacg ttgatcggca cgtaagaggt tccaactttc accataatga 1260

aataagatca ctaccgggcg tattttttga gttgtcgaga ttttcaggag ctaaggaagc 1320

taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa 1380

agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct 1440

ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt 1500

tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttacgtatgg caatgaaaga 1560

cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac 1620

tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat 1680

atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat 1740

tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa 1800

cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca 1860

aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt 1920

ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc 1980

gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggttgctacg cctgaattcg 2040

agctcggtac ccggggatcc tctagtcgac gatgaccagg tttttgacga aagtgatcca 2100

gatgatccag ctctacactg gttcatgtgc cccagggcct gatcccttcg accgacggct 2160

tcgcctggca acgcggata 2179

<210> 8

<211> 2488

<212> DNA

<213>Artificial sequence

<220>

<223>PCR targetin segments 2

<400> 8

gggcgatcgc cgcgaagaag gcctccagct ccggcgtcgg gatgcgacca ttggctgagc 60

cattcgagtg ctgggttgtt gtctctggac accgatccat gggaaactac tcagcaccat 120

ctctagtcga cctgcaggca tgcaagcttc gattggctag gtctagcgga gtgtatactg 180

gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc aggagaaaaa 240

aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct tcctcgctca 300

ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg 360

gagatttcct ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa 420

gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca 480

gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct 540

cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg 600

tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact 660

gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg 720

agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta 780

gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt 840

gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc 900

gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca 960

aaacgatctc aagaagatca tcttattaat cagataaaat atttctagag tcgacctgca 1020

gctaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 1080

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 1140

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 1200

tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 1260

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 1320

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 1380

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 1440

gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg 1500

ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac 1560

atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg 1620

acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa 1680

ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt 1740

atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc 1800

actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa 1860

aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat 1920

tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac 1980

ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc 2040

tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat 2100

ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga 2160

cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag 2220

ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg 2280

aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcga 2340

attcgagctc ggtacccggg gatcctctag agattgacca ggtttttgac gaaactgatc 2400

cagatgatcc agctctacac tggttcatgt gcgctagcca tgtcgtctct acgggggatg 2460

gggaatccgt gcgcccgcct gatacagg 2488

<210> 9

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223> attB0

<400> 9

ggcttgtcga cgacggcggt ctccgtcgtc aggatcat 38

<210> 10

<211> 48

<212> DNA

<213>Artificial sequence

<220>

<223> attP0

<400> 10

ggtttgtctg gtcaaccacc gcggtctcag tggtgtacgg tacaaacc 48

<210> 11

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223> attB6

<400> 11

ggcttgtcga cgacggcgct ctccgtcgtc aggatcat 38

<210> 12

<211> 48

<212> DNA

<213>Artificial sequence

<220>

<223> attP6

<400> 12

ggtttgtctg gtcaaccacc gcgctctcag tggtgtacgg tacaaacc 48

<210> 13

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223> attB7

<400> 13

ggcttgtcga cgacggcgaa ctccgtcgtc aggatcat 38

<210> 14

<211> 48

<212> DNA

<213>Artificial sequence

<220>

<223> attP7

<400> 14

ggtttgtctg gtcaaccacc gcgaactcag tggtgtacgg tacaaacc 48

<210> 15

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223> attB13

<400> 15

ggcttgtcga cgacggcgca ctccgtcgtc aggatcat 38

<210> 16

<211> 48

<212> DNA

<213>Artificial sequence

<220>

<223> attP13

<400> 16

ggtttgtctg gtcaaccacc gcgcactcag tggtgtacgg tacaaacc 48

<210> 17

<211> 38

<212> DNA

<213>Artificial sequence

<220>

<223> attB15

<400> 17

ggcttgtcga cgacggcgcc ctccgtcgtc aggatcat 38

<210> 18

<211> 48

<212> DNA

<213>Artificial sequence

<220>

<223> attP15

<400> 18

ggtttgtctg gtcaaccacc gcgccctcag tggtgtacgg tacaaacc 48

<210> 19

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> BSD-F

<400> 19

ctcgcggggg tatcgcttcc 20

<210> 20

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> BSD-R

<400> 20

tcagccaatc gactggcgag 20

<210> 21

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> accA1-F

<400> 21

atgcgcaagg tgctcatcgc 20

<210> 22

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> accA1-R

<400> 22

tcagtccttg atctcgcaga 20

<210> 23

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> pccA-F

<400> 23

atgatcactt ccgtcctcgt 20

<210> 24

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> pccA-R

<400> 24

tcagtcggac tcgacgaccg 20

<210> 25

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> pccB-F

<400> 25

atgtccgagc cggaagagca 20

<210> 26

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> pccB-R

<400> 26

ttacaggggg atgttgccgt 20

<210> 27

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> epi-F

<400> 27

atgctgacgc gaatcgacca 20

<210> 28

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> epi-R

<400> 28

tcagtgctca ggtgactcaa 20

<210> 29

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> MatB-F

<400> 29

atgtcctctc tcttcccggc 20

<210> 30

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> MatB-R

<400> 30

tcagtcacgg ttcagcgccc 20

<210> 31

<211> 1773

<212> DNA

<213>Streptomyces coelicolor（S. coelicolor A3(2)）

<400> 31

atgcgcaagg tgctcatcgc caatcgtggc gaaatcgctg tccgcgtggc ccgggcctgc 60

cgggacgccg ggatcgcgag cgtggccgtc tacgcggatc cggaccggga cgcgttgcac 120

gtccgtgccg ctgatgaggc gttcgccctg ggtggtgaca cccccgcgac cagctatctg 180

gacatcgcca aggtcctcaa agccgcgcgc gagtcgggcg cggacgccat ccaccccggc 240

tacggattcc tctcggagaa cgccgagttc gcgcaggcgg tcctggacgc cggcctgatc 300

tggatcggcc cgcccccgca cgccatccgc gacctgggcg acaaggtcgc cgcccgccac 360

atcgcccagc gggccggcgc ccccctggtc gccggcaccc ccgaccccgt ctccggcgcg 420

gacgaggtcg tcgccttcgc caaggagcac ggcctgccca tcgccatcaa ggccgccttc 480

ggcggcggcg ggcgcggcct caaggtcgcc cgcaccctcg aagaggtgcc ggagctgtac 540

gactccgccg tccgcgaggc cgtggccgcc ttcggccgcg gggagtgctt cgtcgagcgc 600

tacctcgaca agccccgcca cgtggagacc cagtgcctgg ccgacaccca cggcaacgtg 660

gtcgtcgtct ccacccgcga ctgctccctc cagcgccgcc accaaaagct cgtcgaggag 720

gcccccgcgc ccttcctctc cgaggcccag acggagcagc tgtactcatc ctccaaggcc 780

atcctgaagg aggccggcta cgtcggcgcc ggcaccgtgg agttcctcgt cggcatggac 840

ggcacgatct ccttcctgga ggtcaacacc cgcctccagg tcgagcaccc ggtcaccgag 900

gaagtcgccg gcatcgacct ggtccgcgag atgttccgca tcgccgacgg cgaggaactc 960

ggctacgacg accccgccct gcgcggccac tccttcgagt tccgcatcaa cggcgaggac 1020

cccggccgcg gcttcctgcc cgcccccggc accgtcaccc tcttcgacgc gcccaccggc 1080

cccggcgtcc gcctggacgc cggcgtcgag tccggctccg tcatcggccc cgcctgggac 1140

tccctcctcg ccaaactgat cgtcaccggc cgcacccgcg ccgaggcact ccagcgcgcg 1200

gcccgcgccc tggacgagtt caccgtcgag ggcatggcca ccgccatccc cttccaccgc 1260

acggtcgtcc gcgacccggc cttcgccccc gaactcaccg gctccacgga ccccttcacc 1320

gtccacaccc ggtggatcga gacggagttc gtcaacgaga tcaagccctt caccacgccc 1380

gccgacaccg agacggacga ggagtcgggc cgggagacgg tcgtcgtcga ggtcggcggc 1440

aagcgcctgg aagtctccct cccctccagc ctgggcatgt ccctggcccg caccggcctg 1500

gccgccgggg cccgccccaa gcgccgcgcg gccaagaagt ccggccccgc cgcctcgggc 1560

gacaccctcg cctccccgat gcagggcacg atcgtcaaga tcgccgtcga ggaaggccag 1620

gaagtccagg aaggcgacct catcgtcgta ctcgaggcga tgaagatgga acagcccctc 1680

aacgcccaca ggtccggcac catcaagggc ctcaccgccg aggtcggcgc ctccctcacc 1740

tccggcgccg ccatctgcga gatcaaggac tga 1773

<210> 32

<211> 1845

<212> DNA

<213>Streptomyces coelicolor（S. coelicolor A3(2)）

<400> 32

atgatcactt ccgtcctcgt cgccaaccgc ggcgagatcg cctgccgcgt cttcagcacc 60

tgccgcgagt cgggcatccg caccgtcgcc gtgcactcgg acgccgacgc gaacgccctc 120

cacgcgcgcg tggccgacgc cgccgtacgc ctgccgggcg cggcccccgc cgacacctat 180

ctgcgcggcg acctgatcgt gaaggccgcc gtcgccgccg gagccgacgc cgtccacccc 240

ggctacggct tcctctccga gaacgccgac ttcgcgcgcg ccgtacggga cgcggggctg 300

gtgtggatcg gaccgccgcc cgaggccatc gaggcgatgg cgtccaagac ccgcgccaag 360

gaactgatgg gcatcgcgcc cctcaccgac gtcaccgagg ccgacctgcc ggtgctggtg 420

aaggcggcgg cgggcggcgg cggacgcggc atgcgcgtcg tacgccgcct cgccgacctc 480

gacgccgaac tgaccgccgc ccgcgcggag gccgcgagcg ccttcggcga cggcgaggtc 540

ttcgtcgagc cgtatgtggt cgacggccgc cacgtcgagg tgcagatcct cgccgacacc 600

cacggcacgg tgtgggtgct cggcacccgc gactgctccc tccaacgccg ccaccagaag 660

gtgatcgagg aggccccggc gcccggcctg acccccgggc tcaccgccga actccacgac 720

ctcgccgtgc gcgccgcccg cgccgtcgac tacgtcggcg cgggcaccgt cgagttcctc 780

gtcgccgacg gcacggcgca cttcctggag atgaacaccc gcctccaggt cgaacacccg 840

gtcacggagg cggtcttcgg catcgacctc gtcgccctcc agctccggat cgccgaaggc 900

cacgccctcg acgacgaccc cccgcgcgcg cgtggccacg ccgtcgaggc ccgcctctac 960

gccgaggacc cggcgaacgg ctgggccccg cagaccggcc gcctgcaccg cctcgccgtg 1020

ccggacggca tccgcctgga caccggctac accggcggcg acgacatcgg cgtccactac 1080

gacccgatgc tcgccaaggc ggtcgcccac gcacccacgc gcgcggaggc cgtccgccga 1140

ctcgccggcg ccctggaacg cgccgcgatc cacggcccgg tcaccaaccg cgacctcctc 1200

gtccgctccc tgcgccacga ggagttcacc tccggccgca tggacacggg cttctacgac 1260

cgccacctcg ccgccctcac cgagccggcc cccgaccccc tcgccccgct ggccgccgcc 1320

ctcgccgacg cgagcacccg tgcgggacgc ttcggcggct ggcgcaacct gccctcgcaa 1380

ccgcaggtca agcggtacgc cgtggcgggc gaggaacacg aggtccgtta cgggcacacc 1440

cgcacgggcc tcaccgccga gggcgtccgc gtcgtccacg cgggccccga ccgggtcgtc 1500

ctcgaagcgg acggcgtaca acgccccttc gacatcgccc gctacggcga ccacgtgcac 1560

gtcaacacca cgcgcctcac cgccctgccc cgcttccccg accccaccac ccagcacgcc 1620

cccggctccc tcctggcccc catgccgggc acggtcgtcc gcgtcgcgga gggcctgacc 1680

gagggcacca ccgtccaggc gggccagccg ttgctgtggc tggaggccat gaagatggaa 1740

cacaggatca ccgccccggt gacagggagg ctgaccgcac tcccggcggg cctcggacga 1800

caagtagaga tgggcgccct cttggcggtc gtcgagtccg actga 1845

<210> 33

<211> 1593

<212> DNA

<213>Streptomyces coelicolor（S. coelicolor A3(2)）

<400> 33

atgtccgagc cggaagagca gcagcccgac atccacacga ccgcgggcaa gctcgcggat 60

ctcaggcgcc gtatcgagga agcgacgcac gccggttccg cacgcgccgt cgagaagcag 120

cacgccaagg gcaagctgac ggctcgtgaa cgcatcgacc tcctcctcga cgagggttcc 180

ttcgtcgagc tggacgagtt cgcccggcac cgctccacca acttcggcct cgacgccaac 240

cgcccctacg gcgacggcgt cgtcaccggc tacggcaccg tcgacggccg ccccgtggcc 300

gtcttctccc aggacttcac cgtcttcggc ggcgcgctgg gcgaggtcta cggccagaag 360

atcgtcaagg tgatggactt cgccctcaag accggctgcc cggtcgtcgg catcaacgac 420

tccggcggcg cccgcatcca ggagggcgtg gcctccctcg gcgcctacgg cgagatcttc 480

cgccgcaaca cccacgcctc cggcgtgatc ccgcagatca gcctggtcgt cggcccgtgt 540

gcgggcggcg cggtgtactc ccccgcgatc accgacttca cggtgatggt ggaccagacc 600

agccacatgt tcatcaccgg tcccgacgtc atcaagacgg tcaccggcga ggacgtcggc 660

ttcgaggagc tgggcggcgc ccgcacccac aactccacct cgggcgtggc ccaccacatg 720

gccggcgacg agaaggacgc ggtcgagtac gtcaagcagc tcctgtcgta cctgccgtcc 780

aacaacctct ccgagccccc cgccttcccg gaggaggcgg acctcgcggt cacggacgag 840

gacgccgagc tggacacgat cgtcccggac tcggcgaacc agccctacga catgcactcc 900

gtcatcgagc acgtcctgga cgacgccgag ttcttcgaga cgcaacccct cttcgcgccg 960

aacatcctca ccggcttcgg ccgcgtggag ggccgcccgg tcggcatcgt cgccaaccag 1020

cccatgcagt tcgccggctg cctggacatc acggcctccg agaaggcggc ccgcttcgtg 1080

cgcacctgcg acgccttcaa cgtccccgtc ctcaccttcg tggacgtccc cggcttcctg 1140

cccggcgtcg accaggagca cgacggcatc atccgccgcg gcgccaagct gatcttcgcc 1200

tacgccgagg ccacggtgcc gctcatcacg gtcatcaccc gcaaggcctt cggcggcgcc 1260

tacgacgtca tgggctccaa gcacctgggc gccgacctca acctggcctg gcccaccgcc 1320

cagatcgccg tcatgggcgc ccaaggcgcg gtcaacatcc tgcaccgccg caccatcgcc 1380

gacgccggtg acgacgccga ggccacccgg gcccgcctga tccaggagta cgaggacgcc 1440

ctcctcaacc cctacacggc ggccgaacgc ggctacgtcg acgccgtgat catgccctcc 1500

gacactcgcc gccacatcgt ccgcggcctg cgccagctgc gcaccaagcg cgagtccctg 1560

cccccgaaga agcacggcaa catccccctg taa 1593

<210> 34

<211> 441

<212> DNA

<213>Streptomyces coelicolor（S. coelicolor A3(2)）

<400> 34

atgctgacgc gaatcgacca catcggaatc gcctgccacg acctcgacgc gaccgtcgag 60

ttctaccgtg ccacctacgg cttcgaggtg ttccacaccg aggtcaacga ggagcagggg 120

gtgcgcgagg ccatgctcaa gatcaacgat acgtcggacg ggggcgcctc gtacctccag 180

ctcctggagc cgacccgcga ggactccgcg gtcggcaagt ggctcgcgaa gaacggcgag 240

ggcgtccacc acatcgcctt cggtacggcg gacgtggacg cggacgccgc ggacatccgc 300

gacaagggcg tacgcgttct gtacgacgag ccccggcgcg gttccatggg gtcgcggatc 360

accttcctgc accccaagga ctgccatggc gtactgacag aactggtcac ttcggcggcc 420

gttgagtcac ctgagcactg a 441

<210> 35

<211> 1458

<212> DNA

<213>Streptomyces coelicolor（S. coelicolor A3(2)）

<400> 35

atgtcctctc tcttcccggc cctctccccg gccccgaccg gcgccccggc cgaccggccc 60

gcgctgcggt tcggcgagcg ctccctgacc tacgcggaac tcgccgcggc ggcgggcgcc 120

acggccgggc ggatcggcgg cgccggccgg gtcgcggtct gggccacccc ggcgatggag 180

accggcgtcg ccgtggtggc ggcgctgctg gccggggtcg ccgccgtacc gctcaacccg 240

aagtccggcg acaaggaact cgcgcacatc ctctccgaca gcgcgccctc gctcgtcctg 300

gcgcccccgg acgcggaact cccgcccgcc ctcggggccc tggagcgcgt cgacgtcgac 360

gtgcgggccc gcggggcggt ccccgaggac ggtgccgacg acggcgaccc cgcgctcgtc 420

gtctacacct cgggcaccac gggaccgccg aagggcgccg tcatcccccg gcgggcgctc 480

gccacgaccc tggacgcgct cgccgacgcg tggcagtgga ccggcgagga cgtgctggtg 540

caggggctgc cgctgttcca cgtgcacggg ctggtcctcg gcatcctcgg cccgctgcgc 600

cggggcgggt ccgtgcggca cctgggccgg ttctccaccg agggtgcggc gcgggagctg 660

aacgacggcg cgaccatgct gttcggggtg ccgacgatgt accaccggat cgccgagacg 720

ctccccgccg acccggagct ggcgaaggcg ctcgccgggg cccggctgct ggtgtcgggg 780

tcggccgcgc tgccggtgca cgaccacgag cgcatcgccg ccgccaccgg gcgccgggtg 840

atcgagcggt acggcatgac cgagacgctg atgaacacca gcgtgcgcgc cgacggcgag 900

ccgcgcgccg ggacggtggg cgtgccgctg cccggtgtgg agctgcggct ggtggaggag 960

gacggcacgc cgatcgcggc gctcgacggg gagagcgtcg gcgagatcca ggttcgcggc 1020

ccgaacctgt tcaccgagta cctgaaccgc cccgacgcca ccgccgccgc cttcaccgag 1080

gacggcttct tccgcaccgg cgacatggcg gtgcgcgacc ccgacggcta tgtccgcatc 1140

gtcggccgca aggccaccga cctgatcaag agcggcggtt acaagatcgg ggccggggag 1200

atcgagaacg ccctgctcga acacccggag gtccgggagg ccgccgtcac cggcgaaccc 1260

gaccccgacc tcggggaacg gatcgtggcc tggatcgtcc cggccgaccc cgccgccccg 1320

cccgccctcg gcacgctggc cgaccacgtc gccgcccggc tcgccccgca caagcggccg 1380

cgcgtcgtcc ggtacctcga cgcggtgccc cgcaacgaca tggggaagat catgaagcgg 1440

gcgctgaacc gtgactga 1458

<210> 36

<211> 73

<212> DNA

<213>Orange-yellow myxobacter（Myxococcus xanthus）

<400> 36

ggtcttgtag ctcaggggat agagcactcg gttgcggacc gagaggccgc aggttcgact 60

cctgccagga cca 73

<210> 37

<211> 74

<212> DNA

<213>Orange-yellow myxobacter（Myxococcus xanthus）

<400> 37

gcgttcgtag ctcaactgga tagagcaccg ggcttcgaac ccgggggttg ggggttcaag 60

tccctccgag cgcg 74

<210> 38

<211> 71

<212> DNA

<213>Orange-yellow myxobacter（Myxococcus xanthus）

<400> 38

tggggaatcg tctaacggca ggacagcaga ctctgactct gcttatctag gttcgaatcc 60

tagttcccca g 71

<210> 39

<211> 72

<212> DNA

<213>Orange-yellow myxobacter（Myxococcus xanthus）

<400> 39

ggccctgtcg tctagcggtt aggacggagc cctctcacgg ctcaaactcg ggttcgaatc 60

ccggcagggt ca

<210> 40

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> tRNA-F

<400> 40

cttctgacac cgcgcctcgt 20

<210> 41

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> tRNA-R

<400> 41

tgggcgtctc agtgtgaccc 20

<210> 42

<211> 200

<212> DNA

<213>Artificial sequence

<220>

<223> PKan

<400> 42

tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg 60

aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact 120

ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct gatcaagaga 180

caggatgagg atcgtttcgc 200

<210> 43

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> epo1A3-F

<400> 43

tttgctcaca tgttctttcc 20

<210> 44

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> epo1A3-R

<400> 44

ggtctgacgc tcagtggaac 20

<210> 45

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223> epo3K5-F

<400> 45

ggaaagaaca tgtgagcaaa ggaagatgcc aggaagatac 40

<210> 46

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> epo3K5-R

<400> 46

gttccactga gcgtcagacc 20

<210> 47

<211> 39

<212> DNA

<213>Artificial sequence

<220>

<223> epoA-F

<400> 47

gaattcgcgg ccgcttctag atggcggatc gtcccatcg 39

<210> 48

<211> 44

<212> DNA

<213>Artificial sequence

<220>

<223> epoA-R

<400> 48

tgcagcggcc gctactagta tcatagggca atgatttccc agtc 44

<210> 49

<211> 44

<212> DNA

<213>Artificial sequence

<220>

<223> epoB-F

<400> 49

gaattcgcgg ccgcttctag atgacgatca atcagcttct gaac 44

<210> 50

<211> 42

<212> DNA

<213>Artificial sequence

<220>

<223> epoB-R

<400> 50

tgcagcggcc gctactagta ttagctacgt ctcctgccct tg 42

<210> 51

<211> 48

<212> DNA

<213>Artificial sequence

<220>

<223> epoC-F

<400> 51

gaattcgcgg ccgcttctag atggaagaac aagattcctc cgctatcg 48

<210> 52

<211> 43

<212> DNA

<213>Artificial sequence

<220>

<223> epoC-R

<400> 52

tgcagcggcc gctactagta tcatgtaagc gccttgaatt tag 43

<210> 53

<211> 74

<212> DNA

<213>Artificial sequence

<220>

<223> epoD-F

<400> 53

ggttgttcgc gttgattgat gagtcactcg cgcgtgcggg aaagaggtga tactagtagc 60

ggccgctgca gtcc 74

<210> 54

<211> 71

<212> DNA

<213>Artificial sequence

<220>

<223> epoD-R

<400> 54

gccgcttgtt tcagcggatt ctgctgtgcc gtaggaccgc gagtagtcat ctagaagcgg 60

ccgcgaattc c 71

<210> 55

<211> 74

<212> DNA

<213>Artificial sequence

<220>

<223> epoE-F

<400> 55

cgttgctcgc cgaaaagctg gcgcagctcg cgcagatcgt tggtgagtaa tactagtagc 60

ggccgctgca gtcc 74

<210> 56

<211> 71

<212> DNA

<213>Artificial sequence

<220>

<223> epoE-R

<400> 56

tctcggtctg tcacgcaatc acctctttcc cgcacgcgcg agtgactcat ctagaagcgg 60

ccgcgaattc c 71

<210> 57

<211> 44

<212> DNA

<213>Artificial sequence

<220>

<223> epoF-F

<400> 57

gaattcgcgg ccgcttctag atggcgacca cgaatgccgg gaag 44

<210> 58

<211> 46

<212> DNA

<213>Artificial sequence

<220>

<223> epoF-R

<400> 58

tgcagcggcc gctactagta tcattttgcc tcgaacgccg ggcctg 46

<210> 59

<211> 41

<212> DNA

<213>Artificial sequence

<220>

<223> PKan-F

<400> 59

ggaattcgcg gccgcttcta gatagcttgc agtgggctta c 41

<210> 60

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223> PKan-R

<400> 60

gactagtacg atcctcatcc tgtctcttg 29

<210> 61

<211> 495

<212> DNA

<213>Artificial sequence

<220>

<223> attP0-ccdB-attB15

<400> 61

ggtttgtctg gtcaaccacc gcggtctcag tggtgtacgg tacaaaccca aagcttcggt 60

tgcgcgctga tttgtgcggc ataagaatat atactgatat gtatacccga agtatgtccg 120

gaagaggtgt gctatgcagt tcaaggttta cacctataaa agagagagcc gctatcgcct 180

gtttgtggat gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct 240

ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat 300

cggggatgaa agctggcgca tgatgaccac ccagatggtc agtgtgccgg tctccgtcat 360

cggagaagaa gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaatct 420

gatgttctgg ggaatataaa aggaaaaaag gatccccggc ttgtcgacga cggcgccctc 480

cgtcgtcagg atcat 495

<210> 62

<211> 17

<212> DNA

<213>Artificial sequence

<220>

<223> ccdB-F

<400> 62

gtaaaacgac ggccagt 17

<210> 63

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> ccdB-R

<400> 63

tgctagttat tgctcagcgg 20

<210> 64

<211> 41

<212> DNA

<213>Artificial sequence

<220>

<223> ccdB-vector-F

<400> 64

ggcactggcc gtcgttttac caatctgtac ctccttaagt c 41

<210> 65

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223> ccdB-vector-R

<400> 65

ccgctgagca ataactagca ggtatcgctt cccgaacctc 40

<210> 66

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223> BSD-ccdB-F

<400> 66

ctgaattggc tatccgcgtg caagagatta cgcgcagacc 40

<210> 67

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223> BSD-ccdB-R

<400> 67

gaagagcaca tacctcagtc gcagctcacg gtaactgatg 40

<210> 68

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> ST-F

<400> 68

gactgaggta tgtgctcttc 20

<210> 69

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223> ST-R

<400> 69

cacgcggata gccaattcag 20

<210> 70

<211> 611

<212> DNA

<213>Artificial sequence

<220>

<223> tRNA

<400> 70

cttctgacac cgcgcctcgt ggctggggcg ctctcgcagt cgcagttccg gtcttgtagc 60

tcaggggata gagcactcgg ttgcggaccg agaggccgca ggttcgactc ctgccaggac 120

cactcctcct tcagttgtct ggttcgatcc cggttggccg cccttcttta cccgccctcg 180

aaattgcgtt cgtagctcaa ctggatagag caccgggctt cgaacccggg ggttgggggt 240

tcaagtccct ccgagcgcgc accttccaag ttgttgcagt cccgaagtgg tattggtaga 300

gaagcgccgc ggtcgagcag gtccttgggg aatcgtctaa cggcaggaca gcagactctg 360

actctgctta tctaggttcg aatcctagtt ccccagcttg tagtcccgca gttgcagtgc 420

tcgttggtcc gaagcaacct gagcaggacg aaacaaaagg gttgacgagg gcagacgaaa 480

aaaggtagaa atcgcgggca gttagcggcg gttgaaacaa gtatcggccc tgtcgtctag 540

cggttaggac ggagccctct cacggctcaa actcgggttc gaatcccggc agggtcacac 600

tgagacgccc a 611

<210> 71

<211> 70

<212> DNA

<213>Artificial sequence

<220>

<223> BSD-epo-F

<400> 71

ggaacatcga atcactcaac gtcatcttga ggccctccaa agctggataa ctcgcggggg 60

tatcgcttcc 70

<210> 72

<211> 70

<212> DNA

<213>Artificial sequence

<220>

<223> BSD-epo-R

<400> 72

acgatcgcaa tcggatcttc ggctgcgcgc tcgatgggac gatccgccac aatctgtacc 60

tccttaagtc 70

<210> 73

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223> epo-vector-F

<400> 73

tactagtagc ggccgctgca gtcc 24

<210> 74

<211> 28

<212> DNA

<213>Artificial sequence

<220>

<223> epo-vector-R

<400> 74

ctagaagcgg ccgcgaattc cagaaatc 28

Claims (34)

1. a kind of method of heterogenous expression Epothilones, which is characterized in that introduce epothilone gene cluster in host strain, simultaneously Supplement Epothilones precursor route of synthesis.

2. the method for heterogenous expression Epothilones as described in claim 1, which is characterized in that the host strain belongs to primary kirschner Zoopagales (Burkholderiales).

3. the method for heterogenous expression Epothilones as described in claim 1, which is characterized in that the host strain is Burkholderia 7029 bacterial strains of mesh DSM.

4. the method for heterogenous expression Epothilones as claimed in claim 3, which is characterized in that the precursor route of synthesis is S- The route of synthesis of methylmalonyl CoA.

5. the method for heterogenous expression Epothilones as claimed in claim 4, which is characterized in that supplement the S- methylmalonyls The route of synthesis of coacetylase is, one or more in supplement PCC approach, MatB approach and mutase-isomery enzymatic pathway.

6. the method for heterogenous expression Epothilones as claimed in claim 4, which is characterized in that supplement the S- methylmalonyls The route of synthesis of coacetylase is supplement PCC approach, MatB approach and mutase-isomery enzymatic pathway.

7. such as the method for heterogenous expression Epothilones described in claim 5 or 6, which is characterized in that the PCC approach is by adding Add propionyl CoA carboxylase to supplement；The MatB approach is by adding malonyl coenzyme A/methylmalonyl CoA synzyme To supplement；The mutase-isomery enzymatic pathway is supplemented by adding methylmalonyl CoA isomerase.

8. the method for heterogenous expression Epothilones as claimed in claim 7, which is characterized in that the propionyl CoA carboxylase For accA1/pccB the or pccA/pccB genes of streptomyces coelicolor (S.coelicolor) A3 (2).

9. the method for heterogenous expression Epothilones as claimed in claim 7, which is characterized in that methylmalonyl CoA isomery Enzyme is the epi genes of streptomyces coelicolor (S.coelicolor) A3 (2).

10. the method for heterogenous expression Epothilones as claimed in claim 7, which is characterized in that malonyl coenzyme A/methyl-prop Two acyl-CoA synthetases are the matB genes of streptomyces coelicolor (S.coelicolor) A3 (2).

11. the method for heterogenous expression Epothilones as claimed in claim 4, which is characterized in that also draw in the host strain TRNA genes are entered.

12. the method for heterogenous expression Epothilones as claimed in claim 11, which is characterized in that the tRNA genes are Arg It is one or more in anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.

13. the method for heterogenous expression Epothilones as claimed in claim 12, which is characterized in that Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC gene sources are in orange-yellow myxobacter (Myxococcus xanthus)DK 1622。

14. the method for heterogenous expression Epothilones as claimed in claim 12, which is characterized in that in the Epothilones gene Promoter sequence is added before one or more of cluster gene.

15. the method for heterogenous expression Epothilones as claimed in claim 14, which is characterized in that in the Epothilones gene Promoter sequence is added before one or more of 6 genes of epoA, epoB, epoC, epoD, epoE and epoF in cluster.

16. the method for heterogenous expression Epothilones as claimed in claim 14, which is characterized in that in the Epothilones gene Promoter sequence is added before each in 6 genes of epoA, epoB, epoC, epoD, epoE and epoF in cluster.

17. the method for the heterogenous expression Epothilones as described in claim 15 or 16, which is characterized in that the promoter is PKan。

18. the method for heterogenous expression Epothilones as claimed in claim 17, which is characterized in that pass through the splicing again of gene Add the promoter sequence.

19. the method for heterogenous expression Epothilones as claimed in claim 18, which is characterized in that spliced using Bxb1 integrases Technology carries out the splicing.

20. a kind of engineering strain of heterogenous expression Epothilones, which is characterized in that introduced in the engineering strain Epothilone gene cluster, while being supplemented with Epothilones precursor route of synthesis.

21. the engineering strain of heterogenous expression Epothilones as claimed in claim 20, which is characterized in that the gene work The basic bacterial strain of journey bacterial strain is 7029 bacterial strains of Burkholderia mesh (Burkholderiales) DSM.

22. the engineering strain of heterogenous expression Epothilones as claimed in claim 21, which is characterized in that the basis bacterium The route of synthesis of Epothilones precursor S- methylmalonyl CoAs is supplemented in strain.

23. the engineering strain of heterogenous expression Epothilones as claimed in claim 22, which is characterized in that the S- methyl The route of synthesis of malonyl coenzyme A includes PCC approach, MatB approach and mutase-isomery enzymatic pathway.

24. the engineering strain of heterogenous expression Epothilones as claimed in claim 23, which is characterized in that on the basis In bacterial strain, the accA1/pccB by adding streptomyces coelicolor (S.coelicolor) A3 (2) supplements the PCC approach；It is logical Cross epi genes supplement mutase-isomery enzymatic pathway of addition streptomyces coelicolor (S.coelicolor) A3 (2)；Pass through addition The matB genes of streptomyces coelicolor (S.coelicolor) A3 (2) supplement MatB approach.

25. the engineering strain of heterogenous expression Epothilones as claimed in claim 24, which is characterized in that on the basis It also added tRNA genes in bacterial strain.

26. the engineering strain of heterogenous expression Epothilones as claimed in claim 25, which is characterized in that the tRNA bases Because of Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC genes.

27. the engineering strain of heterogenous expression Epothilones as claimed in claim 26, which is characterized in that described angstrom rich mould It is added to promoter sequence before one or more genes of plain gene cluster.

28. the engineering strain of heterogenous expression Epothilones as claimed in claim 27, which is characterized in that described angstrom rich mould Promoter sequence is added before each in 6 genes of epoA, epoB, epoC, epoD, epoE and epoF in plain gene cluster Row.

29. the engineering strain of heterogenous expression Epothilones as claimed in claim 28, which is characterized in that by spelling again The mode connect is spliced successively according to the sequence of epoA, epoB, epoC, epoD, epoE to epoF to add the promoter sequence Row.

30. the engineering strain of heterogenous expression Epothilones as claimed in claim 29, which is characterized in that the promoter For PKan.

31. a kind of engineering strain of heterogenous expression Epothilones, which is characterized in that the engineering strain is that short spore is more Capsule bacterium (Polyangium brachysporum) MMR11, deposit number CCTCC M 2017037 was protected on January 19th, 2017 It is hidden in China typical culture collection administrative center.

32. a kind of method producing Epothilones, which is characterized in that provide one plant as described in any one of claim 19-30 Bacterial strain, ferment at 30 ± 2 DEG C in the fermentation medium.

33. the method for production Epothilones as claimed in claim 32, which is characterized in that the fermentation medium is sent out for CYMG Ferment culture medium, every liter of formula are junket peptone 8g, yeast extract 4g, magnesium chloride hexahydrate 4.06g, 50% glycerine 10ml, micro member Plain 1ml, sodium acetate 50mg, sodium propionate 100mg, methylmalonic acid 100mg, Cys2 .5mg, serine 5mg, XAD-16 are big Macroporous adsorbent resin weight in wet base 1%, adjustment pH 7.0-7.5.

34. the method for production Epothilones as claimed in claim 32, which is characterized in that the fermentation time is 3 days.