pHT01枯草胞内质粒

基本信息

启动子:	Lac
复制子:	ColE1 ori
质粒分类:	广宿主系列，枯草杆菌载体
质粒大小:	7956bp
原核抗性:	Amp
筛选标记:	Chl
克隆菌株:	DH5α
培养条件:	37℃，有氧 LB
表达宿主:	枯草芽孢杆菌
诱导方式:	IPTG诱导
5'测序引物:	根据序列设计引物
3'测序引物:	根据序列设计引物

质粒简介

    All vectors use the strong promoter preceding the groESL operon of Bacillus subtilis fused to the lac operator allowing their induction by addition of IPTG. While the background level of expression of these expression cassettes is very low in the absence of the inducer, an induction factor of about 1,300 was measured using the bgaB reporter gene. The amount of recombinant protein produced after addition of IPTG may represent 10 and 13%, respectively, of the total cellular protein. High level secretion of amyQ α-amylase and cellulase A and B of Clostridium thermocellum was demonstrated. An efficient Shine-Dalgarno sequence as well as a multiple cloning site (BamH I, Xba I, AatII, SmaI) were also inserted. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an α-amylase was fused to the SD sequence of pHT01, thereby constructing pHT43.
   大肠杆菌-枯草芽孢杆菌穿梭质粒的表达载体pHT01可以在枯草芽孢杆菌中高效表达重组外源蛋白。载体基于强σA-依赖性启动子的枯草杆菌groE操纵子，通过添加lac操纵子改造成为一种高效可控的（IPTG诱导的）启动子。

质粒图谱

质粒序列

LOCUS       Exported File           7956 bp ds-DNA    circular SYN 07-2-2015
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7956)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported 2015-2-7 from MLCC

FEATURES             Location/Qualifiers
     source          1..7956
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1239..1255
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(1298..2380)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKTAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    2764..2788
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        6033..6137
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             6138..6998
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7169..7757
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa
       61 tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa
      121 gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat
      181 aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt
      241 tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat
      301 ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag
      361 gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt
      421 ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt
      481 tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc
      541 cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga
      601 aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa
      661 tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct
      721 cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa
      781 tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc
      841 ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat
      901 ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa
      961 aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc
     1021 ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac
     1081 tatggcgtgc tgctagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     1141 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     1201 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
     1261 cgagctcagg ccttaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1321 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1381 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1441 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1501 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1561 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1621 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1681 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1741 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1801 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     1861 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     1921 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     1981 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     2041 gattgtgcac cgccgtttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     2101 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     2161 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     2221 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2281 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2341 cggcatactc tgcgacatcg tataacgtta ctggtttcat caaaatcgtc tccctccgtt
     2401 tgaatatttg attgatcgta accagatgaa gcactctttc cactatccct acagtgttat
     2461 ggcttgaaca atcacgaaac aataattggt acgtacgatc tttcagccga ctcaaacatc
     2521 aaatcttaca aatgtagtct ttgaaagtat tacatatgta agatttaaat gcaaccgttt
     2581 tttcggaagg aaatgatgac ctcgtttcca ccggaattag cttggtacca gctattgtaa
     2641 cataatcggt acgggggtga aaaagctaac ggaaaaggga gcggaaaaga atgatgtaag
     2701 cgtgaaaaat tttttatctt atcacttgaa attggaaggg agattcttta ttataagaat
     2761 tgtggaattg tgagcggata acaattccca attaaaggag gaaggatcct ctagagtcga
     2821 cgtccccggg gcagcccgcc taatgagcgg gcttttttca cgtcacgcgt ccatggagat
     2881 ctttgtctgc aactgaaaag tttatacctt acctggaaca aatggttgaa acatacgagg
     2941 ctaatatcgg cttattagga atagtccctg tactaataaa atcaggtgga tcagttgatc
     3001 agtatatttt ggacgaagct cggaaagaat ttggagatga cttgcttaat tccacaatta
     3061 aattaaggga aagaataaag cgatttgatg ttcaaggaat cacggaagaa gatactcatg
     3121 ataaagaagc tctaaaacta ttcaataacc ttacaatgga attgatcgaa agggtggaag
     3181 gttaatggta cgaaaattag gggatctacc tagaaagcca caaggcgata ggtcaagctt
     3241 aaagaaccct tacatggatc ttacagattc tgaaagtaaa gaaacaacag aggttaaaca
     3301 aacagaacca aaaagaaaaa aagcattgtt gaaaacaatg aaagttgatg tttcaatcca
     3361 taataagatt aaatcgctgc acgaaattct ggcagcatcc gaagggaatt catattactt
     3421 agaggatact attgagagag ctattgataa gatggttgag acattacctg agagccaaaa
     3481 aactttttat gaatatgaat taaaaaaaag aaccaacaaa ggctgagaca gactccaaac
     3541 gagtctgttt ttttaaaaaa aatattagga gcattgaata tatattagag aattaagaaa
     3601 gacatgggaa taaaaatatt ttaaatccag taaaaatatg ataagattat ttcagaatat
     3661 gaagaactct gtttgttttt gatgaaaaaa caaacaaaaa aaatccacct aacggaatct
     3721 caatttaact aacagcggcc aaactgagaa gttaaatttg agaaggggaa aaggcggatt
     3781 tatacttgta tttaactatc tccattttaa cattttatta aaccccatac aagtgaaaat
     3841 cctcttttac actgttcctt taggtgatcg cggagggaca ttatgagtga agtaaaccta
     3901 aaaggaaata cagatgaatt agtgtattat cgacagcaaa ccactggaaa taaaatcgcc
     3961 aggaagagaa tcaaaaaagg gaaagaagaa gtttattatg ttgctgaaac ggaagagaag
     4021 atatggacag aagagcaaat aaaaaacttt tctttagaca aatttggtac gcatatacct
     4081 tacatagaag gtcattatac aatcttaaat aattacttct ttgatttttg gggctatttt
     4141 ttaggtgctg aaggaattgc gctctatgct cacctaactc gttatgcata cggcagcaaa
     4201 gacttttgct ttcctagtct acaaacaatc gctaaaaaaa tggacaagac tcctgttaca
     4261 gttagaggct acttgaaact gcttgaaagg tacggtttta tttggaaggt aaacgtccgt
     4321 aataaaacca aggataacac agaggaatcc ccgattttta agattagacg taaggttcct
     4381 ttgctttcag aagaactttt aaatggaaac cctaatattg aaattccaga tgacgaggaa
     4441 gcacatgtaa agaaggcttt aaaaaaggaa aaagagggtc ttccaaaggt tttgaaaaaa
     4501 gagcacgatg aatttgttaa aaaaatgatg gatgagtcag aaacaattaa tattccagag
     4561 gccttacaat atgacacaat gtatgaagat atactcagta aaggagaaat tcgaaaagaa
     4621 atcaaaaaac aaatacctaa tcctacaaca tcttttgaga gtatatcaat gacaactgaa
     4681 gaggaaaaag tcgacagtac tttaaaaagc gaaatgcaaa atcgtgtctc taagccttct
     4741 tttgatacct ggtttaaaaa cactaagatc aaaattgaaa ataaaaattg tttattactt
     4801 gtaccgagtg aatttgcatt tgaatggatt aagaaaagat atttagaaac aattaaaaca
     4861 gtccttgaag aagctggata tgttttcgaa aaaatcgaac taagaaaagt gcaataaact
     4921 gctgaagtat ttcagcagtt ttttttattt agaaatagtg aaaaaaatat aatcagggag
     4981 gtatcaatat ttaatgagta ctgatttaaa tttatttaga ctggaattaa taattaacac
     5041 gtagactaat taaaatttaa tgagggataa agaggataca aaaatattaa tttcaatccc
     5101 tattaaattt taacaagggg gggattaaaa tttaattaga ggtttatcca caagaaaaga
     5161 ccctaataaa atttttacta gggttataac actgattaat ttcttaatgg gggagggatt
     5221 aaaatttaat gacaaagaaa acaatctttt aagaaaagct tttaaaagat aataataaaa
     5281 agagctttgc gattaagcaa aactctttac tttttcattg acattatcaa attcatcgat
     5341 ttcaaattgt tgttgtatca taaagttaat tctgttttgc acaacctttt caggaatata
     5401 aaacacatct gaggcttgtt ttataaactc agggtcgcta aagtcaatgt aacgtagcat
     5461 atgatatggt atagcttcca cccaagttag cctttctgct tcttctgaat gtttttcata
     5521 tacttccatg ggtatctcta aatgattttc ctcatgtagc aaggtatgag caaaaagttt
     5581 atggaattga tagttcctct ctttttcttc aactttttta tctaaaacaa acactttaac
     5641 atctgagtca atgtaagcat aagatgtttt tccagtcata atttcaatcc caaatctttt
     5701 agacagaaat tctggacgta aatcttttgg tgaaagaatt tttttatgta gcaatatatc
     5761 cgatacagca ccttctaaaa gcgttggtga atagggcatt ttacctatct cctctcattt
     5821 tgtggaataa aaatagtcat attcgtccat ctacctatcc tattatcgaa cagttgaact
     5881 ttttaatcaa ggatcagtcc tttttttcat tattcttaaa ctgtgctctt aactttaaca
     5941 actcgatttg tttttccaga tctcgagggt aactagcctc gccgatcccg caagaggccc
     6001 ggcagtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     6061 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     6121 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6181 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6241 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6301 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6361 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     6421 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6481 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6541 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6601 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6661 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6721 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6781 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6841 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6901 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6961 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     7021 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     7081 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     7141 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7201 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     7261 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     7321 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7381 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7441 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7501 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7561 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7621 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7681 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7741 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7801 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7861 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7921 tgagcgagga agcggaagag cgcccaatac gcatgc