pHT01枯草芽孢杆菌表达载体

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 http://www.biovector.net
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
//