pHT43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: 8057bp
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
    所有在枯草芽孢杆菌的groESL操纵子之前使强启动子与lac操纵子融合的载体都可通过加入IPTG进行诱导。尽管当未添加诱导物时表达组件的背景表达水平很低，还是成功从约1300种诱导因子中筛选出一种来使用bgaB报道基因（Phan等，2005）。当分别将htpG和pbpE基因融合到groE启动子时，加入IPTG后，表达的重组蛋白可能分别占细胞总蛋白的10%和13%（Phan等，2005）。热纤梭菌的amyQα-淀粉酶和纤维素酶A、B的高水平表达实验证实。该载体还插入了一个高效SD序列以及一个多克隆位点（BamH I, Xba I, Aat II, Sma I）。编码α-淀粉酶的amyQ基因的信号肽编码区域与pHT01的SD序列融合，构成了pHT43，以此获得分泌的重组蛋白。

质粒图谱
质粒序列
LOCUS       Exported File           8057 bp ds-DNA    circular SYN 07-2-2015
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    pHT43
SOURCE      synthetic DNA construct
 ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8057)
 AUTHORS   .
 TITLE     Direct Submission
 JOURNAL   Exported 2015-2-7 from http://www.biovector.net
FEATURES             Location/Qualifiers
    source          1..8057
                    /organism="synthetic DNA construct"
                    /mol_type="other DNA"
    primer_bind     1238..1254
                    /note="M13 fwd"
                    /note="common sequencing primer, one of multiple similar
                    variants"
    CDS             complement(1297..2379)
                    /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
                    EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                    EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                    MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                    YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                    ALADSLMQLARQVSRLESGQ"
    protein_bind    2763..2787
                    /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        6134..6238
                    /gene="bla"
                    /note="AmpR promoter"
    CDS             6239..7099
                    /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      7270..7858
                    /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 gaataaagac cacattaaaa
     961 aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct
    1021 tatcttgata ataagggtaa ctattgccga tcgtccattc cgacagcatc gccagtcact
    1081 atggcgtgct gctagcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg
    1141 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
    1201 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc
    1261 gagctcaggc cttaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
    1321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
    1381 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
    1441 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
    1501 aaaatcctgt ttgatggtgg ttgacggcgg gatataacat gagctgtctt cggtatcgtc
    1561 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
    1621 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
    1681 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
    1741 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
    1801 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
    1861 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
    1921 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
    1981 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
    2041 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
    2101 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
    2161 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
    2221 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
    2281 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
    2341 ggcatactct gcgacatcgt ataacgttac tggtttcatc aaaatcgtct ccctccgttt
    2401 gaatatttga ttgatcgtaa ccagatgaag cactctttcc actatcccta cagtgttatg
    2461 gcttgaacaa tcacgaaaca ataattggta cgtacgatct ttcagccgac tcaaacatca
    2521 aatcttacaa atgtagtctt tgaaagtatt acatatgtaa gatttaaatg caaccgtttt
    2581 ttcggaagga aatgatgacc tcgtttccac cggaattagc ttggtaccag ctattgtaac
    2641 ataatcggta cgggggtgaa aaagctaacg gaaaagggag cggaaaagaa tgatgtaagc
    2701 gtgaaaaatt ttttatctta tcacttgaaa ttggaaggga gattctttat tataagaatt
    2761 gtggaattgt gagcggataa caattcccaa ttaaaggagg aaggatcaat gattcaaaaa
    2821 cgaaagcgga cagtttcgtt cagacttgtg cttatgtgca cgctgttatt tgtcagtttg
    2881 ccgattacaa aaacatcagc cgtaggatcc tctagagtcg acgtccccgg ggcagcccgc
    2941 ctaatgagcg ggcttttttc acgtcacgcg tccatggaga tctttgtctg caactgaaaa
    3001 gtttatacct tacctggaac aaatggttga aacatacgag gctaatatcg gcttattagg
    3061 aatagtccct gtactaataa aatcaggtgg atcagttgat cagtatattt tggacgaagc
    3121 tcggaaagaa tttggagatg acttgcttaa ttccacaatt aaattaaggg aaagaataaa
    3181 gcgatttgat gttcaaggaa tcacggaaga agatactcat gataaagaag ctctaaaact
    3241 attcaataac cttacaatgg aattgatcga aagggtggaa ggttaatggt acgaaaatta
    3301 ggggatctac ctagaaagcc acaaggcgat aggtcaagct taaagaaccc ttacatggat
    3361 cttacagatt ctgaaagtaa agaaacaaca gaggttaaac aaacagaacc aaaaagaaaa
    3421 aaagcattgt tgaaaacaat gaaagttgat gtttcaatcc ataataagat taaatcgctg
    3481 cacgaaattc tggcagcatc cgaagggaat tcatattact tagaggatac tattgagaga
    3541 gctattgata agatggttga gacattacct gagagccaaa aaacttttta tgaatatgaa
    3601 ttaaaaaaaa gaaccaacaa aggctgagac agactccaaa cgagtctgtt tttttaaaaa
    3661 aaatattagg agcattgaat atatattaga gaattaagaa agacatggga ataaaaatat
    3721 tttaaatcca gtaaaaatat gataagatta tttcagaata tgaagaactc tgtttgtttt
    3781 tgatgaaaaa acaaacaaaa aaaatccacc taacggaatc tcaatttaac taacagcggc
    3841 caaactgaga agttaaattt gagaagggga aaaggcggat ttatacttgt atttaactat
    3901 ctccatttta acattttatt aaaccccata caagtgaaaa tcctctttta cactgttcct
    3961 ttaggtgatc gcggagggac attatgagtg aagtaaacct aaaaggaaat acagatgaat
    4021 tagtgtatta tcgacagcaa accactggaa ataaaatcgc caggaagaga atcaaaaaag
    4081 ggaaagaaga agtttattat gttgctgaaa cggaagagaa gatatggaca gaagagcaaa
    4141 taaaaaactt ttctttagac aaatttggta cgcatatacc ttacatagaa ggtcattata
    4201 caatcttaaa taattacttc tttgattttt ggggctattt tttaggtgct gaaggaattg
    4261 cgctctatgc tcacctaact cgttatgcat acggcagcaa agacttttgc tttcctagtc
    4321 tacaaacaat cgctaaaaaa atggacaaga ctcctgttac agttagaggc tacttgaaac
    4381 tgcttgaaag gtacggtttt atttggaagg taaacgtccg taataaaacc aaggataaca
    4441 cagaggaatc cccgattttt aagattagac gtaaggttcc tttgctttca gaagaacttt
    4501 taaatggaaa ccctaatatt gaaattccag atgacgagga agcacatgta aagaaggctt
    4561 taaaaaagga aaaagagggt cttccaaagg ttttgaaaaa agagcacgat gaatttgtta
    4621 aaaaaatgat ggatgagtca gaaacaatta atattccaga ggccttacaa tatgacacaa
    4681 tgtatgaaga tatactcagt aaaggagaaa ttcgaaaaga aatcaaaaaa caaataccta
    4741 atcctacaac atcttttgag agtatatcaa tgacaactga agaggaaaaa gtcgacagta
    4801 ctttaaaaag cgaaatgcaa aatcgtgtct ctaagccttc ttttgatacc tggtttaaaa
    4861 acactaagat caaaattgaa aataaaaatt gtttattact tgtaccgagt gaatttgcat
    4921 ttgaatggat taagaaaaga tatttagaaa caattaaaac agtccttgaa gaagctggat
    4981 atgttttcga aaaaatcgaa ctaagaaaag tgcaataaac tgctgaagta tttcagcagt
    5041 tttttttatt tagaaatagt gaaaaaaata taatcaggga ggtatcaata tttaatgagt
    5101 actgatttaa atttatttag actggaatta ataattaaca cgtagactaa ttaaaattta
    5161 atgagggata aagaggatac aaaaatatta atttcaatcc ctattaaatt ttaacaaggg
    5221 ggggattaaa atttaattag aggtttatcc acaagaaaag accctaataa aatttttact
    5281 agggttataa cactgattaa tttcttaatg ggggagggat taaaatttaa tgacaaagaa
    5341 aacaatcttt taagaaaagc ttttaaaaga taataataaa aagagctttg cgattaagca
    5401 aaactcttta ctttttcatt gacattatca aattcatcga tttcaaattg ttgttgtatc
    5461 ataaagttaa ttctgttttg cacaaccttt tcaggaatat aaaacacatc tgaggcttgt
    5521 tttataaact cagggtcgct aaagtcaatg taacgtagca tatgatatgg tatagcttcc
    5581 acccaagtta gcctttctgc ttcttctgaa tgtttttcat atacttccat gggtatctct
    5641 aaatgatttt cctcatgtag caaggtatga gcaaaaagtt tatggaattg atagttcctc
    5701 tctttttctt caactttttt atctaaaaca aacactttaa catctgagtc aatgtaagca
    5761 taagatgttt ttccagtcat aatttcaatc ccaaatcttt tagacagaaa ttctggacgt
    5821 aaatcttttg gtgaaagaat ttttttatgt agcaatatat ccgatacagc accttctaaa
    5881 agcgttggtg aatagggcat tttacctatc tcctctcatt ttgtggaata aaaatagtca
    5941 tattcgtcca tctacctatc ctattatcga acagttgaac tttttaatca aggatcagtc
    6001 ctttttttca ttattcttaa actgtgctct taactttaac aactcgattt gtttttccag
    6061 atctcgaggg taactagcct cgccgatccc gcaagaggcc cggcagtcag gtggcacttt
    6121 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
    6181 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
    6241 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
    6301 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
    6361 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
    6421 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
    6481 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
    6541 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
    6601 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
    6661 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
    6721 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
    6781 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
    6841 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
    6901 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
    6961 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
    7021 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
    7081 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
    7141 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
    7201 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
    7261 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
    7321 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
    7381 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
    7441 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
    7501 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
    7561 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
    7621 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
    7681 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
    7741 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
    7801 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
    7861 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
    7921 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
    7981 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
    8041 gcgcccaata cgcatgc
//