pHT43枯草分泌质粒

基本信息

启动子:	grac promoter
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:	氨苄青霉素Amp
筛选标记:	氯霉素Chl
克隆菌株:	大肠杆菌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 MLCC
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