pHT01 Plasmid

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  • Model: PVT5001
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pHT01

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pHT01,Plasmid pHT01,pHT01 vector

 

pHT01 Information

Bacterial Resistance:Ampicillin/ chloramphenicol

Promoter: Lac

Replicon: ColE1 ori

Plasmid classification: the wide host series, Bacillus subtilis carrier

Plasmid size: 7956bp

Prokaryotic resistance: Amp

Screening markers: Chl

Cloned strain: DH5 alpha

Culture conditions: 37 centigrade, aerobic LB

Expression host: Bacillus subtilis

Induction mode: IPTG induction

5'sequencing primers: primers designed according to sequence

3'sequencing primers: primers designed according to sequence

Use: Bacillus subtilis

 

pHT01 Description

Escherichia coli bacillus subtilis shuttle plasmid pHT01 can efficiently express recombinant foreign protein in Bacillus subtilis. The carrier of Bacillus subtilis groE operon based on strong Sigma A- dependent promoter is transformed into a highly efficient and controllable (IPTG induced) promoter by adding lac operon.

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.

pHT01

 

 

pHT01 Sequence

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  

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

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