pIEFBPR

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  • Model: PVT11306
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pIEFBPR

Catalog No. PVT11306
Packing 2ug

 

pIEFBPR Information

Replicon: ColE1 Ori

Plasmid classification: Bacillus subtilis plasmid; hay integration plasmid; hay secretory plasmid.

Plasmid size: 8965bp

Prokaryotic resistance: ampicillin Amp

Cloned strains of Escherichia coli, DH5 A and other Escherichia coli

Culture conditions: 37 centigrade, aerobic, LB

 

pIEFBPR Description

pIEFBPR  is a subtype integrated secretion vector.

ColE1 ori, ColE1 replication origin; amp, ampicillin-resistance marker; spc, spectinomycin-resistance marker; lacI, lac-repressor-encoding gene; Pspac, Pspac promoter; mazF, E.coli toxin gene; Plpp, promoter of E.coli lpp gene; mazE, E.coli antitoxin gene; DR, directly-repeated sequence; amyE-front and amyE-back, upstream and downstream sequences of the B.subtilis amyE gene, respectively; bpr-front and bpr-back, upstream and downstream sequences of the B.subtilis bpr gene, respectively. All of the DNA sequences located between the amyE-front and amyE-back or the bpr-front and bpr-back allow integration into the chromosome of B.subtilis via a double-crossover event, resulting in selection for spectinomycin-resistance. The unique restriction enzyme sites are highlighted in blue.

 

pIEFBPR Reference

mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis

 

pIEFBPR Sequence

LOCUS       Exported                8965 bp ds-DNA     circular SYN 15-SEP-2017

DEFINITION  synthetic circular DNA

SOURCE      synthetic DNA construct

  ORGANISM  synthetic DNA construct

REFERENCE   1  (bases 1 to 8965)

  TITLE     Direct Submission

  JOURNAL   Exported Sep 15, 2017

FEATURES             Location/Qualifiers

     source          1..8965

                     /organism="synthetic DNA construct"

                     /mol_type="other DNA"

     rep_origin      4..459

                     /direction=RIGHT

                     /label=f1 ori

                     /note="f1 bacteriophage origin of replication; arrow 

                     indicates direction of (+) strand synthesis"

     promoter        626..644

                     /label=T7 promoter

                     /note="promoter for bacteriophage T7 RNA polymerase"

     misc_feature    692..1697

                     /label=bpr-front

                     /note="upstream of Bacillus subtilis bpr gene"

     misc_feature    1728..1852

                     /label=DR sequence

     CDS             complement(1900..2982)

                     /codon_start=1

                     /gene="lacI"

                     /product="lac repressor"

                     /label=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"

     CDS             complement(3232..3567)

                     /codon_start=1

                     /label=mazF

                     /note="Escherichia coli toxin MazF"

                     /translation="MVSRYVPDMGDLIWVDFDPTKGSEQAGHRPAVVLSPFMYNNKTGM

                     CLCVPCTTQSKGYPFEVVLSGQERDGVALADQVKSIAWRARGATKKGTVAPEELQLIKA

                     KINVLIG"

     CDS             4179..4931

                     /codon_start=1

                     /product="spectinomycin adenyltransferase; selectable 

                     either in Escherichia coli or Bacillus subtilis"

                     /label=spc

                     /translation="LNTYEQINKVKKILRKHLKNNLIGTYMFGSGVESGLKPNSDLDFL

                     VVVSEPLTDQSKEILIQKIRPISKKIGDKSNLRYIELTIIIQQEMVPWNHPPKQEFIYG

                     EWLQELYEQGYIPQKELNSDLTIMLYQAKRKNKRIYGNYDLEELLPDIPFSDVRRAIMD

                     SSEELIDNYQDDETNSILTLCRMILTMDTGKIIPKDIAGNAVAESSPLEHRERILLAVR

                     SYLGENIEWTNENVNLTINYLNNRLKKL"

     misc_feature    4933..5057

                     /label=DR sequence

     misc_feature    5197..6235

                     /label=bpr-back

                     /note="downstream of Bacillus subtilis bpr gene; homologous

                     arm for recombination"

     misc_feature    6450..6758

                     /label=mazE

     CDS             complement(7218..7806)

                     /codon_start=1

                     /label=ColE1 ori

                     /note="high-copy-number colE1/pMB1/pBR322/pUC origin of 

                     replication"

                     /translation="LRSFFSARNLLLANKKTTATSGGLFAGSRATNSFSEGNWLQQSAD

                     TKYCPSSVAVVRPPLQELCSTAYIPRSANPVTSGCCQWR*VVSYRVGLKTIVTG*GAAV

                     GLNGGFVHTAQLGANDLHRTEIPTA*AMRKRHASRREKGGQVSGKRQGRNRRAHEGASR

                     GKRLVSL*SCRVSPPLT*ASIFVMLVRGAEPME"

     CDS             complement(7977..8837)

                     /codon_start=1

                     /gene="bla"

                     /product="beta-lactamase"

                     /label=AmpR

                     /note="confers resistance to ampicillin, carbenicillin, and

                     related antibiotics"

                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI

                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS

                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW

                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA

                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS

                     LIKHW"

     promoter        complement(8838..8942)

                     /gene="bla"

                     /label=AmpR promoter

ORIGIN

        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga

       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg

      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat

      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg

      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata

      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt

      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat

      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg

      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg

      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg

      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg

      661 gccccccctc gagttaacct gcaggcgcgc ctttgatggg tatcggtatt gctactgggg

      721 aaaatcgcgc ggcagaggca gcaaaaaaag caatttccag cccgcttctt gaagcggcca

      781 ttgacggtgc gcaaggcgtc ctcatgaaca tcactggagg aacaaacctc agcctatatg

      841 aggttcagga agcagcagac attgtcgctt cggcgtctga tcaagacgta aacatgattt

      901 tcggttctgt tattaatgaa aatctaaaag atgagattgt ggtgacagtg attgcaaccg

      961 gctttatcga acaagagaag gacgtgacga agcctcagcg cccaagctta aatcaaagca

     1021 tcaaaacaca caatcaaagt gttccgaagc gtgacgcaaa acgtgaggaa cctcagcagc

     1081 agaacacagt aagccgtcat acttcacagc cggctgatga tacgcttgac atcccgacat

     1141 tcttaagaaa ccgtaataaa cgcggctaat gtaaaggaca aaatcgtttt cgattttgtc

     1201 ttttttgttt ttctcttcac acttccttct tataaagtct ttttccctat tgctttcctt

     1261 cgcttagtaa caaaacagat aattagaccc atttattttt gtgacatttt tatcattttc

     1321 atatatatgg aaattgaatg acatgaaacg acaatatctg taattcagat tgtctacagt

     1381 taatatacag cgatgttctg acaaaccatt cattattaaa aggagggacg acactttttt

     1441 taaaaagcat gttgaaaaag ggggatgaaa atgaggaaaa aaacgaaaaa cagactcatc

     1501 agctctgttt taagtacagt tgtcatcagt tcactgctgt ttccgggagc agccggggca

     1561 agcagtaaag tcacctcacc ttctgttaaa aaggagcttc aatctgcgga atccattcaa

     1621 aacaagattt cgagttcatt aaagaaaagc tttaaaaaga aagaaaaaac gacttttctg

     1681 attaaattta aagatctatc gatgctagcc atggtcgacg cggccgcaaa attgaaaaaa

     1741 tggtggaaac acttttttca atttttttgt tttattattt aatatttggg aaatattcat

     1801 tctaattggt aatcagattt tagaaaacaa taaacccttg cataggggga tctccggagt

     1861 ttaaaccgcg gatcctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg

     1921 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg

     1981 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc

     2041 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag

     2101 gcgaaaatcc tgtttgatgg tggttgacgg cgggatataa catgagctgt cttcggtatc

     2161 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg

     2221 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc

     2281 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc

     2341 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg

     2401 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac

     2461 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg

     2521 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc

     2581 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag

     2641 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac

     2701 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc

     2761 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg

     2821 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg

     2881 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac

     2941 accggcatac tctgcgacat cgtataacgt tactggtttc atcaaaatcg tctccctccg

     3001 tttgaatatt tgattgatcg taaccagatg aagcactctt tccactatcc ctacagtgtt

     3061 atggcttgaa caatcacgaa acaataattg gtacgtacga tctttcagcc gactcaaaca

     3121 tcaaatctta caaatgtagt ctttgaaagt attacatatg taagatttaa atgcaaccgt

     3181 tttttcggaa ggaaatgatg acctcgtttc caccggaatt agcttgcatg cctacccaat

     3241 cagtacgtta attttggctt taatgagttg taattcctct ggggcaactg ttcctttctt

     3301 cgttgctcct cttgcccgcc aggcgatact ttttacctga tcagctaacg ctacgccatc

     3361 acgttcctga ccggataaaa caacttcgaa cggatatcct tttgattgcg ttgtacaagg

     3421 aacacacaga cacatacctg ttttgttgtt gtacatgaaa ggactcagga caacagctgg

     3481 acgatgtcca gcttgctcgc taccttttgt cgggtcaaaa tcaacccaaa tcagatcgcc

     3541 catatcgggt acgtatcggc ttaccatacc tgcttcctcc ttaagcttaa ttgttatccg

     3601 ctcacaattc cacacattat gccacacctt gtagataaag tcaacaactt tttgcaaaat

     3661 ttttcaggaa ttttagcaga ggttgttctg gatgtagaac aaaacatctt tccgctcttg

     3721 tgctgttagg atatctttct tggaagctag gtaggcaagg gctacctcaa ataaatcttc

     3781 ttcagggtgc gctattttta aggtgcctag tgaggtcttg accacttcac ccataatttc

     3841 agtgccgaat agtctggact gggctgtgta gaattcctgc agccctggcg aatggcgatt

     3901 ttcgttcgtg aatacatgtt ataataacta taactaataa cgtaacgtga ctggcaagag

     3961 atatttttaa aacaatgaat aggtttacac ttactttagt tttatggaaa tgaaagatca

     4021 tatcatatat aatctagaat aaaattaact aaaataatta ttatctagat aaaaaattta

     4081 gaagccaatg aaatctataa ataaactaaa ttaagtttat ttaattaaca actatggata

     4141 taaaataggt actaatcaaa atagtgagga ggatatattt gaatacatac gaacaaatta

     4201 ataaagtgaa aaaaatactt cggaaacatt taaaaaataa ccttattggt acttacatgt

     4261 ttggatcagg agttgagagt ggactaaaac caaatagtga tcttgacttt ttagtcgtcg

     4321 tatctgaacc attgacagat caaagtaaag aaatacttat acaaaaaatt agacctattt

     4381 caaaaaaaat aggagataaa agcaacttac gatatattga attaacaatt attattcagc

     4441 aagaaatggt accgtggaat catcctccca aacaagaatt tatttatgga gaatggttac

     4501 aagagcttta tgaacaagga tacattcctc agaaggaatt aaattcagat ttaaccataa

     4561 tgctttacca agcaaaacga aaaaataaaa gaatatacgg aaattatgac ttagaggaat

     4621 tactacctga tattccattt tctgatgtga gaagagccat tatggattcg tcagaggaat

     4681 taatagataa ttatcaggat gatgaaacca actctatatt aactttatgc cgtatgattt

     4741 taactatgga cacgggtaaa atcataccaa aagatattgc gggaaatgca gtggctgaat

     4801 cttctccatt agaacatagg gagagaattt tgttagcagt tcgtagttat cttggagaga

     4861 atattgaatg gactaatgaa aatgtaaatt taactataaa ctatttaaat aacagattaa

     4921 aaaaattata aaaaaattga aaaaatggtg gaaacacttt tttcaatttt tttgttttat

     4981 tatttaatat ttgggaaata ttcattctaa ttggtaatca gattttagaa aacaataaac

     5041 ccttgcatag ggggatctcg acatggatga gcgatgatga tatccgttta ggctgggcgg

     5101 tgatagcttc tcgttcaggc agtacgcctc ttttcttttc cagacctgag ggaggcggaa

     5161 atggtgtgag gttcccgggc cggccaattg atgcatcagt tcaaaaggga ttatggtcga

     5221 taaagatttt tatctcgtat atatccagtc gaaacctgat ccgtattcac ctggattggc

     5281 aatggatgaa accggccaga attccggccg caactggcag tatatagatg gaaagtggca

     5341 gccaggtgac aaagcggatg gcaactatat gattcgcgca ttagttgatt atgaagctgc

     5401 tgtacctgag attacttcac cgacagacaa atcatacaca aataaggata gcgtcactgt

     5461 aaaaggaaac gcttctcctg gcacaacggt acacatttat aatggagaga aagaagcagg

     5521 agaaacgaaa gctgctgcgg atggcacgtt ccatgcaggc atcatactca acaagggtga

     5581 aaatgagctg acggcaactg catcaactga caacggaaca acagatgcct ccagcccaat

     5641 cacggtcacg cttgatcaag aaaagcctga attaacactg gacaatccaa aggatggcgg

     5701 gaaaacaaat aaagaaacgc tgactgtcaa aggggctgta tccgatgaca atctgaaaga

     5761 cgtcaaggtg aatggcaaaa aagcaacagt agctgatggt tcatactcag cccgtattct

     5821 tttggaaaat ggaagaaatg aaatcaaggt aattgctaca gacttggcag gcaacaaaac

     5881 gacgaaaaag acagtcattg atgtgaactt tgacaagcct gtcatttccg gcttaattcc

     5941 gggagaggat aaaaacttaa aagccggtga atctgtgaaa atcgctttct caagcgctga

     6001 ggatttagat gcaacgttta ccattcgtat gccgctgacc aatgcaagag cgagtgtgca

     6061 aaatgccacc gaactcccgt taagagaaat ctctccgggg agatatgaag gctattggac

     6121 tgccacttct tctattaaag caaaaggagc aaaagtagaa gtgatcgtcc gagatgatta

     6181 tggaaatgaa acaagaaaaa ctgcgaatgg aaaacttaat atgaacacag aaaataccgg

     6241 tcctagggcg atcgcactag tcgataacca gaagcaataa aaaatcaaat cggatttcac

     6301 tatataatct cactttatct aagatgaatc cgatggaagc atcctgtttt ctctcaattt

     6361 ttttatctaa aacccagcgt tcgatgcttc tttgagcgaa cgatcaaaaa taagtgcctt

     6421 cccatcaaaa aaatattctc aacataaaaa actttgtgta atacttgtaa cgctacatgg

     6481 agattaactc aatctagagg gtattaataa tgatccacag tagcgtaaag cgttggggaa

     6541 attcaccggc ggtgcggatt ccggctacgt taatgcaggc gctcaatctg aatattgatg

     6601 atgaagtgaa gattgacctg gtggatggca aattaattat tgagccagtg cgtaaagagc

     6661 ccgtatttac gcttgctgaa ctggtcaacg acatcacgcc ggaaaacctc cacgagaata

     6721 tcgactgggg agagccgaaa gataaggaag tctggtaaga gctccagctt ttgttccctt

     6781 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat

     6841 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg

     6901 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag

     6961 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt

     7021 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg

     7081 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg

     7141 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag

     7201 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga

     7261 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct

     7321 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc

     7381 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg

     7441 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc

     7501 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca

     7561 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag

     7621 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct

     7681 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc

     7741 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga

     7801 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca

     7861 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat

     7921 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac

     7981 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt

     8041 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt

     8101 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag

     8161 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct

     8221 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt

     8281 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc

     8341 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt

     8401 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg

     8461 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg

     8521 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct

     8581 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc

     8641 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt

     8701 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt

     8761 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg

     8821 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat

     8881 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg

     8941 cgcacatttc cccgaaaagt gccac

//

 

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