LOCUS       V004424                10746 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004424
VERSION     V004424
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10746)
  AUTHORS   Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC,
            Minton NP.
  TITLE     The ClosTron: Mutagenesis in Clostridium refined and streamlined
  JOURNAL   J. Microbiol. Methods 80 (1), 49-55 (2010)
   PUBMED   19891996
REFERENCE   2  (bases 1 to 10746)
  AUTHORS   Heap JT, Kuehne KA, Ehsaan M, Cartman ST, Cooksley CM, Minton NP.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-SEP-2010) School of Molecular Medical Sciences, The
            University of Nottingham, Centre for Biomolecular Sciences,
            Nottingham, Nottinghamshire NG72RD, UK
REFERENCE   3  (bases 1 to 10746)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 10746)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J.
            Microbiol. Methods"; date: "2010"; volume: "80"; issue: "1"; pages:
            "49-55"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (28-SEP-2010) School of Molecular Medical Sciences, The University
            of Nottingham, Centre for Biomolecular Sciences, Nottingham,
            Nottinghamshire NG72RD, UK"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             233..406
                     /label="lacZ-alpha"
                     /note="LacZ-alpha fragment of beta-galactosidase"
     protein_bind    complement(814..847)
                     /label="FRT (minimal)"
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     CDS             complement(864..1598)
                     /gene="ermBP"
                     /label="rRNA adenine N-6-methyltransferase"
                     /note="rRNA adenine N-6-methyltransferase from Enterococcus
                     faecalis. Accession#: P0A4D5"
     protein_bind    complement(2101..2134)
                     /label="FRT (minimal)"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     RBS             2392..2414
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2606..4402
                     /gene="ltrA"
                     /label="Group II intron-encoded protein LtrA"
                     /note="Group II intron-encoded protein LtrA from
                     Lactococcus lactis subsp. cremoris (strain MG1363).
                     Accession#: P0A3U1"
     terminator      4590..4676
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4768..4795
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     CDS             8390..9010
                     /gene="catP"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from Clostridium
                     perfringens. Accession#: P26826"
     rep_origin      9190..9778
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     oriT            10099..10208
                     /label="oriT"
                     /note="incP origin of transfer"
     CDS             10241..10609
                     /label="traJ"
                     /note="oriT-recognizing protein"
ORIGIN
        1 cctgcagggt gtagtagcct gtgaaataag taaggaaaaa aaagaagtaa gtgttatata
       61 tgatgattat tttgtagatg tagataggat aatagaatcc atagaaaata taggttatac
      121 agttatataa aaattacttt aaaaattaat aaaaacatgg taaaatataa atcgtataaa
      181 gttgtgtaat ttttaagctt gagctcataa caatttcaca caggaaacag ctatgaccat
      241 gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
      301 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
      361 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gctaataaag
      421 atcttgtaca atctgtagga gaacctatgg gaacgaaacg aaagcgatgc cgagaatctg
      481 aatttaccaa gacttaacac taactgggga taccctaaac aagaatgcct aatagaaagg
      541 aggaaaaagg ctatagcact agagcttgaa aatcttgcaa gggtacggag tactcgtagt
      601 agtctgagaa gggtaacgcc ctttacatgg caaaggggta cagttattgt gtactaaaat
      661 taaaaattga ttagggagga aaacctcaaa atgaaaccaa caatggcaat tttagaaaga
      721 atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta tcgttatctt
      781 ttacgtccag atatttatta cgtggcgacg cgtgaagttc ctatactttc tagagaatag
      841 gaacttcgcg actcatagaa ttatttcctc ccgttaaata atagataact attaaaaata
      901 gacaatactt gctcataagt aacggtactt aaattgttta ctttggcgtg tttcattgct
      961 tgatgaaact gatttttagt aaacagttga cgatattctc gattgaccca ttttgaaaca
     1021 aagtacgtat atagcttcca atatttatct ggaacatctg tggtatggcg ggtaagtttt
     1081 attaagacac tgtttacttt tggtttagga tgaaagcatt ccgctggcag cttaagcaat
     1141 tgctgaatcg agacttgagt gtgcaagagc aaccctagtg ttcggtgaat atccaaggta
     1201 cgcttgtaga atccttcttc aacaatcaga tagatgtcag acgcatggct ttcaaaaacc
     1261 acttttttaa taatttgtgt gcttaaatgg taaggaatac tcccaacaat tttatacctc
     1321 tgtttgttag ggaattgaaa ctgtagaata tcttggtgaa ttaaagtgac acgagtattc
     1381 agttttaatt tttctgacga taagttgaat agatgactgt ctaattcaat agacgttacc
     1441 tgtttactta ttttagccag tttcgtcgtt aaatgccctt tacctgttcc aatttcgtaa
     1501 acggtatcgg tttcttttaa attcaattgt tttattattt ggttgagtac tttttcactc
     1561 gttaaaaagt tttgagaata ttttatattt ttgttcatac cagcaccaga agcaccagca
     1621 tctcttgggt taattgaggc ctgagtataa ggtgacttat acttgtaatc tatctaaacg
     1681 gggaacctct ctagtagaca atcccgtgct aaattgtagg actgcccttt aataaatact
     1741 tctatattta aagaggtatt tatgaaaagc ggaatttatc agattaaaaa tactttctct
     1801 agagaaaatt tcgtctggat tagttactta tcgtgtaaaa tctgataaat ggaattggtt
     1861 ctacataaat gcctaacgac tatccctttg gggagtaggg tcaagtgact cgaaacgata
     1921 gacaacttgc tttaacaagt tggagatata gtctgctctg catggtgaca tgcagctgga
     1981 tataattccg gggtaagatt aacgacctta tctgaacata atgccatatg aatccctcct
     2041 aatttatacg ttttctctaa caacttaatt atacccacta ttattatttt tatcaatata
     2101 gaagttccta tactttctag agaataggaa cttcacgcgt tgggaaatgg caatgatagc
     2161 gaaacaacgt aaaactcttg ttgtatgctt tcattgtcat cgtcacgtga ttcataaaca
     2221 caagtgaatg tcgacagtga atttttacga acgaacaata acagagccgt atactccgag
     2281 aggggtacgt acggttcccg aagagggtgg tgcaaaccag tcacagtaat gtgaacaagg
     2341 cggtacctcc ctacttcacc atatcatttt ctgcagcccc ctagaaataa ttttgtttaa
     2401 ctttaagaag gagatataca tatatggcta gatcgtccat tccgacagca tcgccagtca
     2461 ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt ctatgcactc gtagtagtct
     2521 gagaagggta acgcccttta catggcaaag gggtacagtt attgtgtact aaaattaaaa
     2581 attgattagg gaggaaaacc tcaaaatgaa accaacaatg gcaattttag aaagaatcag
     2641 taaaaattca caagaaaata tagacgaagt ttttacaaga ctttatcgtt atcttttacg
     2701 tccagatatt tattacgtgg cgtatcaaaa tttatattcc aataaaggag cttccacaaa
     2761 aggaatatta gatgatacag cggatggctt tagtgaagaa aaaataaaaa agattattca
     2821 atctttaaaa gacggaactt actatcctca acctgtacga agaatgtata ttgcaaaaaa
     2881 gaattctaaa aagatgagac ctttaggaat tccaactttc acagataaat tgatccaaga
     2941 agctgtgaga ataattcttg aatctatcta tgaaccggta ttcgaagatg tgtctcacgg
     3001 ttttagacct caacgaagct gtcacacagc tttgaaaaca atcaaaagag agtttggcgg
     3061 cgcaagatgg tttgtggagg gagatataaa aggctgcttc gataatatag accacgttac
     3121 actcattgga ctcatcaatc ttaaaatcaa agatatgaaa atgagccaat tgatttataa
     3181 atttctaaaa gcaggttatc tggaaaactg gcagtatcac aaaacttaca gcggaacacc
     3241 tcaaggtgga attctatctc ctcttttggc caacatctat cttcatgaat tggataagtt
     3301 tgttttacaa ctcaaaatga agtttgaccg agaaagtcca gaaagaataa cacctgaata
     3361 tcgggagctc cacaatgaga taaaaagaat ttctcaccgt ctcaagaagt tggagggtga
     3421 agaaaaagct aaagttcttt tagaatatca agaaaaacgt aaaagattac ccacactccc
     3481 ctgtacctca cagacaaata aagtattgaa atacgtccgg tatgcggacg acttcattat
     3541 ctctgttaaa ggaagcaaag aggactgtca atggataaaa gaacaattaa aactttttat
     3601 tcataacaag ctaaaaatgg aattgagtga agaaaaaaca ctcatcacac atagcagtca
     3661 acccgctcgt tttctgggat atgatatacg agtaaggaga tctggaacga taaaacgatc
     3721 tggtaaagtc aaaaagagaa cactcaatgg gagtgtagaa ctccttattc ctcttcaaga
     3781 caaaattcgt caatttattt ttgacaagaa aatagctatc caaaagaaag atagctcatg
     3841 gtttccagtt cacaggaaat atcttattcg ttcaacagac ttagaaatca tcacaattta
     3901 taattctgaa ctccgcggga tttgtaatta ctacggtcta gcaagtaatt ttaaccagct
     3961 caattatttt gcttatctta tggaatacag ctgtctaaaa acgatagcct ccaaacataa
     4021 gggaacactt tcaaaaacca tttccatgtt taaagatgga agtggttcgt gggggatccc
     4081 gtatgagata aagcaaggta agcagcgccg ttattttgca aattttagtg aatgtaaatc
     4141 cccttatcaa tttacggatg agataagtca agctcctgta ttgtatggct atgcccggaa
     4201 tactcttgaa aacaggttaa aagctaaatg ttgtgaatta tgtgggacgt ctgatgaaaa
     4261 tacttcctat gaaattcacc atgtcaataa ggtcaaaaat cttaaaggca aagaaaaatg
     4321 ggaaatggca atgatagcga aacaacgtaa aactcttgtt gtatgctttc attgtcatcg
     4381 tcacgtgatt cataaacaca agtgaatgtc gagcacccgt tctcggagca ctgtccgacc
     4441 gctttggccg ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga
     4501 tcatggcgac cacacccgtc ctgtggatcg ccaagccgcc gatggtagtg tggggtctcc
     4561 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     4621 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     4681 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     4741 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     4801 ttctacaaac tcttcctgtc gtcatatcta caagccatcc ccccacagat acgggcgcgc
     4861 ccgcccttaa gtctaaaaat taggggagat gtaaggattt gggaaaaata gaagatgtta
     4921 taatcataaa tatggtattc gtaggcttaa agtcaaaaag gaggtgaaat ataaatagat
     4981 ttttagctaa attaagtaag aaataggagg agatttattg aacaaaaaat tagaaaaacc
     5041 atttgtatat aagagagagt acgatttgac tggatatgat gttgaaattt tacaaaaata
     5101 tgagttagaa caagcaatat atgtttatgt tgggagtagt tgtgcatata acatgagagc
     5161 tagaagtagt aaatggagat accatataag aacaaataat aagtctatat gttgtaacat
     5221 taaaaatttt atacataact tggaattgtt ttataaaatg gaattaaagt tgtcagataa
     5281 tattattaat gataagctat actatagcaa tatagcagag tttgaagaat ttgaaacact
     5341 agaaaaagct agagaggtag aaagtactat tataagtcaa tatcaatttt tagattctat
     5401 aaatcacatg ttaaaacaaa aaataatttt attgagtaat aaggatagtg tgttaaacat
     5461 aactaaaaat ggaaatacaa attatttgaa agtaaaaaat aaatacatag aaaaacataa
     5521 gaacaagcca ataatgagat accatatcaa ctgtcaattc aatacagatg gaagtgtcaa
     5581 aagtattaca caggagtttg aaccaatatt ggaattaaac aaaaaaaata ccctaagccg
     5641 accaagcaga gtatttttaa aataatattt taagataaca acaaaatgag ataatactac
     5701 tagacaatga caactcaact accaattgag tttatggagc taccaactcc aatatcggtc
     5761 taactgatta agtatctgta gttatataat aatattgcta tcaattttag catcttaaca
     5821 atattattat acatactaag ctaaaattat tcaatagttg taaaagttga ttagtcaata
     5881 agtatatatt taatgtagtg ttatctctta aaaaaactag ataaggagat aataaatata
     5941 tggaacaatt agattcaaaa tataagttga aaaaatttct aatggcagta tttagagatg
     6001 gtataggaca aggaaataat cttattgata atgaatatgt tagagtattt caaaataata
     6061 aaagtaatag taaacaatta gaactcggag aagaatttaa agaatatagt aaaacaactt
     6121 tttttaaaaa tatagatgat atagtagaat ttaccttcgc aaaaaatatt tattatgaaa
     6181 atacattttt taacctatgt actactgatg gaaaagcagg aaccaatgaa aacttaataa
     6241 atagatatgc attaggattt gattttgaca aaaaagaatt aggacaaggt tttaattata
     6301 aagatataat taatttattt actaagatag gattacatta tcatatccta gttgatagtg
     6361 gaaatggatt ccatgtttat gtgctaatta ataaaactaa taacattaag ttagtatcag
     6421 aagttacaaa tacattaata aataaattgg gtgcagataa acaagcaaat ttatctactc
     6481 aagtattaag agtaccttat acatataata ttaaaaatac tactaaacaa gtaaaaataa
     6541 tacaccaaga caaaaatata tatagatatg acatagaaaa gttagctaaa aaatattgca
     6601 aagatgtaaa aacagtaggt aatactaata caaaatatat attagatagt aagctaccaa
     6661 attgtatagt agatatttta aaaaatggta gtaaagatgg acataaaaac ctagatttgc
     6721 aaaaaatagt tgtgacttta agattgagga ataaaagttt aagtcaagta atatccgttg
     6781 ctagagaatg gaactatata tcacaaaata gtctttcaaa tagtgagcta gaatatcaag
     6841 tcaagtatat gtatgagaaa cttaaaacgg ttaattttgg ttgtactggt tgtgagttta
     6901 atagtgattg ttggaataaa atagaatcag attttatata tagtgatgaa gatactttgt
     6961 tcaatatgcc acataagcac tcaaaggatt tgaaatataa gaataggaaa ggggttaaaa
     7021 taatgactgg taatcaattg tttatctata atgtgttact taacaataaa gatagagaat
     7081 taaacataga cgatataatg gagctgataa cctataaacg taagaagaaa gttaaaaaca
     7141 ttgttatgag tgaaaagaca ttaagagaaa cattaaaaga acttcaacat aatgattata
     7201 ttacaaaaac aaaaggtgtt acaaagctag gaataaaaga tacatacaat gtaaaagaag
     7261 ttagatgtaa tatagataaa caatatacta ttagttactt tgttaccatg gcagtaattt
     7321 ggggaataat ttcaactgaa gaattaagat tatatactca catgagatat aagcaagatt
     7381 tattggtcaa agatgataaa ataaaaggaa atatattaag aattaatcaa gaggaattag
     7441 caaaagattt aggagtaaca cagcaaagaa tttcaaatat gatagaatct ttattagata
     7501 ctaaaatttt agatgtatgg gaaactaaaa taaatgatag aggatttatg tactatacat
     7561 atagattaaa caagtagatt tttgatagga ttagaattga ttttctagtc ctatttttat
     7621 gcaaaaaaac taattaataa aaatttcttt tggtaaaata attgtacgag aattgcaaaa
     7681 aaaaaatggc atcaaagtat tgaaattaag ccgttttaaa aatttctttt ggtaaaataa
     7741 ttctacatat atatgtagta tatatatata tgttttttag agaatgtata actagaatat
     7801 agagctagaa tatagagaat gtataactag aatatagagc tagaatatag agaatgtata
     7861 actagaatat agagctagaa tatagagaat gtataactag aatatagagc tagaatatag
     7921 agaatgtata actagaatat agagctagaa tatagagaat gtataactag aatatagagc
     7981 tagaatatag agaatgtata actagaatat agagctagaa tcctaattag taggtgcttt
     8041 tttaaaacaa gttaaaaatc aaaaatagta ttagtaagca ttggaaatgc tagattctaa
     8101 aatagaaaag taaaaaattg gtgcactatc taaacttatc tatatcgctt tttccgtcgt
     8161 ttggttctct agttacgata caggggatat gcttatattg agttatagta ctaatcagtg
     8221 cttaatatag ttaataaaat tatagttacc atagtttagt aactatgatg tatgttagtt
     8281 agaaacttgc atttcggccg gccagtgggc aagttgaaaa attcacaaaa atgtggtata
     8341 atatctttgt tcattagagc gataaacttg aatttgagag ggaacttaga tggtatttga
     8401 aaaaattgat aaaaatagtt ggaacagaaa agagtatttt gaccactact ttgcaagtgt
     8461 accttgtacc tacagcatga ccgttaaagt ggatatcaca caaataaagg aaaagggaat
     8521 gaaactatat cctgcaatgc tttattatat tgcaatgatt gtaaaccgcc attcagagtt
     8581 taggacggca atcaatcaag atggtgaatt ggggatatat gatgagatga taccaagcta
     8641 tacaatattt cacaatgata ctgaaacatt ttccagcctt tggactgagt gtaagtctga
     8701 ctttaaatca tttttagcag attatgaaag tgatacgcaa cggtatggaa acaatcatag
     8761 aatggaagga aagccaaatg ctccggaaaa catttttaat gtatctatga taccgtggtc
     8821 aaccttcgat ggctttaatc tgaatttgca gaaaggatat gattatttga ttcctatttt
     8881 tactatgggg aaatattata aagaagataa caaaattata cttcctttgg caattcaagt
     8941 tcatcacgca gtatgtgacg gatttcacat ttgccgtttt gtaaacgaat tgcaggaatt
     9001 gataaatagt taacttcagg tttgtctgta actaaaaaca agtatttaag caaaaacatc
     9061 gtagaaatac ggtgtttttt gttaccctaa gtttaaactc ctttttgata atctcatgac
     9121 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     9181 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     9241 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     9301 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     9361 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     9421 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     9481 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     9541 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     9601 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     9661 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     9721 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     9781 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     9841 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     9901 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     9961 gcgcccaata cgcagggccc cctgcttcgg ggtcattata gcgatttttt cggtatatcc
    10021 atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact ttccttggtg
    10081 tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga gcgggtgttc
    10141 cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg ctctgcgagg
    10201 ctggccggct accgccggcg taacagatga gggcaagcgg atggctgatg aaaccaagcc
    10261 aaccaggaag ggcagcccac ctatcaaggt gtactgcctt ccagacgaac gaagagcgat
    10321 tgaggaaaag gcggcggcgg ccggcatgag cctgtcggcc tacctgctgg ccgtcggcca
    10381 gggctacaaa atcacgggcg tcgtggacta tgagcacgtc cgcgagctgg cccgcatcaa
    10441 tggcgacctg ggccgcctgg gcggcctgct gaaactctgg ctcaccgacg acccgcgcac
    10501 ggcgcggttc ggtgatgcca cgatcctcgc cctgctggcg aagatcgaag agaagcagga
    10561 cgagcttggc aaggtcatga tgggcgtggt ccgcccgagg gcagagccat gactttttta
    10621 gccgctaaaa cggccggggg gtgcgcgtga ttgccaagca cgtccccatg cgctccatca
    10681 agaagagcga cttcgcggag ctggtgaagt acatcaccga cgagcaaggc aagaccgatc
    10741 gggccc
//
