LOCUS       V005201                 7202 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V005201
VERSION     V005201
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7202)
  AUTHORS   Al-Hinai MA, Fast AG, Papoutsakis ET.
  TITLE     Novel system for efficient isolation of clostridium double-crossover
            allelic exchange mutants enabling markerless chromosomal gene
            deletions and DNA integration
  JOURNAL   Appl. Environ. Microbiol. 78 (22), 8112-8121 (2012)
   PUBMED   22983967
REFERENCE   2  (bases 1 to 7202)
  AUTHORS   Al-Hinai MA, Papoutsakis ET, Fast AG.
  TITLE     Direct Submission
  JOURNAL   Submitted (06-SEP-2012) Biological Sciences, University of Delaware,
            15 Innovation Way, Newark, DE 19711, USA
REFERENCE   3  (bases 1 to 7202)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7202)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
            Environ. Microbiol."; date: "2012"; volume: "78"; issue: "22";
            pages: "8112-8121"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (06-SEP-2012) Biological Sciences, University of Delaware, 15
            Innovation Way, Newark, DE 19711, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..693
                     /label="CmR"
                     /note="chloramphenicol acetyltransferase"
     CDS             1038..1340
                     /label="ccdB"
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     protein_bind    complement(1384..1508)
                     /label="attR2"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1533..1973)
                     /codon_start=1
                     /gene="repL"
                     /product="gram positive replication protein"
                     /label="repL"
                     /note="RepL"
                     /protein_id="AFS68389.1"
                     /translation="MKERYGTVYKGSQRLIDEESGEVIEVDKLYRKQTSGNFVKAYIVQ
                     LISMLDMIGGKKLKIVNYILDNVHLSNNTMIATTREIAKATGTSLQTVITTLKILEEGN
                     IIKRKTGVLMLNPELLMRGDDQKQKYLLLEFGNFEQEANEID"
     gene            complement(1533..1973)
                     /gene="repL"
                     /label="repL"
     CDS             complement(2584..3423)
                     /codon_start=1
                     /gene="bgaR"
                     /product="beta-galactosidase regulator"
                     /label="bgaR"
                     /note="BgaR"
                     /protein_id="AFS68386.1"
                     /translation="MQILWKKYVKENFEMNVDECGIEQGIPGLGYNYEVLKNAVIHYVT
                     KGYGTFKFNGKVYNLKQGDIFILLKGMQVEYVASIDDPWEYYWIGFSGSNANEYLNRTS
                     ITNSCVANCEENSKIPQIILNMCEISKTYNPSRSDDILLLKELYSLLYALIEEFPKPFE
                     YKDKELHTYIQDALNFINSNYMHSITVQEIADYVNLSRSYLYKMFIKNLGISPQRYLIN
                     LRMYKATLLLKSTKLPIGEVASSVGYSDSLLFSKTFSKHFSMSPLNYRNNQVNKPSI"
     gene            complement(2584..3423)
                     /gene="bgaR"
                     /label="bgaR"
     protein_bind    3854..3870
                     /label="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)."
     CDS             3889..4221
                     /gene="mazF"
                     /label="Endoribonuclease toxin MazF"
                     /note="Endoribonuclease toxin MazF from Escherichia coli
                     (strain K12). Accession#: P0AE70"
     CDS             complement(4364..5095)
                     /gene="ermC'"
                     /label="rRNA adenine N-6-methyltransferase"
                     /note="rRNA adenine N-6-methyltransferase from Bacillus
                     subtilis. Accession#: P13956"
     primer_bind     complement(5627..5643)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(5651..5667)
                     /label="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        complement(5675..5705)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5720..5741)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(6029..6617)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     protein_bind    7006..7130
                     /label="attR1"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        7155..7185
                     /label="lac UV5 promoter"
                     /note="E. coli lac promoter with an 'up' mutation"
ORIGIN
        1 gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
       61 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
      121 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
      181 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
      241 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
      301 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
      361 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
      421 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
      481 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
      541 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
      601 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
      661 caacagtact gcgatgagtg gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa
      721 gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat
      781 atgtataccc gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt acagtgacag
      841 ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt
      901 aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa
      961 tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga
     1021 gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc
     1081 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc
     1141 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     1201 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     1261 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     1321 acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc
     1381 gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa
     1441 atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac
     1501 aaagtggtga tctacgtggt gttattggga ggtcaatcta tttcatttgc ctcttgctca
     1561 aagttcccaa attcgagtaa gaggtatttt tgtttttggt cgtcgcctct cattagtagt
     1621 tcagggttta acattaatac tccagttttt ctttttataa tatttccttc ttctaagatt
     1681 ttaagtgttg ttattactgt ttgtagactt gttcctgtag cttttgctat ttctcttgtt
     1741 gtagctatca ttgtattgtt acttaagtgg acattatcta ggatatagtt aacgatttta
     1801 agtttttttc cgccaatcat atctaacata cttattaatt gcactatata tgcctttacg
     1861 aagttaccag acgtttgttt acggtataac ttgtctacct ctatgacttc tccactttct
     1921 tcgtctatga gcctctgaga gcctttatag actgttccat atctttcttt catctttttc
     1981 tcactcctta ttttaaacta ttctaactat atcataactg ttctaaaaaa aaaagaacat
     2041 ttgttaaaag aaattagaac aaaatgagtg aaaaattaga acaaacaaat tccttataaa
     2101 ccttatcatc tcaacctata ttaagatttt acctagttga atcttctttt ctatataaag
     2161 cgtcggagca tatcaggggg ttatctaacg taaatgctac ccttcggctc gctttcgctc
     2221 ggcattgacg tcagatactg caccccctga acccccatgc tccaacagca aaaaggaaac
     2281 tttttgctgc ttttccgacg cttattcgct tcgctcatat ttatatagaa aagaagtgaa
     2341 tgcgcaaaag acataatcga tcctcgagat gaaaagtatt agggctaatt tagatattaa
     2401 ttctaaatta agtgaaatta atatagtaat tatattaaaa gtctaattaa gactttagaa
     2461 aataaatttc atataatttt aattcttata aaagatttaa aaagtataac agaggtttta
     2521 atttacgcct ctgttatact ttttattttt gaaatttttt tgttttaaag ctgtatttta
     2581 aatttatata cttggtttat ttacttgatt atttctgtaa tttagtggag acattgaaaa
     2641 atgttttgaa aaagtttttg aaaataacag ggagtcacta taacctacac tacttgcgac
     2701 ttctcctata ggaagtttag tgctttttaa taaaagggtg gctttgtaca ttctaaggtt
     2761 tattaaatat ctttgaggag aaattccaag gttttttatg aacattttat ataaataact
     2821 tctacttaag ttcacataat cagcaatttc ttgaacagtt atgctatgca tgtaattaga
     2881 attaatgaaa ttaagagcat cttgaatata tgtgtgtaat tccttatctt tgtattcaaa
     2941 aggttttggg aattcttcta taagtgcgta caataatgag taaagttctt ttagtaatag
     3001 tatgtcatca gatcttgaag gattataagt ttttgatatt tcgcacatat ttaatattat
     3061 ctgtggaatt tttgagtttt cttcacaatt agcaacacag gagttagtaa tagaagttct
     3121 atttaaatac tcattagcat ttgaaccact aaatcctatc cagtagtatt cccaaggatc
     3181 atcaatagaa gccacatact caacttgcat accttttagt agtataaaaa tatcaccttg
     3241 ttttaagtta tataccttac cattaaattt aaaagttcca tatcccttag ttacgtaatg
     3301 aataacagca tttttcaata cttcatagtt atatcctaat cctggtatac cttgttctat
     3361 accacattca tctacattca tttcaaagtt ttctttaaca tactttttcc acaatatttg
     3421 catttctacc tcctaaccta taaaattagc caattttata gtagtcttat attaaacatt
     3481 tacatgagag ctttgcaaag cagtttatca acataaaagc tttttatttt aaaataaatt
     3541 cttctaaata taagaatatt ttaaagaaat atctttatat attagttatt aaaatttata
     3601 agattataag aaacattata acatatttta gaacttttta actattctaa aagattaatt
     3661 tacatattaa catttaatta tgggtaaaaa ctattttgaa aaatgattta tatggaatta
     3721 tgtttcttaa atatacaatc atgtttcatg aatacataat tattttaaat gtattgggag
     3781 ggtaaaatga tattaaaaaa tgaataccat gaagatactg cagaatctag aatccgcggt
     3841 agtcgacgtg gaattgtgag cggataacaa tttcacagga gggctgaaat ggtaagcagg
     3901 tacgtacctg atatgggaga tttaatatgg gttgattttg atcctacaaa aggaagtgag
     3961 caagctggac atagaccagc tgttgtttta agtcctttta tgtataataa taaaacagga
     4021 atgtgtttat gtgttccttg tacaacacaa tcaaaaggat atccttttga agttgtttta
     4081 tcaggacaag aaagagatgg agtagcatta gctgatcaag taaaaagtat agcatggaga
     4141 gcaagaggag caacaaaaaa aggaacagtt gcaccagagg aattacaatt aataaaagca
     4201 aaaataaatg tattaatagg atagtaaaaa taagagttac cttaaatggt aactcttatt
     4261 tttttaatat tgtttcatag tattcgtcaa tcgggatcga ttcacaaaaa ataggtacac
     4321 gaaaaacaag ttaagggatg cagtttatgc atcccttaac ttacttatta aataatttat
     4381 agctattgaa aagagataag aattgttcaa agctaatatt gtttaaatcg tcaattcctg
     4441 catgttttaa ggaattgtta aattgatttt ttgtaaatat tttcttgtat tctttgttaa
     4501 cccatttcat aacgaaataa ttatacttct gtttatcttt gtgtgatatt cttgattttt
     4561 ttctatttaa tctgataagt gagctattca ctttaggttt aggatgaaaa tattctcttg
     4621 gaaccatact taatatagaa atatcaactt ctgccattaa aaataatgcc aatgagcgtt
     4681 ttgtatttaa taatctttta gcaaacccgt attccacgat taaataaatc tcatcagcta
     4741 tactatcaaa aacaattttg cgtattatat ccgtacttat gttataaggt atattaccaa
     4801 atattttata ggattggttt ttaggaaatt taaactgcaa tatatccttg tttaaaactt
     4861 ggaaattatc gtgatcaaca agtttatttt ctgtagtttt gcataattta tggtctattt
     4921 caatggcagt tacgaaatta cacctctgta ctaattcaag ggtaaaatgc ccttttcctg
     4981 agccgatttc aaagatatta tcatgttcat ttaatcttat atttgtcatt attttatcta
     5041 tattatgttt tgaagtaata aagttttgac tgtgttttat atttttctcg ttcattataa
     5101 ccctctttat tttttcctcc ttataaaatt agtataatta tagcacgagc tctgataaat
     5161 atgaacatga tgagtgatcg ttaaatttat actgcaatct gatgcgatta ttgaataaaa
     5221 gatatgagag atttatctag tttctttttt tacaagaaaa aagaaagttc ttaaaggttt
     5281 tatacttttg gtcgtagagc acacggttta acgacttaat tacgaagtaa ataagtctag
     5341 tgtgttagac tttaatgttt ttttaaggca ttagtgcatt taagcgtcag agcatggctt
     5401 tatgccgaga aaactattgg ttggaatggc gtgtgtgtta gccaaagctc cgcgccactt
     5461 aatgatttgc cagtaaaaga gattgtttct agctctcaca ttcttgcaga tataatattg
     5521 cctagagctg aagttatata tgattatctt aagtaataaa aataagagtt accttaaatg
     5581 gtaactctta tttttttaat gtcgactcat agaattcgta atcatggtca tagctgtttc
     5641 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5701 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5761 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5821 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5881 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5941 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6001 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6061 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6121 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     6181 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaaagctca cgctgtaggt
     6241 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     6301 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     6361 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     6421 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     6481 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6541 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6601 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6661 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6721 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6781 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6841 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6901 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6961 caataaacca gccagccgga agggccgagc gcagaagtgg tcatcacaag tttgtacaaa
     7021 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     7081 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat
     7141 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt
     7201 ag
//
