LOCUS       V004408                10413 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V004408
VERSION     V004408
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10413)
  AUTHORS   Mearls EB, Olson DG, Herring CD, Lynd LR.
  TITLE     Development of a regulatable plasmid-based gene expression system
            for Clostridium thermocellum
  JOURNAL   Appl. Microbiol. Biotechnol. 99 (18), 7589-7599 (2015)
   PUBMED   25994254
REFERENCE   2  (bases 1 to 10413)
  AUTHORS   Argyros A, Mearls EB.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-MAR-2013) Engineering, Dartmouth College, 14
            Engineering Dr, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 10413)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 10413)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
            Microbiol. Biotechnol."; date: "2015"; volume: "99"; issue: "18";
            pages: "7589-7599"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (13-MAR-2013) Engineering, Dartmouth College, 14 Engineering Dr,
            Hanover, NH 03755, USA"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(294..1094)
                     /label="URA3"
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(1095..1315)
                     /label="URA3 promoter"
     misc_feature    1343..1846
                     /label="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     misc_recomb     1888..3099
                     /label="spo0A 3' flank for homologous recombination"
                     /note="spo0A 3' flank for homologous recombination"
     misc_recomb     3100..4192
                     /label="spo0A 5' flank for homologous recombination"
                     /note="spo0A 5' flank for homologous recombination"
     CDS             complement(4311..4856)
                     /codon_start=1
                     /gene="hpt"
                     /product="Hpt"
                     /label="hpt"
                     /note="hypoxanthine phosphoribosyl transferase"
                     /protein_id="AGT95731.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     gene            complement(4311..4856)
                     /gene="hpt"
                     /label="hpt"
     CDS             complement(4882..5529)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     regulatory      complement(5530..6057)
                     /gene="gapD"
                     /regulatory_class="promoter"
     gene            complement(5530..6057)
                     /gene="gapD"
                     /label="gapD"
     misc_feature    6058..6800
                     /note="fragment of Spo0A; sporulation master regulator-like
                     protein"
     CDS             complement(6801..7379)
                     /codon_start=1
                     /gene="tdk"
                     /product="Tdk"
                     /label="tdk"
                     /note="thymidine kinase"
                     /protein_id="AGT95733.1"
                     /translation="MYGPKDHGYIEVVTGPMFSGKSEELIRRIKRAKIARQKVQVFKPA
                     IDDRYSIDKVVSHNGDNMHAIAIVKASDILAYAEEDTDVFAIDEVQFFDSEIVDIVKEI
                     ADSGKRVICAGLDMDFRGEPFGPTPELMAIAEFVDKLTAICMKCGNPATRTQRLINGKP
                     ANYDDPIIMVGAKESYEARCRKCHEVPRT"
     gene            complement(6801..7379)
                     /gene="tdk"
                     /label="tdk"
     regulatory      complement(7380..8000)
                     /gene="cbp"
                     /regulatory_class="promoter"
     gene            complement(7380..8000)
                     /gene="cbp"
                     /label="cbp"
     CDS             complement(8110..9111)
                     /label="repB"
                     /note="RepB replication protein"
     rep_origin      complement(9803..10391)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
ORIGIN
        1 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
       61 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
      121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
      181 agcgatctgt caattcgcgg ccgccgaaaa gtgggtaata actgatataa ttaaattgaa
      241 gctctaattt gtgagtttag tatacatgca tttacttata atacagtttt ttagttttgc
      301 tggccgcatc ttctcaaata tgcttcccag cctgcttttc tgtaacgttc accctctacc
      361 ttagcatccc ttccctttgc aaatagtcct cttccaacaa taataatgtc agatcctgta
      421 gagaccacat catccacggt tctatactgt tgacccaatg cgtctccctt gtcatctaaa
      481 cccacaccgg gtgtcataat caaccaatcg taaccttcat ctcttccacc catgtctctt
      541 tgagcaataa agccgataac aaaatctttg tcgctcttcg caatgtcaac agtaccctta
      601 gtatattctc cagtagatag ggagcccttg catgacaatt ctgctaacat caaaaggcct
      661 ctaggttcct ttgttacttc ttctgccgcc tgcttcaaac cgctaacaat acctgggccc
      721 accacaccgt gtgcattcgt aatgtctgcc cattctgcta ttctgtatac acccgcagag
      781 tactgcaatt tgactgtatt accaatgtca gcaaattttc tgtcttcgaa gagtaaaaaa
      841 ttgtacttgg cggataatgc ctttagcggc ttaactgtgc cctccatgga aaaatcagtc
      901 aagatatcca catgtgtttt tagtaaacaa attttgggac ctaatgcttc aactaactcc
      961 agtaattcct tggtggtacg aacatccaat gaagcacaca agtttgtttg cttttcgtgc
     1021 atgatattaa atagcttggc agcaacagga ctaggatgag tagcagcacg ttccttatat
     1081 gtagctttcg acatgattta tcttcgtttc ctgcaggttt ttgttctgtg cagttgggtt
     1141 aagaatactg ggcaatttca tgtttcttca acactacata tgcgtatata taccaatcta
     1201 agtctgtgct ccttccttcg ttcttccttc tgttcggaga ttaccgaatc aaaaaaattt
     1261 caaagaaacc gaaatcaaaa aaaagaataa aaaaaaaatg atgaattgaa ttgaaaagct
     1321 agcttatcga tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt
     1381 aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt
     1441 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct
     1501 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc
     1561 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt
     1621 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt
     1681 aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt
     1741 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc
     1801 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatcgcgg ccgcggtacc
     1861 cggggatcct ctagagtcga cctgcagagt caactccgtt cagcacaata tcaataattt
     1921 ctgtcgaatt aacattttca ggcttttctt cgcgaaggta aaagaacaga tcttcaatag
     1981 aatgggcaag ttttgatata tcgtcaaaaa gcatcattgc cgctgagcct tttattgtat
     2041 gcatgattct gaaaatctca tctatcgaag attcaaaccc actttctttt tcactgttaa
     2101 tcaaagactg ctctaactga tctaataact ggagtgtttc aaatataaac atgtccagca
     2161 tcggctcacg gctaacatta tcagtcaatt tttccacctc catgtgttta tattactact
     2221 tccatgctat tataattttt caatagcctg tataacataa tcttctttga aaggttttac
     2281 aatgaatcct tttgcacctg ccaaaattgc gtcccttaca aagcactcct ggcccatgga
     2341 agaaatcatt atgacttttg catcgggatc aaactcttta atcatcttaa ccgcctttat
     2401 cccgtcacat accggcatgg ttatatccat cgtaactata tccggcctga tttccttata
     2461 ttttgtgacc gcaaccaaac cgttttcagc ttctccaacc acctgatgtc catttctttc
     2521 caacatcatt ttaagtgacg ccctcataaa tcccgcatcg tcaaccacta aaattctttt
     2581 catttcctca cactcccacg tttttagttt tgttgaatac taaatctcat ggtttaaaat
     2641 tgtgttattt ccccttctat attggaaatt tagaataaac aatcacaaga cttgcgctcc
     2701 cctcttttta tgaacaaatc aagatgtgta tatttgacca ataaaatact ttatataaat
     2761 attatgttat tatttttcaa tttaatcaag taaaaagttt tatttgatgt ttatatcgaa
     2821 atctataata ctattattaa cagtaaattt ttattatttt gtagaatatc atgttgaata
     2881 cctcgtaatc acctaaaaaa ctcaggaaat tgacatgact aaaatccgct aaaataatca
     2941 gatttttaaa gccaacataa atgaagcagt aataaattct aagcttataa cttaaaattt
     3001 attactgctt ccacacctta ttactgtttt gtattttttt gcattttttt attgttttat
     3061 gtagccattt tcatttccaa tctcaatttg tctgccaccg acccgaatct tatttgatgt
     3121 caaattgact cccccttaat tttttgtaaa agactgctag ttaatttata ccattattat
     3181 attgtctatt cgacaaaatt tcaatatact gggtaatgaa attactcttt atataacttg
     3241 ttttacattt ataatactat ataatcattt actgtaagta ctggtatttc tggattttgt
     3301 gacataaaga aaccttttat gaattttttt attctaaaag caatatctta aaattttttg
     3361 gaaatatgca aaacccggga aatatgcaat ttatgtaaaa tatgtgtatc ctccttacga
     3421 aaatacacat atttttataa tcttacaaat ttattatttc gttaatcttt cttcttaaaa
     3481 aactttttaa gaagcattgg ccatttcaac attttgcaaa tttgcatcag tcatgttttt
     3541 aatcatccac tcaatcagta tgccatagcc tcttgcaggg tcattcacca gtacatgggt
     3601 tacagctccc accagtttgc catcctgtaa aatagggctg cctgacattc cctgaacaat
     3661 acctcccgtt gcctcaagaa gtctgtcatc agtcactctg ataaccattc ccttcaaacc
     3721 attggcgctt tttcttgaaa ctttttcaat ttcaatactg tattcttcaa cttttttgcc
     3781 gtctatattg gcaagtatgg tggcaggtcc cacctttata tcgtttctca aagctatagg
     3841 atattttacg ttcggaaatt tatccaacgc cgcgtcattc aaagtaccat atatgccata
     3901 atggctgttt ttcactattg ttccaagaac accgctgtct tctatcaata cacctttaag
     3961 ttccccggga ttgcctttgg tacctttttt tacggtcaaa atatttgatt cgacaagctc
     4021 acctctttga acgggcataa tggctcccgt atcaatatcg gtgattccgt gtcccaaagc
     4081 cccaaagcct ttagtcacag ggtcgtaaaa tgtcaacgtt ccgattcctg ccgtgctgtc
     4141 tctcacccac attccgacac ggtacttatt gtcttcagcc gatttgaccg gcgcgataat
     4201 tatcagtatc catataacga tacctctgat cgttttattc aaatatggcc ctcccttcaa
     4261 agccgtatca cttaatttat aataacatat accatataaa aaaacaaatt ttagctgtac
     4321 atttcaggtt tcaaaacgcc tataaaagga aggttcctgt acttttcatc aaagtccaat
     4381 ccataaccca cgacaaactt atcaggtatc ttaaatccta cataatcgac ttttacagat
     4441 gcttctcttc tctccggttt gtctaatatt gtgcatattt tcaggctttt tggcttcctt
     4501 ccaagtaaag tttcccttaa gtaagacaaa gtcaaaccgc tgtcaattat gtcttccaca
     4561 atcagaacat ctttgccttc tatgcttata tcaagatctt tgattatctt tactattcct
     4621 gatgaatgag ttgagcttcc atagctggaa acagccataa aatcaagtga taaaggcaaa
     4681 tctatgtgtc ttgacaaatc agccataaac attaaagcac cttttaaaac tcctaccaac
     4741 atcaaatttt tccctttgta gtcttttgtg atttgcctcc caagctcttt tatcttttcc
     4801 ttaagttctt cttctgtgat caaaatttca tcgatgtctt ttgataaatt ttccatcaaa
     4861 tttcctcctt aatcttgttt ataaaagcca gtcattaggc ctatctgaca attcctgaat
     4921 agagttcata aacaatcctg catgataacc atcacaaaca gaatgatgta cctgtaaaga
     4981 tagcggtaaa tatattgaat tacctttatt aatgaatttt cctgctgtaa taatgggtag
     5041 aaggtaatta ctattattat tgatatttaa gttaaaccca gtaaatgaag tccatggaat
     5101 aatagaaaga gaaaaagcat tttcaggtat aggtgttttg ggaaacaatt tccccgaacc
     5161 attatatttc tctacatcag aaaggtataa atcataaaac tctttgaagt cattctttac
     5221 aggagtccaa ataccagaga atgttttaga tacaccatca aaaattgtat aaagtggctc
     5281 taacttatcc caataaccta actctccgtc gctattgtaa ccagttctaa aagctgtatt
     5341 tgagtttatc acccttgtca ctaagaaaat aaatgcaggg taaaatttat atccttcttg
     5401 ttttatgttt cggtataaaa cactaatatc aatttctgtg gttatactaa aagtcgtttg
     5461 ttggttcaaa taatgattaa atatctcttt tctcttccaa ttgtctaaat caattttatt
     5521 aaagttcatc attaatatcg cctcctattg taaattaaaa tttaatttaa tgcctctatt
     5581 agctgccata aagcatgaat gcagtaaaat attcatcctc tggcaagtat ctcttgcact
     5641 atgattcatt ttatattatt ttattttcat tttcaatagt ttagataaaa aataattaat
     5701 tttttaacaa agtattttta tcttcctttg atgtaattct ttgttttttt gtccattaat
     5761 attatggaca atttttttat ttctagtcaa taattttaat ctttattaaa acctggcaat
     5821 gccagagaga tacagtaatt attgcatata aaagaaaaca gacgcgccct gtttgtttcc
     5881 tgaaaaacca cctcgtcctc caaagtatcc agctttttaa aatccatcct cattcccaga
     5941 cgaaaaagtt ttgacaaagc ctcttttctt gtccatagaa ttgtactctg cacacaatat
     6001 tcatcagtat ttccaaaact ctttagaatc ttttcctctc tttcggaaaa gaaatacatt
     6061 gctataaatt ccgaatttgt gggtttacct tttccaacat ttatcgtata gccaaacaac
     6121 gaatctattg tatcaatctg tcccctgctc cacgccactt caatagcatg tcttattgcc
     6181 ctttcaactc tgctgggtgt ggtgttgtat tccttcgcta ttgtaggata cagctgcttt
     6241 gtaattgaat ttatcacgtc aaggtctttg actaccatta ttatggcatc cctcagatac
     6301 tgataccctt ttatatgggc cggtacgcct atttcatgca taatatttgt tacttcagct
     6361 tcaagatttt ttggctgcgg aggatgataa gacgacttga cttcaccgga caaaccgtcc
     6421 tgtcttataa cattgggctg gttcacgttc ttgagttgtc ttattctcga aattaaaact
     6481 tccatgtcga aaggctttac aacatagtat tcagctccga gtgccaatgc tctttgtgtt
     6541 atcttgtcct ggcccactgc cgaaagaata atgaacaaag gtcttttact gattgcagtc
     6601 gcgcctattt tctccaaaac acccaaaccg tccaaatgag gcatgataat atcaagtata
     6661 gcaatgtcag gaagtttttc aactataagc tcataagctt caataccgtc tcttgccacc
     6721 cctaccactt caatatcttc ctgattgtta aggtattcat ataaaatatc gccaaattcc
     6781 ctgttatcat cagcaattac ttaagtccgc gggacttcat ggcactttct acaccttgct
     6841 tcataagact cctttgctcc aaccattata atgggatcgt cgtaattggc aggcttccca
     6901 tttataagcc tttgcgtgcg agtagcagga ttgccacact tcatgcatat agcagtaagc
     6961 ttatcgacaa attcagctat ggccatcaat tctggagtcg gaccaaatgg ttcacctcta
     7021 aagtccatgt caagccctgc acatatgact cttttcccgc tatcggcaat ctcttttacg
     7081 atgtcgacta tttcagaatc aaaaaattga acttcatcta tagcaaatac atccgtatct
     7141 tcttcagcat aagccaatat gtcagaagcc tttactatgg caatggcgtg catgttgtcg
     7201 ccgttatgag atacgacttt gtctatggaa tacctatcgt ctatagccgg tttgaaaacc
     7261 tgaacttttt gcctggcaat cttagctctc tttatccttc ttataagttc ctcacttttt
     7321 ccactgaaca tgggaccagt tacaacttct atgtacccgt ggtctttagg cccatacatc
     7381 tttcgtcctc cttaaaattt tcgtttattt gttttttatt tagtgtgcct aaaagcacca
     7441 tcttatacat tcgtacctta tatctcttat cttgtatctc ttacctcata tctcttatta
     7501 ttaactaccg aagtatattt tcataacgga atcttatgac ttatttttct gactgccgtt
     7561 ttaccatacg gttaatttat acggaaacag ctaatctaac cttgctgaag gcatgtttac
     7621 caaaatatta tatctacttt accatattaa ttcaagtggt cattttgata ttttacgcca
     7681 aaatttttag cgttccttta cttgataatt tatccaaaaa ccttcattcg tcaaaacaat
     7741 tatgatataa ttaatttatg atatgattaa ttaatattta cggataacta atcatttcaa
     7801 tgcaatagat caacatgcaa atcaatatcc accttactcc gaacattttg caccaacgaa
     7861 tgcccagcac tatatcccgg caagcaatat aacgtctata attttttatg ataacgataa
     7921 aattaaattt tatgaaaggg gtaaaagcat gagaaaaata gctgtattgt taattacttt
     7981 gacgttctta gtcacgactc ttgatatgcc tcctaaattt ttatctaaag tgaatttagg
     8041 aggcttactt gtctgctttc ttcattagaa tcaatccttt tttaaaagtc aatcccgttt
     8101 gttgaactac tctttaataa aataattttt ccgttcccaa ttccacattg caataataga
     8161 aaatccatct tcatcggctt tttcgtcatc atctgtatga atcaaatcgc cttcttctgt
     8221 gtcatcaagg tttaattttt tatgtatttc ttttaacaaa ccaccatagg agattaacct
     8281 tttacggtgt aaaccttcct ccaaatcaga caaacgtttc aaattctttt cttcatcatc
     8341 ggtcataaaa tccgtatcct ttacaggata ttttgcagtt tcgtcaattg ccgattgtat
     8401 atccgattta tatttatttt tcggtcgaat catttgaact tttacatttg gatcatagtc
     8461 taatttcatt gcctttttcc aaaattgaat ccattgtttt tgattcacgt agttttctgt
     8521 attcttaaaa taagttggtt ccacacatac caatacatgc atgtgctgat tataagaatt
     8581 atctttatta tttattgtca cttccgttgc acgcataaaa ccaacaagat ttttattaat
     8641 ttttttatat tgcatcattc ggcgaaatcc ttgagccata tctgacaaac tcttatttaa
     8701 ttcttcgcca tcataaacat ttttaactgt taatgtgaga aacaaccaac gaactgttgg
     8761 cttttgttta ataacttcag caacaacctt ttgtgactga atgccatgtt tcattgctct
     8821 cctccagttg cacattggac aaagcctgga tttacaaaac cacactcgat acaactttct
     8881 ttcgcctgtt tcacgatttt gtttatactc taatatttca gcacaatctt ttactctttc
     8941 agccttttta aattcaagaa tatgcagaag ttcaaagtaa tcaacattag cgattttctt
     9001 ttctctccat ggtctcactt ttccactttt tgtcttgtcc actaaaaccc ttgatttttc
     9061 atctgaataa atgctactat taggacacat aatattaaaa gaaaccccca tctatttagt
     9121 tatttgtttg gtcacttata actttaacag atggggtttt tctgtgcaac caattttaag
     9181 ggttttcaat actttaaaac acatacatac caacacttca acgcaccttt cagcaactaa
     9241 aataaaaatg acgttatttc tatatgtatc aagaatagaa agaactcgtt tttcgctacg
     9301 ctcaaaacgc aaaaaaagca ctcattcgag tgctttttct tatcgctcca aatcatgcga
     9361 ttttttcctc tttgcttttc tttgctcacg aagttctcga tcacgctgca aaacatcttg
     9421 aagcgaaaaa gtattcttct tttcttccga tcgctcatgc tgacgcacga aaagccctct
     9481 aggcgcatag gaacaactcc taaatgcatg tgaggggttt tctcgtccat gtgaacagtc
     9541 gcatacgcaa tattttgttt cccatactgc attaatgaat cggccaacgc gcggggagag
     9601 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     9661 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     9721 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     9781 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     9841 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     9901 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     9961 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
    10021 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
    10081 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
    10141 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
    10201 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
    10261 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
    10321 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
    10381 aaggatctca agaagatcct ttgatctttt cta
//
