LOCUS       40924_48483        9963 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pZJ20, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9963)
  AUTHORS   Zhou J, Olson DG, Lanahan AA, Tian L, Murphy SJ, Lo J, Lynd LR.
  TITLE     Physiological roles of pyruvate ferredoxin oxidoreductase and 
            pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum 
            JW/SL-YS485
  JOURNAL   Biotechnol Biofuels 8, 138 (2015)
  PUBMED    26379770
REFERENCE   2  (bases 1 to 9963)
  AUTHORS   Zhou J.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-OCT-2014) Thayer School of Engineering, Dartmouth 
            College, 14 Engineering Drive, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 9963)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 9963)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biotechnol 
            Biofuels 8, 138 (2015)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-OCT-2014) Thayer School of Engineering, Dartmouth College, 14 
            Engineering Drive, Hanover, NH 03755, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..9963
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            complement(347..456)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     terminator      complement(877..904)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(996..1082)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     misc_feature    1144..2059
                     /label=upstream of Tsac_0628
                     /note="upstream of Tsac_0628"
     CDS             2209..3267
                     /codon_start=1
                     /gene="pta"
                     /product="phosphate acetyltransferase"
                     /label=pta
                     /note="negative selective marker in Thermoanaerobacter 
                     saccharolyticum"
                     /protein_id="AJP77044.1"
                     /translation="MYTIYFFLVRGMYKNKYFKGRDDLMSIIQNIIEKAKSDKKKIVLP
                     EGAEPRTLKAAEIVLKEGIADLVLLGNEDEIRNAAKDLDISKAEIIDPVKSEMFDRYAN
                     DFYELRKNKGITLEKARETIKDNIYFGCMMVKEGYADGLVSGAIHATADLLRPAFQIIK
                     TAPGAKIVSSFFIMEVPNCEYGENGVFLFADCAVNPSPNAEELASIAVQSANTAKNLLG
                     FEPKVAMLSFSTKGSASHELVDKVRKATEIAKELMPDVAIDGELQLDAALVKEVAELKA
                     PGSKVAGCANVLIFPDLQAGNIGYKLVQRLAKANAIGPITQGMGAPVNDLSRGCSYRDI
                     VDVIATTAVQAQ"
     gene            2209..3267
                     /gene="pta"
                     /label=pta
     CDS             3285..4496
                     /codon_start=1
                     /gene="ack"
                     /product="acetate kinase"
                     /label=ack
                     /note="negative selective marker in Thermoanaerobacter 
                     saccharolyticum"
                     /protein_id="AJP77043.1"
                     /translation="MKIMKILVINCGSSSLKYQLIESTDGNVLAKGLAERIGINDSMLT
                     HNANGEKIKIKKDMKDHKDAIKLVLDALVNSDYGVIKDMSEIDAVGHRVVHGGESFTSS
                     VLINDEVLKAITDCIELAPLHNPANIEGIKACQQIMPNVPMVAVFDTAFHQTMPDYAYL
                     YPIPYEYYTKYRIRRYGFHGTSHKYVSNRAAEILNKPIEDLKIITCHLGNGSSIAAVKY
                     GKSIDTSMGFTPLEGLAMGTRSGSIDPSIISYLMEKENISAEEVVNILNKKSGVYGISG
                     ISSDFRDLEDAAFKNGDERAQLALNVFAYRVKKTIGAYAAAMGGVDVIVFTAGVGENGP
                     EIREFILDGLEFLGFSLDKEKNKVRGKETIISTPNSKVSVMVVPTNEEYMIAKDTEKIV
                     KSIK"
     gene            3285..4496
                     /gene="ack"
                     /label=ack
     regulatory      4624..5154
                     /label=kanamycin promoter
                     /note="kanamycin promoter"
                     /regulatory_class="promoter"
     CDS             5155..5946
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    6285..7187
                     /label=downstream of Tsac_0628
                     /note="downstream of Tsac_0628"
     rep_origin      complement(7597..8142)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     promoter        8254..8282
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     CDS             complement(8359..9159)
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(9160..9380)
                     /label=URA3 promoter
     misc_feature    9408..9911
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
ORIGIN
        1 ctagagtcga tcttcgccag cagggcgagg atcgtggcat caccgaaccg cgccgtgcgc
       61 gggtcgtcgg tgagccagag tttcagcagg ccgcccaggc ggcccaggtc gccattgatg
      121 cgggccagct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg
      181 acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct
      241 cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg
      301 gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc
      361 agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac
      421 ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa
      481 ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat
      541 accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc
      601 cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac
      661 tgaggggaca ggcgagagac gatgccaaag agctacaccg acgagctggc cgagtgggtt
      721 gaatcccgcg cggccaagaa gcgccggcgt gatgaggctg cggttgcgtt cctggcggtg
      781 agggcggatg tcgatatgcg taaggagaaa ataccgcatc aggcgcatat ttgaatgtat
      841 ttagaaaaat aaacaaaaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc
      901 ttctgcttaa tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc
      961 cgttgcttcg caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc
     1021 accgacaaac aacagataaa acgaaaggcc cagtctttcg actgagcctt tcgttttatt
     1081 tgatgcctgg ctcatcgagg tatccaagcg attcaatagt aacagtcctt gtatgccctc
     1141 tttttaattc tcgccattcc ccttctttta aattttcatc taattttaat tggcctattg
     1201 aaagcctttt taagtagacg acttttgagc ctatcgcttc aaacattctt tttatttgat
     1261 gatatttacc ttctctgatt gagacatata ctttcgatgt actgcctgaa gatattattt
     1321 ccagttttgc cggcatagtc ttgtaaccat cgtctaatag tatgccatct gaaaacaaag
     1381 atacgtcatc ttcatcgata aaacccaaaa cttctgcata gtattttttg aaaacgtgtt
     1441 tttttggcga taagagctta tgagatagtt caccgtcatt tgtaattaaa agtaatccct
     1501 ctgtgtcttt atcaagcctt cctgctggga aaacctttct tgcctttata tggtgtggca
     1561 aaagatctac aacggttttt tctgatggat catatgttgc acagattaca cctttcggtt
     1621 tattcatcat tatatatatg tattctttgt acgatatttt ttcacttcta aacgtgatta
     1681 tatctttatc aggttgtact gcaaaaccgg ggtcgtcaat cgtcacatta tttattgcaa
     1741 caaggccttc ttttataaaa tttttaattt cttttcttgt gccataaccc atatttgata
     1801 aaagcttatc tattctcatt tttgacatct taaattcctc ctaaacaata tgactgtgct
     1861 tcttagtaaa ttatatccca aaaatataaa atttgtagca aaaatgtgat atatatcata
     1921 ttttttgcgt tttcctgatg atacaattaa gatgatgttt caagataata aattttctga
     1981 agtgtataca gtatattgac tacaaagaac aaaatagcta taataaaacc ataataaatc
     2041 gttaggggga ataaatatgt aaaccgctaa ggcatgatag ctaaagcgtg cccattgtga
     2101 agtggattgt attctacaat taaacctaat acgctcataa tatgcgcctt tctaaaaaat
     2161 tattaattgt acttattatt ttataaaaaa tatgttaaaa tgtaaaatgt gtatacaata
     2221 tatttcttct tagtaagagg aatgtataaa aataaatatt ttaaaggaag ggacgatctt
     2281 atgagcatta ttcaaaacat cattgaaaaa gctaaaagcg ataaaaagaa aattgttctg
     2341 ccagaaggtg cagaacccag gacattaaaa gctgctgaaa tagttttaaa agaagggatt
     2401 gcagatttag tgcttcttgg aaatgaagat gagataagaa atgctgcaaa agacttggac
     2461 atatccaaag ctgaaatcat tgaccctgta aagtctgaaa tgtttgatag gtatgctaat
     2521 gatttctatg agttaaggaa gaacaaagga atcacgttgg aaaaagccag agaaacaatc
     2581 aaggataata tctattttgg atgtatgatg gttaaagaag gttatgctga tggattggta
     2641 tctggcgcta ttcatgctac tgcagattta ttaagacctg catttcagat aattaaaacg
     2701 gctccaggag caaagatagt atcaagcttt tttataatgg aagtgcctaa ttgtgaatat
     2761 ggtgaaaatg gtgtattctt gtttgctgat tgtgcggtca acccatcgcc taatgcagaa
     2821 gaacttgctt ctattgccgt acaatctgct aatactgcaa agaatttgtt gggctttgaa
     2881 ccaaaagttg ccatgctatc attttctaca aaaggtagtg catcacatga attagtagat
     2941 aaagtaagaa aagcgacaga gatagcaaaa gaattgatgc cagatgttgc tatcgacggt
     3001 gaattgcaat tggatgctgc tcttgttaaa gaagttgcag agctaaaagc gccgggaagc
     3061 aaagttgcgg gatgtgcaaa tgtgcttata ttccctgatt tacaagctgg taatatagga
     3121 tataagcttg tacagaggtt agctaaggca aatgcaattg gacctataac acaaggaatg
     3181 ggtgcaccgg ttaatgattt atcaagagga tgcagctata gagatattgt tgacgtaata
     3241 gcaacaacag ctgtgcaggc tcaataaaat gtaaagtatg gaggatgaaa attatgaaaa
     3301 tactggttat taattgcgga agttcttcgc taaaatatca actgattgaa tcaactgatg
     3361 gaaatgtgtt ggcaaaaggc cttgctgaaa gaatcggcat aaatgattcc atgttgacac
     3421 ataatgctaa cggagaaaaa atcaagataa aaaaagacat gaaagatcac aaagacgcaa
     3481 taaaattggt tttagatgct ttggtaaaca gtgactacgg cgttataaaa gatatgtctg
     3541 agatagatgc tgtaggacat agagttgttc acggaggaga atcttttaca tcatcagttc
     3601 tcataaatga tgaagtgtta aaagcgataa cagattgcat agaattagct ccactgcaca
     3661 atcctgctaa tatagaagga attaaagctt gccagcaaat catgccaaac gttccaatgg
     3721 tggcggtatt tgatacagcc tttcatcaga caatgcctga ttatgcatat ctttatccaa
     3781 taccttatga atactacaca aagtacagga ttagaagata tggatttcat ggcacatcgc
     3841 ataaatatgt ttcaaatagg gctgcagaga ttttgaataa acctattgaa gatttgaaaa
     3901 tcataacttg tcatcttgga aatggctcca gcattgctgc tgtcaaatat ggtaaatcaa
     3961 ttgacacaag catgggattt acaccattag aaggtttggc tatgggtaca cgatctggaa
     4021 gcatagaccc atccatcatt tcgtatctta tggaaaaaga aaatataagc gctgaagaag
     4081 tagtaaatat attaaataaa aaatctggtg tttacggtat ttcaggaata agcagcgatt
     4141 ttagagactt agaagatgcc gcctttaaaa atggagatga aagagctcag ttggctttaa
     4201 atgtgtttgc atatcgagta aagaagacga ttggcgctta tgcagcagct atgggaggcg
     4261 tcgatgtcat tgtatttaca gcaggtgttg gtgaaaatgg tcctgagata cgagaattta
     4321 tacttgatgg attagagttt ttagggttca gcttggataa agaaaaaaat aaagtcagag
     4381 gaaaagaaac tattatatct acgccgaatt caaaagttag cgtgatggtt gtgcctacta
     4441 atgaagaata catgattgct aaagatactg aaaagattgt aaagagtata aaatagcatt
     4501 cttgacaaat gtttacccca ttagtataat taattttggc aattatattg gggtgagaaa
     4561 atgaaaattg atttatcaaa aattaaagga cataggggcc gcagcatcga agtcaactac
     4621 gtaaaaccca gcgaaccatt tgaggtgata ggtaagatta taccgaggta tgaaaacgag
     4681 aattggacct ttacagaatt actctatgaa gcgccatatt taaaaagcta ccaagacgaa
     4741 gaggatgaag aggatgagga ggcagattgc cttgaatata ttgacaatac tgataagata
     4801 atatatcttt tatatagaag atatcgccgt atgtaaggat ttcagggggc aaggcatagg
     4861 cagcgcgctt atcaatatat ctatagaatg ggcaaagcat aaaaacttgc atggactaat
     4921 gcttgaaacc caggacaata accttatagc ttgtaaattc tatcataatt gtggtttcaa
     4981 aatcggctcc gtcgatacta tgttatacgc caactttcaa aacaactttg aaaaagctgt
     5041 tttctggtat ttaaggtttt agaatgcaag gaacagtgaa ttggagttcg tcttgttata
     5101 attagcttct tggggtatct ttaaatactg tagaaaagag gaaggaaata ataaatggct
     5161 aaaatgagaa tatcaccgga attgaaaaaa ctgatcgaaa aataccgctg cgtaaaagat
     5221 acggaaggaa tgtctcctgc taaggtatat aagctggtgg gagaaaatga aaacctatat
     5281 ttaaaaatga cggacagccg gtataaaggg accacctatg atgtggaacg ggaaaaggac
     5341 atgatgctat ggctggaagg aaagctgcct gttccaaagg tcctgcactt tgaacggcat
     5401 gatggctgga gcaatctgct catgagtgag gccgatggcg tcctttgctc ggaagagtat
     5461 gaagatgaac aaagccctga aaagattatc gagctgtatg cggagtgcat caggctcttt
     5521 cactccatcg acatatcgga ttgtccctat acgaatagct tagacagccg cttagccgaa
     5581 ttggattact tactgaataa cgatctggcc gatgtggatt gcgaaaactg ggaagaagac
     5641 actccattta aagatccgcg cgagctgtat gattttttaa agacggaaaa gcccgaagag
     5701 gaacttgtct tttcccacgg cgacctggga gacagcaaca tctttgtgaa agatggcaaa
     5761 gtaagtggct ttattgatct tgggagaagc ggcagggcgg acaagtggta tgacattgcc
     5821 ttctgcgtcc ggtcgatcag ggaggatatc ggggaagaac agtatgtcga gctatttttt
     5881 gacttactgg ggatcaagcc tgattgggag aaaataaaat attatatttt actggatgaa
     5941 ttgttttagt acctagattt agatgtctaa aaagcttttt agacatctaa tcttttctga
     6001 agtacatccg caactgtcca tactctgatg ttttatatct tttctaaaag ttcgctagat
     6061 aggggtcccg agcgcctacg aggaatttgt atcggatccg caagagatta tatcgagtgc
     6121 ctttaagaag gctaaaaatt acgaagatgt gatacacaaa aaggcaaaag attacggcaa
     6181 aaacataccg gatagtcaag ttaaaggagt attgaaacag atagagatta ctgccttaaa
     6241 ccatgtagac aagattgtcg ctgctgaaaa gacgatgcag atagtatcat ggggcttttc
     6301 ctttgaatta atttgataaa gggtgtaaaa ttatcatgtg atgatgtgat tttggaggta
     6361 atcgcatgaa tttaaataag ataaatagaa acacgtacta catagataat cctacgaata
     6421 ttggcgttta tgcctataaa aataaaaatt gtctattagt agatactggt ataaacaatg
     6481 gacaagcgag aaagattgac aatgtattag tagaaaatgg attacatccc aaatatatca
     6541 taaacaccca caaccacatg gaccactgtg gcggtaatat atactttaag actcagtacc
     6601 ctggatgcga aatctataca tcaaataagg agcgattata tatagaaaat ccagagttaa
     6661 gagatattgt actattttct tcatgcccaa taagagatct tgatacgact aataaaacat
     6721 tttctgtaga ttttgtcctt gattatggta tctcaaaaat tggcgatgaa aagtttgata
     6781 taatatcttt ggtggggcat tcaatagatc aaatcggcgt cattacacct gatagagtat
     6841 gttttttggg agacagtgta tttagcgagg atacaataaa aaaatattca cttccatatt
     6901 tgtttaatat agaaaaaagc attgaaactt taaaaaagct aaaagaggtg gatgcagact
     6961 atttcgtaat aagtcacata gatagagtgc ttaataagga tgagcttgat gtcctgatag
     7021 aaaagaatat ttcaaatata gaagataatg ttgaaattat actggaatta ttggaacaac
     7081 ctcagacaag ggaaggatta ttgcaaaact tagtaatatt aaacgatctg ccattaaatt
     7141 ttactcagta ttatatttat ttttcatcgg tttccgcatt tttgagcagt taggatgttg
     7201 gcagacgctt gttgatacga ggcttaaaat tgctgaggta atcggtaaat tcgacccgtc
     7261 tatcagctgt ccctcctgtt cagctactga cggggtggtg cgtaacggca aaagcaccgc
     7321 cggacatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc
     7381 agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt
     7441 gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     7501 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     7561 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     7621 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     7681 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     7741 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     7801 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     7861 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     7921 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     7981 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     8041 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     8101 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaat
     8161 cagataaaat atttctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc
     8221 agccccatac gatataagtt gtaattctca tgtttgacag cttatcatcg ataagcttta
     8281 atgcggtagt ttatcacagt taaattgcta acgcagtcag gcacctatac atgcatttac
     8341 ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc
     8401 ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc
     8461 aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc
     8521 caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc
     8581 ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct
     8641 cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga
     8701 caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt
     8761 caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc
     8821 tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa
     8881 ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac
     8941 tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt
     9001 gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc
     9061 acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg
     9121 atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca
     9181 ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact
     9241 acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc
     9301 ggagattacc gaatcaaaaa aatttcaaag aaaccgaaat caaaaaaaag aataaaaaaa
     9361 aaatgatgaa ttgaattgaa aagctagctt atcgatgggt ccttttcatc acgtgctata
     9421 aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata gaaagtaaaa
     9481 aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact ctagggggat
     9541 cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa tgccgaattg
     9601 tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac attttactta
     9661 tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat atatatgtaa
     9721 agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt cattccgtaa
     9781 ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa aataaataaa
     9841 cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt gtaagttaca
     9901 ggcaagcgat ctctaagaaa ccattattat catgacatta acctataaaa aaggcctctc
     9961 gag
//
