LOCUS       V015288                 8597 bp    DNA     circular SYN 16-FEB-2021
DEFINITION  Exported.
ACCESSION   V015288
VERSION     V015288
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8597)
  AUTHORS   Yayo J, Kuil T, Olson DG, Lynd LR, Holwerda EK, van Maris AJA.
  TITLE     Laboratory evolution and reverse engineering of Clostridium
            thermocellum for growth on glucose and fructose
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8597)
  AUTHORS   Yayo J, Kuil T, Olson DG, Lynd LR, Holwerda EK, van Maris AJA.
  TITLE     Direct Submission
  JOURNAL   Submitted (09-DEC-2020) Industrial Biotechnology, KTH Royal
            Institute of Technology, Roslagstullsbacken 21, Stockholm 11421,
            Sweden
REFERENCE   3  (bases 1 to 8597)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName:
            "Unpublished"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (09-DEC-2020) Industrial Biotechnology, KTH Royal Institute of
            Technology, Roslagstullsbacken 21, Stockholm 11421, Sweden"
FEATURES             Location/Qualifiers
     source          1..8597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..455
                     /label="C. thermocellum origin of replication"
                     /note="C. thermocellum origin of replication"
     CDS             456..1457
                     /label="repB"
                     /note="RepB replication protein"
     regulatory      1536..2156
                     /label="C. thermocellum cbp promoter"
                     /note="C. thermocellum cbp promoter"
                     /regulatory_class="promoter"
     gene            2157..2735
                     /gene="tdk"
                     /label="tdk"
     CDS             2157..2735
                     /codon_start=1
                     /transl_table=11
                     /gene="tdk"
                     /product="thymidine kinase"
                     /label="tdk"
                     /protein_id="QRQ89494.1"
                     /translation="MYGPKDHGYIEVVTGPMFSGKSEELIRRIKRAKIARQKVQVFKPA
                     IDDRYSIDKVVSHNGDNMHAIAIVKASDILAYAEEDTDVFAIDEVQFFDSEIVDIVKEI
                     ADSGKRVICAGLDMDFRGEPFGPTPELMAIAEFVDKLTAICMKCGNPATRTQRLINGKP
                     ANYDDPIIMVGAKESYEARCRKCHEVPRT"
     rep_origin      complement(3002..3547)
                     /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."
     terminator      complement(3597..3785)
                     /label="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     misc_feature    complement(3821..4339)
                     /label="gene target internal homology recombination flank"
                     /note="gene target internal homology recombination flank"
     gene            complement(4524..5069)
                     /gene="hpt"
                     /label="hpt"
     CDS             complement(4524..5069)
                     /codon_start=1
                     /transl_table=11
                     /gene="hpt"
                     /product="hypoxanthine phosphoribosyltransferase"
                     /label="hpt"
                     /protein_id="QRQ89495.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     CDS             complement(5090..5737)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     regulatory      complement(5738..6312)
                     /label="C. thermocellum gapDH promoter"
                     /note="C. thermocellum gapDH promoter"
                     /regulatory_class="promoter"
     misc_feature    complement(6316..6896)
                     /label="downstream homology recombination 3' flank"
                     /note="downstream homology recombination 3' flank"
     misc_feature    complement(6897..7437)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     promoter        7530..7634
                     /label="AmpR promoter"
     CDS             7635..8492
                     /label="AmpR"
                     /note="beta-lactamase"
ORIGIN
        1 tatgggaaac aaaatattgc gtatgcgact gttcacatgg acgagaaaac ccctcacatg
       61 catttaggag ttgttcctat gcgcctagag ggcttttcgt gcgtcagcat gagcgatcgg
      121 aagaaaagaa gaatactttt tcgcttcaag atgttttgca gcgtgatcga gaacttcgtg
      181 agcaaagaaa agcaaagagg aaaaaatcgc atgatttgga gcgataagaa aaagcactcg
      241 aatgagtgct ttttttgcgt tttgagcgta gcgaaaaacg agttctttct attcttgata
      301 catatagaaa taacgtcatt tttattttag ttgctgaaag gtgcgttgaa gtgttggtat
      361 gtatgtgttt taaagtattg aaaaccctta aaattggttg cacagaaaaa ccccatctgt
      421 taaagttata agtgaccaaa caaataacta aatagatggg ggtttctttt aatattatgt
      481 gtcctaatag tagcatttat tcagatgaaa aatcaagggt tttagtggac aagacaaaaa
      541 gtggaaaagt gagaccatgg agagaaaaga aaatcgctaa tgttgattac tttgaacttc
      601 tgcatattct tgaatttaaa aaggctgaaa gagtaaaaga ttgtgctgaa atattagagt
      661 ataaacaaaa tcgtgaaaca ggcgaaagaa agttgtatcg agtgtggttt tgtaaatcca
      721 ggctttgtcc aatgtgcaac tggaggagag caatgaaaca tggcattcag tcacaaaagg
      781 ttgttgctga agttattaaa caaaagccaa cagttcgttg gttgtttctc acattaacag
      841 ttaaaaatgt ttatgatggc gaagaattaa ataagagttt gtcagatatg gctcaaggat
      901 ttcgccgaat gatgcaatat aaaaaaatta ataaaaatct tgttggtttt atgcgtgcaa
      961 cggaagtgac aataaataat aaagataatt cttataatca gcacatgcat gtattggtat
     1021 gtgtggaacc aacttatttt aagaatacag aaaactacgt gaatcaaaaa caatggattc
     1081 aattttggaa aaaggcaatg aaattagact atgatccaaa tgtaaaagtt caaatgattc
     1141 gaccgaaaaa taaatataaa tcggatatac aatcggcaat tgacgaaact gcaaaatatc
     1201 ctgtaaagga tacggatttt atgaccgatg atgaagaaaa gaatttgaaa cgtttgtctg
     1261 atttggagga aggtttacac cgtaaaaggt taatctccta tggtggtttg ttaaaagaaa
     1321 tacataaaaa attaaacctt gatgacacag aagaaggcga tttgattcat acagatgatg
     1381 acgaaaaagc cgatgaagat ggattttcta ttattgcaat gtggaattgg gaacggaaaa
     1441 attattttat taaagagtag ttcaacaaac gggattgact tttaaaaaag gattgattct
     1501 aatgaagaaa gcagacaagt aagcctccta aattcgagtc gtgactaaga acgtcaaagt
     1561 aattaacaat acagctattt ttctcatgct tttacccctt tcataaaatt taattttatc
     1621 gttatcataa aaaattatag acgttatatt gcttgccggg atatagtgct gggcattcgt
     1681 tggtgcaaaa tgttcggagt aaggtggata ttgatttgca tgttgatcta ttgcattgaa
     1741 atgattagtt atccgtaaat attaattaat catatcataa attaattata tcataattgt
     1801 tttgacgaat gaaggttttt ggataaatta tcaagtaaag gaacgctaaa aattttggcg
     1861 taaaatatca aaatgaccac ttgaattaat atggtaaagt agatataata ttttggtaaa
     1921 catgccttca gcaaggttag attagctgtt tccgtataaa ttaaccgtat ggtaaaacgg
     1981 cagtcagaaa aataagtcat aagattccgt tatgaaaata tacttcggta gttaataata
     2041 agagatatga ggtaagagat acaagataag agatataagg tacgaatgta taagatggtg
     2101 cttttaggca cactaaataa aaaacaaata aacgaaaatt ttaaggagga cgaaagatgt
     2161 atgggcctaa agaccacggg tacatagaag ttgtaactgg tcccatgttc agtggaaaaa
     2221 gtgaggaact tataagaagg ataaagagag ctaagattgc caggcaaaaa gttcaggttt
     2281 tcaaaccggc tatagacgat aggtattcca tagacaaagt cgtatctcat aacggcgaca
     2341 acatgcacgc cattgccata gtaaaggctt ctgacatatt ggcttatgct gaagaagata
     2401 cggatgtatt tgctatagat gaagttcaat tttttgattc tgaaatagtc gacatcgtaa
     2461 aagagattgc cgatagcggg aaaagagtca tatgtgcagg gcttgacatg gactttagag
     2521 gtgaaccatt tggtccgact ccagaattga tggccatagc tgaatttgtc gataagctta
     2581 ctgctatatg catgaagtgt ggcaatcctg ctactcgcac gcaaaggctt ataaatggga
     2641 agcctgccaa ttacgacgat cccattataa tggttggagc aaaggagtct tatgaagcaa
     2701 ggtgtagaaa gtgccatgaa gtcccgcgga cttaagcgga tttttttgta tactggctta
     2761 ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct
     2821 gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac
     2881 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat
     2941 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
     3001 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     3061 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc
     3121 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
     3181 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
     3241 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3301 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga
     3361 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg
     3421 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa
     3481 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg
     3541 atctcaagaa gatcatctta ttaatcagat aaaagccgca gcttgcaaat taaagccttc
     3601 gagcgtccca aaaccttctc aagcaaggtt ttcagtataa tgttacatgc gtacacgcgt
     3661 ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg ttcttaatac taacataact
     3721 ataaaaaaat aaatagggac ctagacttca ggttgtctaa ctccttcctt ttcggttaga
     3781 gcggcattat ccctgatttt ttcactacta ttagcagctg gctgattatt tgggcattac
     3841 tcgacaggca gtggcgtcgt atgagcttgc taaaagggaa ccggactatg aagttcttaa
     3901 aaagctcgca gattattttg acgtatccat tgactatttg cttggaagaa ccaattgcag
     3961 agatgctacg gcagcaatta taggtaagaa tattgatctt ataaggggaa atcttacata
     4021 caaggaattg tcagaggata ttagtgccaa attgggagcg ttgatttttc ctgagatgct
     4081 tgagttatat gctcgtggtg aaagaatgcc ctttgtcgga acgataaaaa tacttgccaa
     4141 atatgcacag gtaagggatt ctttttttta cacccataac actccagaga cctatgaaag
     4201 agagaaagaa ttgtacagac tggaaatgag ccagcagaaa atggcagagg agattgaaga
     4261 gacaaaaggt ttgcttgatt ttaccggtga tgtggaacta aagaaatgga ttttaaaaga
     4321 gagcagtctg ccttatatcc tgaactactg ggccaggtat gtattaaagc atggggaggg
     4381 cgtgaatgta agcgtgacat aactaattac atgatgcggc cctctaggat cagcgggttt
     4441 aaacgctgag gcgcgccgaa ttcggtacca aaacaaaagg cccagtcttc cgactgagcc
     4501 ttttgttttc tcgaggcccg ggcttagctg tacatttcag gtttcaaaac gcctataaaa
     4561 ggaaggttcc tgtacttttc atcaaagtcc aatccataac ccacgacaaa cttatcaggt
     4621 atcttaaatc ctacataatc gacttttaca gatgcttctc ttctctccgg tttgtctaat
     4681 attgtgcata ttttcaggct ttttggcttc cttccaagta aagtttccct taagtaagac
     4741 aaagtcaaac cgctgtcaat tatgtcttcc acaatcagaa catctttgcc ttctatgctt
     4801 atatcaagat ctttgattat ctttactatt cctgatgaat gagttgagct tccatagctg
     4861 gaaacagcca taaaatcaag tgataaaggc aaatctatgt gtcttgacaa atcagccata
     4921 aacattaaag caccttttaa aactcctacc aacatcaaat ttttcccttt gtagtctttt
     4981 gtgatttgcc tcccaagctc ttttatcttt tccttaagtt cttcttctgt gatcaaaatt
     5041 tcatcgatgt cttttgataa attttccatg acgtcgacct cctttattat aaaagccagt
     5101 cattaggcct atctgacaat tcctgaatag agttcataaa caatcctgca tgataaccat
     5161 cacaaacaga atgatgtacc tgtaaagata gcggtaaata tattgaatta cctttattaa
     5221 tgaattttcc tgctgtaata atgggtagaa ggtaattact attattattg atatttaagt
     5281 taaacccagt aaatgaagtc catggaataa tagaaagaga aaaagcattt tcaggtatag
     5341 gtgttttggg aaacaatttc cccgaaccat tatatttctc tacatcagaa aggtataaat
     5401 cataaaactc tttgaagtca ttctttacag gagtccaaat accagagaat gttttagata
     5461 caccatcaaa aattgtataa agtggctcta acttatccca ataacctaac tctccgtcgc
     5521 tattgtaacc agttctaaaa gctgtatttg agtttatcac ccttgtcact aagaaaataa
     5581 atgcagggta aaatttatat ccttcttgtt ttatgtttcg gtataaaaca ctaatatcaa
     5641 tttctgtggt tatactaaaa gtcgtttgtt ggttcaaata atgattaaat atctcttttc
     5701 tcttccaatt gtctaaatca attttattaa agttcattaa tatcgcctcc tattgtaaat
     5761 taaaatttaa tttaatgcct ctattagctg ccataaagca tgaatgcagt aaaatattca
     5821 tcctctggca agtatctctt gcactatgat tcattttata ttattttatt ttcattttca
     5881 atagtttaga taaaaaataa ttaatttttt aacaaagtat ttttatcttc ctttgatgta
     5941 attctttgtt tttttgtcca ttaatattat ggacaatttt tttatttcta gtcaataatt
     6001 ttaatcttta ttaaaacctg gcaatgccag agagatacag taattattgc atataaaaga
     6061 aaacagacgc gccctgtttg tttcctgaaa aaccacctcg ccctccaaag tatccagctt
     6121 tttaaaatcc atcctcattc ccagacgaaa aagttttgac aaagcctctt ttcttgtcca
     6181 tagaattgta ctctgcacac aatattcatc agtatttcca aaactcttta gaatcttttc
     6241 ctctctttcg gaaaagaaat actttatcaa tgcatcctcg ggcaaaaaaa tcttttccat
     6301 gcctattccc acgatcttac ggagttaagc atgtatccat agcgtctttt cctgagatga
     6361 aggataagac ggtggttata aacggctttt ccaaagccta tgccatgacg ggctggagac
     6421 ttggatattg cttggggcat cctgatttga tttcacagat gtacaagata catcaatatg
     6481 ccataatgtg ttctccgaca actgcccaat atgcagccat agaagcttta agaaacagtg
     6541 atgatgatgt ggaagcaatg gtcagagagt acaaccgaag aagaagaatt atggttgaag
     6601 gtttcaggag tgcagggctt gaatgttttg agccgttggg tgctttttat gtttttccgt
     6661 gcatagaatc aacaggaatg acttcgtatg agttttccga aaggctttta atggaggaaa
     6721 aagttgcggt agttccgggg actgcttttg gagaatgtgg agaaggatat gtacgttcgt
     6781 gctatgccag ctccatggac aatataattg aagcaatgaa aagaattaaa agatttgtcg
     6841 aaagaaatag aaagaagtaa aaaatagaaa taaaaagact ctggttttgg aaagctttgt
     6901 gatataattg taattctttt atttcatttc ggataattta tatcagggtg caaaatttta
     6961 taatttaata gtttggacca attcacgccg gttgcattat ggtcttaaag gctaaatact
     7021 accttacaga gattattgga atttagttgc caaatatttt aattaaattt tatgatagac
     7081 attatttctt atatgttgta aaataaaccg taatgaaatt taaatattga ttggaggagt
     7141 tgatgtaagt gctggagctt gaacagctta aacttgaaat cgatgatttt aagcaaaatt
     7201 tagatgaaat ggggaaatct ctttgacatc gccagtatag gcaatcaaat tgaggaactt
     7261 gaacaaaaag cttcagaacc tgatttctgg aatgataccg agaattctca gaaaattttg
     7321 caaaagatta aatctttaag aagcaaagtg gaaagataca acaagctggt ttcccagtgg
     7381 gaagatttga tcactttgtg tgagcttgga attgaagagc aggatgaaag tgtgattgat
     7441 atcgcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     7501 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     7561 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     7621 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     7681 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     7741 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     7801 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     7861 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     7921 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     7981 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     8041 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     8101 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     8161 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     8221 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     8281 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     8341 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag cgctcccgta
     8401 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     8461 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     8521 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     8581 ttgataatcg cggccgc
//
