LOCUS       V016341                 8716 bp    DNA     circular SYN 16-FEB-2021
DEFINITION  Exported.
ACCESSION   V016341
VERSION     V016341
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8716)
  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  JOURNAL   Unpublished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 8716)
  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 8716)
  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            "Unpublished"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..8716
                     /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="QRQ89509.1"
                     /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..4369)
                     /label="gene target internal homology recombination flank"
                     /note="gene target internal homology recombination flank"
     gene            complement(4554..5099)
                     /gene="hpt"
                     /label="hpt"
     CDS             complement(4554..5099)
                     /codon_start=1
                     /transl_table=11
                     /gene="hpt"
                     /product="hypoxanthine phosphoribosyltransferase"
                     /label="hpt"
                     /protein_id="QRQ89510.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     CDS             complement(5120..5767)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     regulatory      complement(5768..6342)
                     /label="C. thermocellum gapDH promoter"
                     /note="C. thermocellum gapDH promoter"
                     /regulatory_class="promoter"
     misc_feature    complement(6346..6967)
                     /label="downstream homology recombination 3' flank"
                     /note="downstream homology recombination 3' flank"
     misc_feature    complement(6968..7556)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     promoter        7649..7753
                     /label="AmpR promoter"
     CDS             7754..8611
                     /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 gaggatgcag ctaaagcaat
     3841 tggcgtaact gcaaaatata ccggtcagac agatactgat gtttcaggtc aggttgcagt
     3901 gcttgaacag gtcattgctc aaaaaccaaa aggtatagca gtaacagcgg ttaattcaac
     3961 agcattggcg gatacaatca attcagctat tgaacaaggt atttcagttg tatgctttga
     4021 ctctgactct cctacttcaa accgttccgc atatcttgga accggtaact atgcagcagg
     4081 tcagaaggcg gctgagttcc tggtaccgtt ggtaaactac aaaggtaaaa ttgcagtatt
     4141 gtatacagtt ggtgcagaaa acagtgaatc ccgtgttcag ggttttgaag actggtgtaa
     4201 acaaaatgcg cccgaagttt ccctcgtaaa agttaatgat gcaggcgata caactgtcgc
     4261 agcggacaac ctggcggctg cattacaggc aaatgatgat attgttggtg tattctgtgt
     4321 agacggtgtt gcaggtactg caggaccgac agcagttgca gaatcaaagc tgaactactg
     4381 ggccaggtat gtattaaagc atggggaggg cgtgaatgta agcgtgacat aactaattac
     4441 atgatgcggc cctctaggat cagcgggttt aaacgctgag gcgcgccgaa ttcggtacca
     4501 aaacaaaagg cccagtcttc cgactgagcc ttttgttttc tcgaggcccg ggcttagctg
     4561 tacatttcag gtttcaaaac gcctataaaa ggaaggttcc tgtacttttc atcaaagtcc
     4621 aatccataac ccacgacaaa cttatcaggt atcttaaatc ctacataatc gacttttaca
     4681 gatgcttctc ttctctccgg tttgtctaat attgtgcata ttttcaggct ttttggcttc
     4741 cttccaagta aagtttccct taagtaagac aaagtcaaac cgctgtcaat tatgtcttcc
     4801 acaatcagaa catctttgcc ttctatgctt atatcaagat ctttgattat ctttactatt
     4861 cctgatgaat gagttgagct tccatagctg gaaacagcca taaaatcaag tgataaaggc
     4921 aaatctatgt gtcttgacaa atcagccata aacattaaag caccttttaa aactcctacc
     4981 aacatcaaat ttttcccttt gtagtctttt gtgatttgcc tcccaagctc ttttatcttt
     5041 tccttaagtt cttcttctgt gatcaaaatt tcatcgatgt cttttgataa attttccatg
     5101 acgtcgacct cctttattat aaaagccagt cattaggcct atctgacaat tcctgaatag
     5161 agttcataaa caatcctgca tgataaccat cacaaacaga atgatgtacc tgtaaagata
     5221 gcggtaaata tattgaatta cctttattaa tgaattttcc tgctgtaata atgggtagaa
     5281 ggtaattact attattattg atatttaagt taaacccagt aaatgaagtc catggaataa
     5341 tagaaagaga aaaagcattt tcaggtatag gtgttttggg aaacaatttc cccgaaccat
     5401 tatatttctc tacatcagaa aggtataaat cataaaactc tttgaagtca ttctttacag
     5461 gagtccaaat accagagaat gttttagata caccatcaaa aattgtataa agtggctcta
     5521 acttatccca ataacctaac tctccgtcgc tattgtaacc agttctaaaa gctgtatttg
     5581 agtttatcac ccttgtcact aagaaaataa atgcagggta aaatttatat ccttcttgtt
     5641 ttatgtttcg gtataaaaca ctaatatcaa tttctgtggt tatactaaaa gtcgtttgtt
     5701 ggttcaaata atgattaaat atctcttttc tcttccaatt gtctaaatca attttattaa
     5761 agttcattaa tatcgcctcc tattgtaaat taaaatttaa tttaatgcct ctattagctg
     5821 ccataaagca tgaatgcagt aaaatattca tcctctggca agtatctctt gcactatgat
     5881 tcattttata ttattttatt ttcattttca atagtttaga taaaaaataa ttaatttttt
     5941 aacaaagtat ttttatcttc ctttgatgta attctttgtt tttttgtcca ttaatattat
     6001 ggacaatttt tttatttcta gtcaataatt ttaatcttta ttaaaacctg gcaatgccag
     6061 agagatacag taattattgc atataaaaga aaacagacgc gccctgtttg tttcctgaaa
     6121 aaccacctcg ccctccaaag tatccagctt tttaaaatcc atcctcattc ccagacgaaa
     6181 aagttttgac aaagcctctt ttcttgtcca tagaattgta ctctgcacac aatattcatc
     6241 agtatttcca aaactcttta gaatcttttc ctctctttcg gaaaagaaat actttatcaa
     6301 tgcatcctcg ggcaaaaaaa tcttttccat gcctattccc acgatggaat acccgttctc
     6361 ttcatcatat ttataataat ttcggcgatt gcatttatct ttatgaagta ttcaaagata
     6421 tgcaggaata tatactacgt cggaagcaat gaaaatgctg caagactttc cggtataaat
     6481 gtagacaggg taaaaatcgg tgtatatgtg acaattgcgt taatctccac attagcaggc
     6541 ttattgtctc tggcacgctt taacgtggca accccggaac ttagcaaggg tgctgaaaca
     6601 acggcaattt ccgcggcagt tatcggcgga acatcgatga caggtggttc cggaggcgta
     6661 ctgggaactg ttttgggtat tttcttactg aagatagtta attcggcact tgttatgatg
     6721 aacgtttcgg tttattggca ggagtttgtt cagggagcaa ttcttgtact ggctgttacc
     6781 attgactatt tgagtcaccg taaaaacggc ggtccaagct ttataatgaa attcttcgga
     6841 ttggacaaat tggcttcaaa aaaattagat gacaaatctt ccaggcaaat ttaattttta
     6901 agaattttgg tgctttatgc actgaaagat aaaaatacat tttatcctta aaaatgtaag
     6961 gaggtaagaa taaaataaaa tcagcatgaa aggaaagtaa gtttatgaaa gcatttaact
     7021 attatgcacc tactgaaatt attttcggat gcggcagagt tcaggaaatt ggatcaataa
     7081 ctgcccaata tggaaagaaa gctttgctgg taactgtacc ggaatttccg gaagtaaagg
     7141 agctctatga aaaagtaaag aagtctctta gggaaaatgg tgtggaagta gtacattttg
     7201 acggtgttat tccaaatccc accactgatg ttgtaactga aggagcaaat atggcaaagg
     7261 ctgccggggt tgatgtagtt attggattgg gaggaggttc ctcaatagat actgcaaaag
     7321 ccattgcagt tgaagcaacc catccgggaa ctgcctggga ttataactgc cacactccgg
     7381 gccccacatc tgctaccctc cctattatcg caataggaac aacagcagga accggcagcc
     7441 agtgtacaca atgtgcagtt atcacaaaaa caagtgaaaa ggataaatcc gcaatttggc
     7501 acaaaaatat tttccctaaa gttgcgatag ttgatcccga agtgacagtc acgatggata
     7561 tcgcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     7621 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     7681 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     7741 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     7801 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     7861 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     7921 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     7981 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     8041 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     8101 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     8161 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     8221 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     8281 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     8341 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     8401 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     8461 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc gctcccgtat
     8521 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     8581 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     8641 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     8701 tgataatcgc ggccgc
//
