LOCUS       V015504                 8679 bp    DNA     circular SYN 16-FEB-2021
DEFINITION  Exported.
ACCESSION   V015504
VERSION     V015504
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8679)
  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  JOURNAL   Unpublished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 8679)
  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 8679)
  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            "Unpublished"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..8679
                     /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="QRQ89499.1"
                     /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..4392)
                     /label="gene target internal homology recombination flank"
                     /note="gene target internal homology recombination flank"
     gene            complement(4577..5122)
                     /gene="hpt"
                     /label="hpt"
     CDS             complement(4577..5122)
                     /codon_start=1
                     /transl_table=11
                     /gene="hpt"
                     /product="hypoxanthine phosphoribosyltransferase"
                     /label="hpt"
                     /protein_id="QRQ89500.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     CDS             complement(5143..5790)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     regulatory      complement(5791..6365)
                     /label="C. thermocellum gapDH promoter"
                     /note="C. thermocellum gapDH promoter"
                     /regulatory_class="promoter"
     misc_feature    complement(6369..6960)
                     /label="downstream homology recombination 3' flank"
                     /note="downstream homology recombination 3' flank"
     misc_feature    complement(6961..7519)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     promoter        7612..7716
                     /label="AmpR promoter"
     CDS             7717..8574
                     /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 ggtttttctt tgtcggagtg
     3841 gatgtaggaa gaacatctgt aagggtagtt gtaatgaaca actgcaggga tgttgtttac
     3901 aaagtgagta agccgacgga atctgttgaa ccagatgaat taataaatca gataaccgaa
     3961 atgactatgg aaagtataaa tgaatcaaaa ttcccccttg acagagttgt tggtataggt
     4021 gttgcaatgc cgggcttaat tgagcggggc accggcaggg taatttttag cccgaatttt
     4081 ggctggaaca atattgcttt acaagacgaa cttaaaaagc accttccttt caatgtactg
     4141 gtggaaaacg caaatcgcgc cctggttata ggagagataa aaaatacaca gccaaatcct
     4201 acttcatgta ttgtcggggt taaccttgga tacggtatcg gatcggcgat agtcttacca
     4261 aatggtttgt attatggggt aagcggaacg agtggtgaaa ttggacatat tattgttgaa
     4321 aaccatggtt catattgttc gtgtggcaat tatgggtgta ttgaatccat tgcaagcgga
     4381 gaagcgatag ctctgaacta ctgggccagg tatgtattaa agcatgggga gggcgtgaat
     4441 gtaagcgtga cataactaat tacatgatgc ggccctctag gatcagcggg tttaaacgct
     4501 gaggcgcgcc gaattcggta ccaaaacaaa aggcccagtc ttccgactga gccttttgtt
     4561 ttctcgaggc ccgggcttag ctgtacattt caggtttcaa aacgcctata aaaggaaggt
     4621 tcctgtactt ttcatcaaag tccaatccat aacccacgac aaacttatca ggtatcttaa
     4681 atcctacata atcgactttt acagatgctt ctcttctctc cggtttgtct aatattgtgc
     4741 atattttcag gctttttggc ttccttccaa gtaaagtttc ccttaagtaa gacaaagtca
     4801 aaccgctgtc aattatgtct tccacaatca gaacatcttt gccttctatg cttatatcaa
     4861 gatctttgat tatctttact attcctgatg aatgagttga gcttccatag ctggaaacag
     4921 ccataaaatc aagtgataaa ggcaaatcta tgtgtcttga caaatcagcc ataaacatta
     4981 aagcaccttt taaaactcct accaacatca aatttttccc tttgtagtct tttgtgattt
     5041 gcctcccaag ctcttttatc ttttccttaa gttcttcttc tgtgatcaaa atttcatcga
     5101 tgtcttttga taaattttcc atgacgtcga cctcctttat tataaaagcc agtcattagg
     5161 cctatctgac aattcctgaa tagagttcat aaacaatcct gcatgataac catcacaaac
     5221 agaatgatgt acctgtaaag atagcggtaa atatattgaa ttacctttat taatgaattt
     5281 tcctgctgta ataatgggta gaaggtaatt actattatta ttgatattta agttaaaccc
     5341 agtaaatgaa gtccatggaa taatagaaag agaaaaagca ttttcaggta taggtgtttt
     5401 gggaaacaat ttccccgaac cattatattt ctctacatca gaaaggtata aatcataaaa
     5461 ctctttgaag tcattcttta caggagtcca aataccagag aatgttttag atacaccatc
     5521 aaaaattgta taaagtggct ctaacttatc ccaataacct aactctccgt cgctattgta
     5581 accagttcta aaagctgtat ttgagtttat cacccttgtc actaagaaaa taaatgcagg
     5641 gtaaaattta tatccttctt gttttatgtt tcggtataaa acactaatat caatttctgt
     5701 ggttatacta aaagtcgttt gttggttcaa ataatgatta aatatctctt ttctcttcca
     5761 attgtctaaa tcaattttat taaagttcat taatatcgcc tcctattgta aattaaaatt
     5821 taatttaatg cctctattag ctgccataaa gcatgaatgc agtaaaatat tcatcctctg
     5881 gcaagtatct cttgcactat gattcatttt atattatttt attttcattt tcaatagttt
     5941 agataaaaaa taattaattt tttaacaaag tatttttatc ttcctttgat gtaattcttt
     6001 gtttttttgt ccattaatat tatggacaat ttttttattt ctagtcaata attttaatct
     6061 ttattaaaac ctggcaatgc cagagagata cagtaattat tgcatataaa agaaaacaga
     6121 cgcgccctgt ttgtttcctg aaaaaccacc tcgccctcca aagtatccag ctttttaaaa
     6181 tccatcctca ttcccagacg aaaaagtttt gacaaagcct cttttcttgt ccatagaatt
     6241 gtactctgca cacaatattc atcagtattt ccaaaactct ttagaatctt ttcctctctt
     6301 tcggaaaaga aatactttat caatgcatcc tcgggcaaaa aaatcttttc catgcctatt
     6361 cccacgatct ttggaagccg ccagaattgc aaaagaagca ggagcaataa tttcatatga
     6421 cccgaattac cggcctgctt tgtggagtaa tgaacacgaa gcacgtaaat acatgttaaa
     6481 ggcagttgaa tatgctgatt tggttaaagt cagtgaagaa gaactttggc ttttggcgga
     6541 aagtgaggat ttttccaaag gggcgcacaa actgcttgcc atgggtccgt cggttgtctt
     6601 tgttacttgt ggtgaaaaag gctcttatgt tttcactgga agttgtaagg cgaatcactg
     6661 tggatacgac gtaaaagtgg ttgatactac cggggcggga gatgcgtttg tgggagccat
     6721 gctatggcag cttaggggta tgtctttgga agaaatatct cagatatgtg agcaaaaatg
     6781 gggagaatta ctggcatttt caaatgctgc aggtactatt gtggctacta aaaaaggtgc
     6841 aattcctgca atgcccgacc tggatacgat aaatactttt atgaggcagt ttcttgaaga
     6901 gaagtaaaaa aggcagagta tgatttgaca gtagatataa tgacggaaag ggaggtgctg
     6961 ctctttagtt aatgcatttc atttttcgct gtatccaatc agttttttat ttataaatat
     7021 taaaaattga aaaaaaatac attgactttc tatataaaaa ggatatataa taaaattgta
     7081 gttagttaag ttaactaagt aatcgtttat taaaaataat cataattttt tcttcttagt
     7141 agtacgaaag atttagtatc aatcaaagta tcattaagta tcaattcaaa ggttgtagtc
     7201 ctgcagtact taatataccc caccgattga tagatgcaac taaagtaaag ttggtaaagc
     7261 aaggaggagc gtaaatggat aatgctttac ttctggaagt taaagggctt tcaaaagtat
     7321 tccctggaac acgtgcacta aatcgtgtca gccttgaagt aagaaaaggt gaagtacatg
     7381 gtttgtgcgg agaaaacggt gcagggaaaa gcactttaat gaacattgta ggtggagttt
     7441 ttcctgcaac tgaaggaatt atgaaattcg acggcaaaat tttcaatccc aaaaacccta
     7501 aggactctca ggatgcaggg atatcgcctc gtgatacgcc tatttttata ggttaatgtc
     7561 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     7621 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     7681 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     7741 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     7801 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     7861 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     7921 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     7981 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     8041 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     8101 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     8161 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     8221 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     8281 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     8341 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     8401 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     8461 ccagatggta agcgctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     8521 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     8581 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     8641 aggatctagg tgaagatcct ttttgataat cgcggccgc
//
