LOCUS       V015695                 8791 bp    DNA     circular SYN 16-FEB-2021
DEFINITION  Exported.
ACCESSION   V015695
VERSION     V015695
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8791)
  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  JOURNAL   Unpublished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 8791)
  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 8791)
  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            "Unpublished"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..8791
                     /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="QRQ89504.1"
                     /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..4419)
                     /label="downstream homology recombination 3' flank"
                     /note="downstream homology recombination 3' flank"
     misc_feature    complement(4420..5037)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     gene            complement(5222..5767)
                     /gene="hpt"
                     /label="hpt"
     CDS             complement(5222..5767)
                     /codon_start=1
                     /transl_table=11
                     /gene="hpt"
                     /product="hypoxanthine phosphoribosyltransferase"
                     /label="hpt"
                     /protein_id="QRQ89505.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     CDS             complement(5788..6435)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     regulatory      complement(6436..7010)
                     /label="C. thermocellum gapDH promoter"
                     /note="C. thermocellum gapDH promoter"
                     /regulatory_class="promoter"
     misc_feature    complement(7014..7631)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     promoter        7724..7828
                     /label="AmpR promoter"
     CDS             7829..8686
                     /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 gccgtacttt tcaatctctt
     3841 caagcctttt ttcggcttct tcaagctttt ttgccatttc ctgaaacttt gaattcacat
     3901 agcttaaaga aaccaaagta tcgtcttccg aaacagattc agctgtgacc gaaccatagg
     3961 ccacctgaac aatatgtatg actatcgcac ataaaacagc caccaaaaca attttccctg
     4021 agtttttatt aaaatttaaa tttttcatct tttgtacccc ctgaaccttt acatatctat
     4081 taatccaaaa cttattgaaa gtgcttcatt aaccttttcc ataagttcgt catcaagatg
     4141 ccctattttc tctctgagtc tctttttgtc aatggttcgt atctgctcca gcagcaccac
     4201 cgaatctttc ggaagtccgt actcctttgc gctcaattcg atgtgtgtgg gcagctttgc
     4261 tttatttatc tgggatgtta tagccgatgc tataaccgta ggactgtatt tgttgccaat
     4321 atcattctga acaataagca caggtctgat tccaccctgt tctgacccaa tgacaggact
     4381 taaatcagca tagaatatat ctccgcgttt aattaccaca agtaaaaccc ccacttcttt
     4441 tttgggttaa gacataaaga aaaattcaaa tgacgcaagc taatgacgtt tacaaagaat
     4501 gcttcaatac agtgtataca ttttctttgt caaaggttac tctacagtta agcggtaatc
     4561 gcgaaggtag taattctata actatatata ttaattataa tatctaagat atgcacgggt
     4621 caaattaaaa tttaattact tgccttcgcg cgcctatgta attgttggca aaatcatttc
     4681 agacttaaca aaggttgaac ccccatgagc ttacctgcag cactgcagta attctgtctc
     4741 taaaattatt tttttgtcat ttaccccata ttatacataa aactttgata tttgttatgg
     4801 attgacagac aaatcatgtg tttgttaacc gaacctgtat attttattat attatcaata
     4861 acattattat gacaatattt caaatacatt ttatttatat tttttctata ttttcgaatt
     4921 atataactcc ttatatcaaa taatttaata cattgtatat cttcccttct ttcaggtaaa
     4981 ctctgggaat tctctttcct attatgctta ccagttcgta atttattgtc ccaacacctg
     5041 aactactggg ccaggtatgt attaaagcat ggggagggcg tgaatgtaag cgtgacataa
     5101 ctaattacat gatgcggccc tctaggatca gcgggtttaa acgctgaggc gcgccgaatt
     5161 cggtaccaaa acaaaaggcc cagtcttccg actgagcctt ttgttttctc gaggcccggg
     5221 cttagctgta catttcaggt ttcaaaacgc ctataaaagg aaggttcctg tacttttcat
     5281 caaagtccaa tccataaccc acgacaaact tatcaggtat cttaaatcct acataatcga
     5341 cttttacaga tgcttctctt ctctccggtt tgtctaatat tgtgcatatt ttcaggcttt
     5401 ttggcttcct tccaagtaaa gtttccctta agtaagacaa agtcaaaccg ctgtcaatta
     5461 tgtcttccac aatcagaaca tctttgcctt ctatgcttat atcaagatct ttgattatct
     5521 ttactattcc tgatgaatga gttgagcttc catagctgga aacagccata aaatcaagtg
     5581 ataaaggcaa atctatgtgt cttgacaaat cagccataaa cattaaagca ccttttaaaa
     5641 ctcctaccaa catcaaattt ttccctttgt agtcttttgt gatttgcctc ccaagctctt
     5701 ttatcttttc cttaagttct tcttctgtga tcaaaatttc atcgatgtct tttgataaat
     5761 tttccatgac gtcgacctcc tttattataa aagccagtca ttaggcctat ctgacaattc
     5821 ctgaatagag ttcataaaca atcctgcatg ataaccatca caaacagaat gatgtacctg
     5881 taaagatagc ggtaaatata ttgaattacc tttattaatg aattttcctg ctgtaataat
     5941 gggtagaagg taattactat tattattgat atttaagtta aacccagtaa atgaagtcca
     6001 tggaataata gaaagagaaa aagcattttc aggtataggt gttttgggaa acaatttccc
     6061 cgaaccatta tatttctcta catcagaaag gtataaatca taaaactctt tgaagtcatt
     6121 ctttacagga gtccaaatac cagagaatgt tttagataca ccatcaaaaa ttgtataaag
     6181 tggctctaac ttatcccaat aacctaactc tccgtcgcta ttgtaaccag ttctaaaagc
     6241 tgtatttgag tttatcaccc ttgtcactaa gaaaataaat gcagggtaaa atttatatcc
     6301 ttcttgtttt atgtttcggt ataaaacact aatatcaatt tctgtggtta tactaaaagt
     6361 cgtttgttgg ttcaaataat gattaaatat ctcttttctc ttccaattgt ctaaatcaat
     6421 tttattaaag ttcattaata tcgcctccta ttgtaaatta aaatttaatt taatgcctct
     6481 attagctgcc ataaagcatg aatgcagtaa aatattcatc ctctggcaag tatctcttgc
     6541 actatgattc attttatatt attttatttt cattttcaat agtttagata aaaaataatt
     6601 aattttttaa caaagtattt ttatcttcct ttgatgtaat tctttgtttt tttgtccatt
     6661 aatattatgg acaatttttt tatttctagt caataatttt aatctttatt aaaacctggc
     6721 aatgccagag agatacagta attattgcat ataaaagaaa acagacgcgc cctgtttgtt
     6781 tcctgaaaaa ccacctcgcc ctccaaagta tccagctttt taaaatccat cctcattccc
     6841 agacgaaaaa gttttgacaa agcctctttt cttgtccata gaattgtact ctgcacacaa
     6901 tattcatcag tatttccaaa actctttaga atcttttcct ctctttcgga aaagaaatac
     6961 tttatcaatg catcctcggg caaaaaaatc ttttccatgc ctattcccac gataagtaaa
     7021 acccccactt cttttttggg ttaagacata aagaaaaatt caaatgacgc aagctaatga
     7081 cgtttacaaa gaatgcttca atacagtgta tacattttct ttgtcaaagg ttactctaca
     7141 gttaagcggt aatcgcgaag gtagtaattc tataactata tatattaatt ataatatcta
     7201 agatatgcac gggtcaaatt aaaatttaat tacttgcctt cgcgcgccta tgtaattgtt
     7261 ggcaaaatca tttcagactt aacaaaggtt gaacccccat gagcttacct gcagcactgc
     7321 agtaattctg tctctaaaat tatttttttg tcatttaccc catattatac ataaaacttt
     7381 gatatttgtt atggattgac agacaaatca tgtgtttgtt aaccgaacct gtatatttta
     7441 ttatattatc aataacatta ttatgacaat atttcaaata cattttattt atattttttc
     7501 tatattttcg aattatataa ctccttatat caaataattt aatacattgt atatcttccc
     7561 ttctttcagg taaactctgg gaattctctt tcctattatg cttaccagtt cgtaatttat
     7621 tgtcccaaca cgatatcgcc tcgtgatacg cctattttta taggttaatg tcatgataat
     7681 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     7741 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     7801 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     7861 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     7921 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     7981 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     8041 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     8101 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     8161 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     8221 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     8281 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     8341 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     8401 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     8461 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     8521 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     8581 taagcgctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     8641 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     8701 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     8761 ggtgaagatc ctttttgata atcgcggccg c
//
