LOCUS       V015364                 9728 bp    DNA     circular SYN 16-FEB-2021
DEFINITION  Exported.
ACCESSION   V015364
VERSION     V015364
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9728)
  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  JOURNAL   Unpublished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 9728)
  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 9728)
  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            "Unpublished"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..9728
                     /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="QRQ89514.1"
                     /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..4409)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     gene            complement(4594..5139)
                     /gene="hpt"
                     /label="hpt"
     CDS             complement(4594..5139)
                     /codon_start=1
                     /transl_table=11
                     /gene="hpt"
                     /product="hypoxanthine phosphoribosyltransferase"
                     /label="hpt"
                     /protein_id="QRQ89515.1"
                     /translation="MENLSKDIDEILITEEELKEKIKELGRQITKDYKGKNLMLVGVLK
                     GALMFMADLSRHIDLPLSLDFMAVSSYGSSTHSSGIVKIIKDLDISIEGKDVLIVEDII
                     DSGLTLSYLRETLLGRKPKSLKICTILDKPERREASVKVDYVGFKIPDKFVVGYGLDFD
                     EKYRNLPFIGVLKPEMYS"
     CDS             complement(5160..5807)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     regulatory      complement(5808..6382)
                     /label="C. thermocellum gapDH promoter"
                     /note="C. thermocellum gapDH promoter"
                     /regulatory_class="promoter"
     misc_feature    complement(6386..7007)
                     /label="downstream homology recombination 3' flank"
                     /note="downstream homology recombination 3' flank"
     gene            7014..7979
                     /gene="cbpA"
                     /label="cbpA"
                     /note="gene target with single-point mutation c.443G>T
                     resulting in G148V"
     CDS             7014..7979
                     /codon_start=1
                     /transl_table=11
                     /gene="cbpA"
                     /product="carbohydrate binding protein A"
                     /label="cbpA"
                     /note="gene target CDS with single-point mutation c.443G>T
                     resulting in G148V"
                     /protein_id="QRQ89517.1"
                     /translation="MKRFLVLLLAISLIFTFTACNSGSGSGNGQAKGYVGDPSDEYYMV
                     TFLSGIDYWKYCFEGFEDAAKAIGVTAKYTGQTDTDVSGQVAVLEQVIAQKPKGIAVTA
                     VNSTALADTINSAIEQGISVVCFDSDSPTSNRSAYLGTGNYAAVQKAAEFLVPLVNYKG
                     KIAVLYTVGAENSESRVQGFEDWCKQNAPEVSLVKVNDAGDTTVAADNLAAALQANDDI
                     VGVFCVDGVAGTAGPTAVAESKKDLRVLAFDVDVTVLDKVKSGEIDGTVAQGMYNMGYW
                     SLMMLYTEANGLSSKALPGNLDTGVVIVTKDNVDEYYPKK"
     misc_feature    complement(7980..8568)
                     /label="upstream homology recombination 5' flank"
                     /note="upstream homology recombination 5' flank"
     promoter        8661..8765
                     /label="AmpR promoter"
     CDS             8766..9623
                     /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 gaataaaata aaatcagcat
     3841 gaaaggaaag taagtttatg aaagcattta actattatgc acctactgaa attattttcg
     3901 gatgcggcag agttcaggaa attggatcaa taactgccca atatggaaag aaagctttgc
     3961 tggtaactgt accggaattt ccggaagtaa aggagctcta tgaaaaagta aagaagtctc
     4021 ttagggaaaa tggtgtggaa gtagtacatt ttgacggtgt tattccaaat cccaccactg
     4081 atgttgtaac tgaaggagca aatatggcaa aggctgccgg ggttgatgta gttattggat
     4141 tgggaggagg ttcctcaata gatactgcaa aagccattgc agttgaagca acccatccgg
     4201 gaactgcctg ggattataac tgccacactc cgggccccac atctgctacc ctccctatta
     4261 tcgcaatagg aacaacagca ggaaccggca gccagtgtac acaatgtgca gttatcacaa
     4321 aaacaagtga aaaggataaa tccgcaattt ggcacaaaaa tattttccct aaagttgcga
     4381 tagttgatcc cgaagtgaca gtcacgatgc tgaactactg ggccaggtat gtattaaagc
     4441 atggggaggg cgtgaatgta agcgtgacat aactaattac atgatgcggc cctctaggat
     4501 cagcgggttt aaacgctgag gcgcgccgaa ttcggtacca aaacaaaagg cccagtcttc
     4561 cgactgagcc ttttgttttc tcgaggcccg ggcttagctg tacatttcag gtttcaaaac
     4621 gcctataaaa ggaaggttcc tgtacttttc atcaaagtcc aatccataac ccacgacaaa
     4681 cttatcaggt atcttaaatc ctacataatc gacttttaca gatgcttctc ttctctccgg
     4741 tttgtctaat attgtgcata ttttcaggct ttttggcttc cttccaagta aagtttccct
     4801 taagtaagac aaagtcaaac cgctgtcaat tatgtcttcc acaatcagaa catctttgcc
     4861 ttctatgctt atatcaagat ctttgattat ctttactatt cctgatgaat gagttgagct
     4921 tccatagctg gaaacagcca taaaatcaag tgataaaggc aaatctatgt gtcttgacaa
     4981 atcagccata aacattaaag caccttttaa aactcctacc aacatcaaat ttttcccttt
     5041 gtagtctttt gtgatttgcc tcccaagctc ttttatcttt tccttaagtt cttcttctgt
     5101 gatcaaaatt tcatcgatgt cttttgataa attttccatg acgtcgacct cctttattat
     5161 aaaagccagt cattaggcct atctgacaat tcctgaatag agttcataaa caatcctgca
     5221 tgataaccat cacaaacaga atgatgtacc tgtaaagata gcggtaaata tattgaatta
     5281 cctttattaa tgaattttcc tgctgtaata atgggtagaa ggtaattact attattattg
     5341 atatttaagt taaacccagt aaatgaagtc catggaataa tagaaagaga aaaagcattt
     5401 tcaggtatag gtgttttggg aaacaatttc cccgaaccat tatatttctc tacatcagaa
     5461 aggtataaat cataaaactc tttgaagtca ttctttacag gagtccaaat accagagaat
     5521 gttttagata caccatcaaa aattgtataa agtggctcta acttatccca ataacctaac
     5581 tctccgtcgc tattgtaacc agttctaaaa gctgtatttg agtttatcac ccttgtcact
     5641 aagaaaataa atgcagggta aaatttatat ccttcttgtt ttatgtttcg gtataaaaca
     5701 ctaatatcaa tttctgtggt tatactaaaa gtcgtttgtt ggttcaaata atgattaaat
     5761 atctcttttc tcttccaatt gtctaaatca attttattaa agttcattaa tatcgcctcc
     5821 tattgtaaat taaaatttaa tttaatgcct ctattagctg ccataaagca tgaatgcagt
     5881 aaaatattca tcctctggca agtatctctt gcactatgat tcattttata ttattttatt
     5941 ttcattttca atagtttaga taaaaaataa ttaatttttt aacaaagtat ttttatcttc
     6001 ctttgatgta attctttgtt tttttgtcca ttaatattat ggacaatttt tttatttcta
     6061 gtcaataatt ttaatcttta ttaaaacctg gcaatgccag agagatacag taattattgc
     6121 atataaaaga aaacagacgc gccctgtttg tttcctgaaa aaccacctcg ccctccaaag
     6181 tatccagctt tttaaaatcc atcctcattc ccagacgaaa aagttttgac aaagcctctt
     6241 ttcttgtcca tagaattgta ctctgcacac aatattcatc agtatttcca aaactcttta
     6301 gaatcttttc ctctctttcg gaaaagaaat actttatcaa tgcatcctcg ggcaaaaaaa
     6361 tcttttccat gcctattccc acgatggaat acccgttctc ttcatcatat ttataataat
     6421 ttcggcgatt gcatttatct ttatgaagta ttcaaagata tgcaggaata tatactacgt
     6481 cggaagcaat gaaaatgctg caagactttc cggtataaat gtagacaggg taaaaatcgg
     6541 tgtatatgtg acaattgcgt taatctccac attagcaggc ttattgtctc tggcacgctt
     6601 taacgtggca accccggaac ttagcaaggg tgctgaaaca acggcaattt ccgcggcagt
     6661 tatcggcgga acatcgatga caggtggttc cggaggcgta ctgggaactg ttttgggtat
     6721 tttcttactg aagatagtta attcggcact tgttatgatg aacgtttcgg tttattggca
     6781 ggagtttgtt cagggagcaa ttcttgtact ggctgttacc attgactatt tgagtcaccg
     6841 taaaaacggc ggtccaagct ttataatgaa attcttcgga ttggacaaat tggcttcaaa
     6901 aaaattagat gacaaatctt ccaggcaaat ttaattttta agaattttgg tgctttatgc
     6961 actgaaagat aaaaatacat tttatcctta aaaatgtaag gaggtaattt ttcatgaaaa
     7021 gatttttagt attattgctt gccatttcgc tgatatttac atttacagca tgcaattccg
     7081 gttcaggctc aggaaatgga caggcaaaag gctatgtagg cgatccttcg gatgaatact
     7141 acatggttac tttcctttcc ggtattgatt actggaagta ctgctttgaa ggttttgagg
     7201 atgcagctaa agcaattggc gtaactgcaa aatataccgg tcagacagat actgatgttt
     7261 caggtcaggt tgcagtgctt gaacaggtca ttgctcaaaa accaaaaggt atagcagtaa
     7321 cagcggttaa ttcaacagca ttggcggata caatcaattc agctattgaa caaggtattt
     7381 cagttgtatg ctttgactct gactctccta cttcaaaccg ttccgcatat cttggaaccg
     7441 gtaactatgc agcagttcag aaggcggctg agttcctggt accgttggta aactacaaag
     7501 gtaaaattgc agtattgtat acagttggtg cagaaaacag tgaatcccgt gttcagggtt
     7561 ttgaagactg gtgtaaacaa aatgcgcccg aagtttccct cgtaaaagtt aatgatgcag
     7621 gcgatacaac tgtcgcagcg gacaacctgg cggctgcatt acaggcaaat gatgatattg
     7681 ttggtgtatt ctgtgtagac ggtgttgcag gtactgcagg accgacagca gttgcagaat
     7741 caaagaaaga tttaagagtt cttgcatttg acgttgatgt gacagttctt gacaaggtta
     7801 agagcggtga aattgatggt acagttgcac agggaatgta taacatgggt tattggagcc
     7861 tgatgatgct ctatactgag gcaaacggtc tttcgtccaa agctcttccg ggcaatcttg
     7921 acacaggtgt tgtaatagta acaaaagata atgttgatga gtactatcca aagaaataag
     7981 aataaaataa aatcagcatg aaaggaaagt aagtttatga aagcatttaa ctattatgca
     8041 cctactgaaa ttattttcgg atgcggcaga gttcaggaaa ttggatcaat aactgcccaa
     8101 tatggaaaga aagctttgct ggtaactgta ccggaatttc cggaagtaaa ggagctctat
     8161 gaaaaagtaa agaagtctct tagggaaaat ggtgtggaag tagtacattt tgacggtgtt
     8221 attccaaatc ccaccactga tgttgtaact gaaggagcaa atatggcaaa ggctgccggg
     8281 gttgatgtag ttattggatt gggaggaggt tcctcaatag atactgcaaa agccattgca
     8341 gttgaagcaa cccatccggg aactgcctgg gattataact gccacactcc gggccccaca
     8401 tctgctaccc tccctattat cgcaatagga acaacagcag gaaccggcag ccagtgtaca
     8461 caatgtgcag ttatcacaaa aacaagtgaa aaggataaat ccgcaatttg gcacaaaaat
     8521 attttcccta aagttgcgat agttgatccc gaagtgacag tcacgatgga tatcgcctcg
     8581 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     8641 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     8701 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     8761 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     8821 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     8881 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     8941 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     9001 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     9061 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     9121 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     9181 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     9241 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     9301 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     9361 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     9421 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     9481 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gcgctcccgt atcgtagtta
     9541 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     9601 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     9661 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     9721 gcggccgc
//
