LOCUS       V009585                 8180 bp    DNA     circular SYN 17-DEC-2018
DEFINITION  Exported.
ACCESSION   V009585
VERSION     V009585
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8180)
  AUTHORS   Keys TG, Wetter M, Hang I, Rutschmann C, Russo S, Mally M, Steffen
            M, Zuppiger M, Muller F, Schneider J, Faridmoayer A, Lin CW, Aebi M.
  TITLE     A biosynthetic route for polysialylating proteins in Escherichia
            coli
  JOURNAL   Metab. Eng. (2017) In press
   PUBMED   29101090
REFERENCE   2  (bases 1 to 8180)
  AUTHORS   Keys TG.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-NOV-2016) Institute of Microbiology, ETH Zurich,
            Vladimir-Prelog-Weg 4, Zurich, ZH 8093, Switzerland
REFERENCE   3  (bases 1 to 8180)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8180)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName: "Metab. Eng.
            (2017) In press"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (21-NOV-2016) Institute of Microbiology, ETH Zurich,
            Vladimir-Prelog-Weg 4, Zurich, ZH 8093, Switzerland"
            SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      28..75
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     CDS             complement(298..954)
                     /label="CmR"
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(955..1057)
                     /label="cat promoter"
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(1583..2127)
                     /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."
     protein_bind    complement(2318..2339)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(2355..3434)
                     /label="lacI"
                     /note="lac repressor"
     promoter        complement(3435..3512)
                     /label="lacI promoter"
     promoter        3550..3580
                     /label="lac UV5 promoter"
                     /note="E. coli lac promoter with an 'up' mutation"
     protein_bind    3584..3608
                     /label="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             3656..4342
                     /codon_start=1
                     /product="SynB"
                     /label="SynB"
                     /protein_id="ATN32179.1"
                     /translation="MEKQNIAVILARQNSKGLPLKNLRKMNGISLLGHTINAAISSKCF
                     DRIIVSTDGGLIAEEAKNFGVEVVLRPAELASDTASSISGVIHALETIGSNSGTVTLLQ
                     PTSPLRTGAHIREAFSLFDEKIKGSVVSACPMEHHPLKTLLQINNGEYAPMRHLSDLEQ
                     PRQQLPQAFRPNGAIYINDTASLIANNCFFIAPTKLYIMSHQDSIDIDTELDLQQAENI
                     LNHKES"
     promoter        4357..4387
                     /label="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      4357..4362
                     /regulatory_class="minus_35_signal"
     regulatory      4381..4387
                     /note="-10 seq (TATAAT; Pribnow box)"
                     /regulatory_class="minus_10_signal"
     protein_bind    4391..4415
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    4393..4413
                     /label="lac operator"
                     /note="lac operator"
     CDS             4463..5242
                     /codon_start=1
                     /product="CstII"
                     /label="CstII"
                     /note="delta-C32 and I53S mutation"
                     /protein_id="ATN32180.1"
                     /translation="MKKVIIAGNGPSLKEIDYSRLPNDFDVFRCNQFYFEDKYYLGKKC
                     KAVFYNPSLFFEQYYTLKHLIQNQEYETELIMCSNYNQAHLENENFVKTFYDYFPDAHL
                     GYDFFKQLKDFNAYFKFHEIYFNQRITSGVYMCAVAIALGYKEIYLSGIDFYQNGSSYA
                     FDTKQKNLLKLAPNFKNDNSHYIGHSKNTDIKALEFLEKTYKIKLYCLCPNSLLANFIG
                     LAPNLNSNFIIQEKNNYTKDILIPSSEAYGKFSKNIN"
     promoter        5254..5284
                     /label="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      5254..5259
                     /regulatory_class="minus_35_signal"
     regulatory      5278..5284
                     /note="-10 seq (TATAAT; Pribnow box)"
                     /regulatory_class="minus_10_signal"
     protein_bind    5288..5312
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    5290..5310
                     /label="lac operator"
                     /note="lac operator"
     CDS             5360..6190
                     /codon_start=1
                     /product="LgtB"
                     /label="LgtB"
                     /protein_id="ATN32181.1"
                     /translation="MSQNHVISLASAAERRAHIADTFGRHGIPFQFFDALMPSERLEQA
                     MAELVPGLSAHPYLSGVEKACFMSHAVLWKQALDEGLPYITVFEDDVLLGEGAEKFLAE
                     DAWLQERFDPDTAFIVRLETMFMHVLTSPSGVADYCGRAFPLLESEHWGTAGYIISRKA
                     MRFFLDRFAALPPEGLHPVDLMMFSDFFDREGMPVCQLNPALCAQELHYAKFHDQNSAL
                     GSLIEHDRLLNRKQQRRDSPANTFKHRLIRALTKISREREKRRQRREQFIVPFQ"
     promoter        6202..6232
                     /label="lac UV5 promoter"
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      6202..6207
                     /regulatory_class="minus_35_signal"
     regulatory      6226..6232
                     /note="-10 seq (TATAAT; Pribnow box)"
                     /regulatory_class="minus_10_signal"
     protein_bind    6236..6260
                     /label="lac operator"
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    6238..6258
                     /label="lac operator"
                     /note="lac operator"
     CDS             6308..8167
                     /note="UDP-glucose:protein N-beta-glucosyltransferase from
                     Actinobacillus pleuropneumoniae serotype 7 (strain AP76).
                     Accession#: B3H2N2"
                     /label="UDP-glucose:protein N-beta-glucosyltransferase"
ORIGIN
        1 taggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
       61 ttgaggggtt ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg
      121 gtagtcaata aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct
      181 gaaccgacga ccgggtcgaa tttgctttcg aatttctgcc attcatccgc ttattatcac
      241 ttattcaggc gtagcaccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc
      301 cccgccctgc cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa
      361 gccatcacag acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg
      421 cgtataatat ttgcccatag tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt
      481 taaatcaaaa ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat
      541 aaacccttta gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat
      601 gtgtagaaac tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt
      661 ttgctcatgg aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc
      721 tttcattgcc atacggaact ccggatgagc attcatcagg cgggcaagaa tgtgaataaa
      781 ggccggataa aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag
      841 ctgaacggtc tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt
      901 acgatgccat tgggatatat caacggtggt atatccagtg atttttttct ccattttagc
      961 ttccttagct cctgaaaatc tcgataactc aaaaaatacg cccggtagtg atcttatttc
     1021 attatggtga aagttggaac ctcttacgtg ccgatcaacg tctcattttc gccaaaagtt
     1081 ggcccagggc ttcccggtat caacagggac accaggattt atttattctg cgaagtgatc
     1141 ttccgtcaca ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttactgat
     1201 ttagtgtatg atggtgtttt tgaggtgctc cagtggcttc tgtttctatc agctgtccct
     1261 cctgttcagc tactgacggg gtggtgcgta acggcaaaag caccgccgga catcagcgct
     1321 agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg aagtgcttca
     1381 tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata caggatatat
     1441 tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg
     1501 gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg
     1561 agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa
     1621 tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1681 ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc
     1741 gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc
     1801 gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg
     1861 gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca
     1921 ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa
     1981 aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta
     2041 gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag
     2101 attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc
     2161 tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat
     2221 aagttgtaat tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     2281 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     2341 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2401 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     2461 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     2521 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     2581 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca
     2641 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     2701 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     2761 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     2821 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     2881 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     2941 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     3001 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     3061 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     3121 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     3181 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     3241 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     3301 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     3361 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     3421 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     3481 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttacataa
     3541 acgctagcct ttacacttta tgcttccggc tcgtataatg tgtggaattg tgagcggata
     3601 acaattcccc actagaaata agcaaaagaa aaagacaagg aggtacctgc taagcatgga
     3661 gaaacaaaac attgctgtta tcctggcgcg tcaaaattcc aaaggtctgc ctttgaagaa
     3721 tctgcgtaag atgaacggca tctccttgtt gggtcacact atcaatgctg caatctccag
     3781 caaatgcttc gatcgtatca tcgtttcgac cgacggtggt ctgattgcgg aagaggcgaa
     3841 aaattttggc gtggaagttg tcctgcgtcc ggccgaatta gcgtctgaca cggcgagctc
     3901 catctctggt gtgatccacg cgctggaaac gattggctcc aactccggta ccgtcacctt
     3961 gctgcaaccg accagcccac tgcgtacggg cgctcatatt cgtgaggcgt ttagcctgtt
     4021 cgatgagaag attaagggca gcgtggtgtc cgcgtgcccg atggaacacc acccgttaaa
     4081 aaccctcctg cagatcaata atggtgaata tgcgccgatg cgccacttgt ccgacttgga
     4141 gcaaccgcgc cagcaactgc cgcaagcttt ccgtccaaat ggcgcaatct atatcaacga
     4201 caccgcaagc ctgatcgcca ataactgctt tttcattgct ccaaccaaac tgtacattat
     4261 gagccaccag gactcgattg atattgatac cgaactggat ctgcagcagg cggaaaacat
     4321 tctcaaccat aaggagagct aagaagacca ctagacttta cactttatgc ttccggctcg
     4381 tataatgtgt ggaattgtga gcggataaca attccccact agaaataatt caacaacaaa
     4441 cttactcaga ggagctaaca tcatgaagaa ggttattatc gctggcaatg gcccgtcgct
     4501 gaaggagatt gactattccc gtttgcctaa tgatttcgac gtgtttcgct gcaaccaatt
     4561 ttatttcgag gacaaatatt acttgggtaa gaaatgtaag gctgtttttt acaacccgag
     4621 cctgttcttt gaacaatact atacgctgaa gcacctgatc cagaatcaag aatacgaaac
     4681 ggagttgatc atgtgcagca attacaacca ggcccacttg gagaatgaaa actttgtgaa
     4741 aaccttttat gactactttc cggatgcgca tctgggttac gatttcttta agcagctcaa
     4801 agatttcaac gcttatttta agttccacga gatttacttc aaccagcgca tcacctctgg
     4861 cgtgtatatg tgcgcggtcg cgatcgcgct gggctacaaa gaaatctacc tctccggtat
     4921 cgacttctac cagaacggta gctcgtacgc gttcgacacc aaacagaaga atctgttgaa
     4981 gctggcaccg aacttcaaaa acgacaattc tcactatatt ggtcatagca agaacaccga
     5041 catcaaagcg ctggaatttt tggaaaaaac ctataagatt aaactgtact gcctgtgtcc
     5101 aaatagcttg ttagcaaact tcattggttt ggcgccgaat ctgaatagca acttcattat
     5161 tcaagagaag aataactaca ccaaggacat cttaattccg tccagcgaag cgtatggcaa
     5221 gttctccaag aacatcaact aagcacacta gtctttacac tttatgcttc cggctcgtat
     5281 aatgtgtgga attgtgagcg gataacaatt ccccactaga aataactctt taatactatt
     5341 tacataagga ggcgagaaca tgtctcaaaa ccatgtgatc tctttggcat ctgcggccga
     5401 gcgtcgcgcg cacatcgcgg ataccttcgg ccgtcatggc attccatttc aattttttga
     5461 cgcgctcatg ccatccgaac gtttagagca agcgatggcg gaactggtgc caggtttgtc
     5521 cgcgcatccg tacctcagcg gcgttgagaa agcttgtttt atgtcgcacg ccgttctgtg
     5581 gaagcaggcg ttagacgagg gtctgccgta tattaccgtt ttcgaggacg acgttttgtt
     5641 gggtgaaggt gcggaaaagt ttttggctga ggacgcttgg ctgcaggagc gttttgatcc
     5701 tgatacggcc ttcattgttc gcctggaaac catgttcatg catgtcttga cctccccgag
     5761 cggtgtcgcc gactactgtg gtcgcgcttt cccgttgctg gagagcgagc actggggcac
     5821 cgcgggttat attatctccc gtaaagcaat gcgtttcttc ctggatcgct tcgctgcgct
     5881 gccgccggag ggcttgcacc cggtggacct gatgatgttc agcgattttt tcgaccgcga
     5941 gggtatgcct gtttgccaac tgaatccggc gttgtgcgcg caggagttac actacgcaaa
     6001 attccacgat cagaacagcg cgctgggtag cctgatcgag catgaccgtc tgctgaaccg
     6061 caaacagcaa cgccgtgaca gcccggctaa cacctttaag caccgtctga tccgcgcgct
     6121 gactaagatc agccgtgaac gtgagaagcg tcgtcagcgt cgcgaacaat tcattgtgcc
     6181 gtttcaataa ttcgcctagg ctttacactt tatgcttccg gctcgtataa tgtgtggaat
     6241 tgtgagcgga taacaattcc ccactagaaa taaaccccca gacccacaga tactcaggag
     6301 gtctcgcatg gagaacgaga ataagcctaa tgttgcaaac tttgaggcgg cagttgctgt
     6361 caaagactac gagaaggctt gctccgagct gttgctgatc ttgtctcagc tggatagcaa
     6421 ctttggcggt attcaagaaa tcgagttcga gtacccggtt caactccagg acctcgaaca
     6481 agagaaaatt gtatacttct gcacccgcat ggcgaccgcg atcacgacct tgttttccga
     6541 ccctgttctg gagatcagcg acctgggtgt tcagcgtttc ctggtttacc aacgttggct
     6601 cgcattgatc tttgcatcgt ccccgtttgt taacgcggat catattctgc aaacctataa
     6661 ccgcgaaccg aaccgtaaaa actctttgga aatccatctg gactctagca aatccagcct
     6721 catcaaattt tgcattttat acttgccgga gtccaacgtc aacctgaact tggacgtgat
     6781 gtggaacatt agcccggagt tgtgcgccag cctgtgcttc gcactgcagt cgccgcgctt
     6841 catcggcacc tctaccgcgt tcaacaagcg tgcgactatt ttacaatggt ttccacgtca
     6901 cctggaccag ctgaaaaatt tgaacaacat tccgagcgcg atttcccacg acgtctatat
     6961 gcattgctct tacgacacct ccgtgaacaa gcatgatgtc aaacgtgcgc tgaaccatgt
     7021 gattcgccgt catatcgagt ccgaatacgg ctggaaagac cgttacgtgg cgcacatcgg
     7081 ctaccgtaac aacaagccgg tgatggttgt tctgctggag cactttcact ctgcacactc
     7141 catctatcgt acccactcga cctccatgat tgcggcccgc gagcactttt acctgatcgg
     7201 tttgggtagc ccgagcgttg atcaagcggg ccaagaggtt tttgacgagt ttcatctcgt
     7261 tgcgggtgat aatatgaagc agaagctgga gttcattcgt tcggtgtgcg agtctaacgg
     7321 tgccgccatc ttctacatgc cgtccattgg catggatatg accaccatct tcgcttcgaa
     7381 tactcgcctt gcgcctatcc aggcgatcgc actgggccac cctgccacca cccacagcga
     7441 ctttatcgag tacgtcatcg ttgaggacga ttacgtgggc tccgaggcat gctttagcga
     7501 aacgttgctg agactgccga aggacgcgct gccgtatgtc ccgtccgcct tggccccgga
     7561 gaaagttgat tatcttttgc gtgagaatcc agaggtcgtt aatattggca ttgcgagcac
     7621 caccatgaag ctgaaccctt atttcttgga agcgctgaag gcgatccgtg atcgtgctaa
     7681 agtcaaagtt cacttccatt ttgcgctggg ccaaagcaac ggtattaccc atccatacgt
     7741 tgagcgtttc attaagtcct acctgggtga tagcgcgacc gctcacccac attccccgta
     7801 tcaccaatac ttgcgcatct tgcataattg cgacatgatg gttaacccgt tcccgtttgg
     7861 taacacgaat ggcatcattg atatggtgac cctcggtctg gttggtgttt gcaaaaccgg
     7921 tgcagaggtc cacgaacaca tcgacgaggg cttgtttaag cgtctgggct tgccggaatg
     7981 gctgattgcc aacaccgttg acgaatatgt ggagcgtgcg gtgcgtttgg cggaaaacca
     8041 ccaggaacgc ttggagctgc gccgctatat tatcgagaat aatggcttga atacgctgtt
     8101 taccggtgac ccgcgtccga tgggtcaggt ttttctggaa aaattgaacg ctttcttgaa
     8161 ggagaactaa tagcgtctct
//
