LOCUS       40924_31100        7583 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pMLBAD_GlycoTagGFP, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7583)
  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 7583)
  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 7583)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7583)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Metab. Eng.
            (2017) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (21-NOV-2016) Institute of Microbiology, ETH Zurich, 
            Vladimir-Prelog-Weg 4, Zurich, ZH 8093, Switzerland"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7583
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        55..83
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             410..643
                     /codon_start=1
                     /label=TpR
                     /note="E. coli plasmid-associated dihydrofolate reductase"
                     /translation="MGQSSDEANAPVAGQFALPLSATFGLGDRVRKKSGAAWQGQVVGW
                     YCTKLTPEGYAVESESHPGSVQIYPVAALERVA"
     CDS             complement(872..1747)
                     /codon_start=1
                     /label=araC
                     /note="L-arabinose regulatory protein"
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1774..2054
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     RBS             2063..2085
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2101..2127
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             2137..2823
                     /codon_start=1
                     /label=superfolder GFP
                     /note="GFP variant that folds robustly even when fused to
                     poorly folded proteins (Nager et al., 2011)"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGIT"
     misc_feature    2833..2889
                     /label=NAT-tag (C-terminal glycosylation-tag)
                     /note="NAT-tag (C-terminal glycosylation-tag)"
     CDS             2890..2913
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     terminator      3150..3236
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3328..3355
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        complement(4210..4312)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             complement(4462..5481)
                     /codon_start=1
                     /gene="mob"
                     /product="Mob"
                     /function="required for plasmid mobilization"
                     /label=mob
                     /protein_id="ATN32196.1"
                     /translation="MAAYAIMRCKKLAKMGNVAASLKHAYRERETPNADASRTPENEHW
                     AASSTDEAMGRLRELLPEKRRKDAVLAVEYVMTASPEWWKSASQEQQAAFFEKAHKWLA
                     DKYGADRIVTASIHRDETSPHMTAFVVPLTQDGRLSAKEFIGNKAQMTRDQTTFAAAVA
                     DLGLQRGIEGSKARHTRIQAFYEALERPPVGHVTISPQAVEPRAYAPQGLAEKLGISKR
                     VETPEAVADRLTKAVRQGYEPALQAAAGAREMRKKADQAQETARDLRERLKPVLDALGP
                     LNRDMQAKAAAIIKAVGEKLLTEQREVQRQKQAQRQQERGRAHFPEKCHLTSKKPLLS"
     gene            complement(4462..5481)
                     /gene="mob"
                     /label=mob
     rep_origin      5705..6474
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             6475..7134
                     /codon_start=1
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 tctacggggt ctgacgctca gtggaacgaa atcgatgagc tcgcacgaac ccagttgaca
       61 taagcctgtt cggttcgtaa actgtaatgc aagtagcgta tgcgctcacg caactggtcc
      121 agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat ggcttgttat
      181 gactgttttt ttgtacagtc tagcctcggg catccaagct agctaagcgc gttacgccgt
      241 gggtcgatgt ttgatgttat ggaacagcaa cgatgttacg cagcagggta gtcgccctaa
      301 aacaaagtta ggcagccgtt gtgctggtgc tttctagtag ttgttgtggg gtaggcagtc
      361 agagctcgat ttgcttgtcg ccataataga ttcacaagaa ggattcgaca tgggtcaaag
      421 tagcgatgaa gccaacgctc ccgttgcagg gcagtttgcg cttcccctga gtgccacctt
      481 tggcttaggg gatcgcgtac gcaagaaatc tggtgccgct tggcagggtc aagtcgtcgg
      541 ttggtattgc acaaaactca ctcctgaagg ctatgcggtc gagtccgaat cccacccagg
      601 ctcagtgcaa atttatcctg tggctgcact tgaacgtgtg gcctaagcga tatcttagga
      661 tctcccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
      721 gccggccacg atgcgtccgg cgtagaggat ctgctcatgt ttgacagctt atcatcgatg
      781 cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca
      841 agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt
      901 tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc
      961 gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc
     1021 gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga
     1081 cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat
     1141 gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac
     1201 aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc
     1261 gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc
     1321 cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat
     1381 ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt
     1441 aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac
     1501 cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt
     1561 ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt
     1621 tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta
     1681 aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt
     1741 cagccatact tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca
     1801 gacattgccg tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc
     1861 ttattaaaag cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa
     1921 gtgtctataa tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct
     1981 atgccatagc atttttatcc ataagattag cggatcctac ctgacgcttt ttatcgcaac
     2041 tctctactgt ttctgaaata attttgttta actttaataa ggagatatac catgggctct
     2101 caccaccatc atcaccacca tcatcaccac ggctcgagca aaggcgagga attattcact
     2161 ggcgtcgtgc caattttggt cgagctcgat ggtgacgtta acggtcacaa attttcggtt
     2221 cgtggcgagg gtgaaggcga tgcaaccaac ggcaaattaa ccctgaaatt catctgcacc
     2281 acgggtaagt tgccagttcc gtggccgacc ttggtcacca ccctgactta cggtgttcaa
     2341 tgcttctccc gctacccgga tcacatgaaa cgtcatgatt tcttcaaatc cgcgatgccg
     2401 gagggctacg tccaggagcg tacgatcagc ttcaaggacg atggtaccta caaaactcgt
     2461 gcggaggtta agttcgaagg cgatacgctc gtgaatcgta tcgagttgaa gggcattgat
     2521 tttaaggagg acggcaacat cctcggtcac aagctggaat ataactttaa ctcgcataac
     2581 gtttacatca ccgctgataa acagaagaac ggtattaaag caaatttcaa aattcgccac
     2641 aatgttgagg atggctctgt gcagttggct gaccattatc agcaaaatac ccctattggt
     2701 gatggtccgg tcttactgcc ggacaatcac tacttatcca cgcagtcggt gctgagcaaa
     2761 gacccgaatg agaagcgtga ccacatggtc ctgctggagt tcgtgacggc ggcgggcatc
     2821 accaaaacta gtgcgcatgc taccgcgtcc ggcgcgcacg cgaccgctgg tagcgcaaac
     2881 gccacggcgt ggtcccatcc tcaatttgag aaataactct agagtcgacc tgcaggcatg
     2941 caagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
     3001 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
     3061 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
     3121 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     3181 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     3241 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     3301 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     3361 ttctacaaac tcttccactc actacagcag agccatttaa acaacatccc ctcccccttt
     3421 ccaccgcgtc agacgcccgt agcagcccgc tacgggcttt ttcatgccct gccctagcgt
     3481 ccaagcctca cggccgcgct cggcctctct ggcggccttc tggcgctgag gtctgcctcg
     3541 tgaagaaggt gttgctgact cataccaggc ctgaatcgcc ccatcatcca gccagaaagt
     3601 gagggagcca cggttgatga gagctttgtt gtaggtggac cagttggtga ttttgaactt
     3661 ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc gtgatctgat ccttcaactc
     3721 agcaaaagtt cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt aatgctctgc
     3781 cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac
     3841 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3901 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3961 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     4021 tcaagcgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagctaaaaa
     4081 ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc
     4141 ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt
     4201 tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg
     4261 tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca
     4321 ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta
     4381 ttctgcgaag tgatcttccg tcacaggtat ttattcgaag acgaaagggc ctcgtgatac
     4441 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     4501 ttcggggaaa tgtgcgcgcc cgcgttcctg ctggcgctgg gcctgtttct ggcgctggac
     4561 ttcccgctgt tccgtcagca gcttttcgcc cacggccttg atgatcgcgg cggccttggc
     4621 ctgcatatcc cgattcaacg gccccagggc gtccagaacg ggcttcaggc gctcccgaag
     4681 gtctcgggcc gtctcttggg cttgatcggc cttcttgcgc atctcacgcg ctcctgcggc
     4741 ggcctgtagg gcaggctcat acccctgccg aaccgctttt gtcagccggt cggccacggc
     4801 ttccggcgtc tcaacgcgct ttgagattcc cagcttttcg gccaatccct gcggtgcata
     4861 ggcgcgtggc tcgaccgctt gcgggctgat ggtgacgtgg cccactggtg gccgctccag
     4921 ggcctcgtag aacgcctgaa tgcgcgtgtg acgtgccttg ctgccctcga tgccccgttg
     4981 cagccctaga tcggccacag cggccgcaaa cgtggtctgg tcgcgggtca tctgcgcttt
     5041 gttgccgatg aactccttgg ccgacagcct gccgtcctgc gtcagcggca ccacgaacgc
     5101 ggtcatgtgc gggctggttt cgtcacggtg gatgctggcc gtcacgatgc gatccgcccc
     5161 gtacttgtcc gccagccact tgtgcgcctt ctcgaagaac gccgcctgct gttcttggct
     5221 ggccgacttc caccattccg ggctggccgt catgacgtac tcgaccgcca acacagcgtc
     5281 cttgcgccgc ttctctggca gcaactcgcg cagtcggccc atcgcttcat cggtgctgct
     5341 ggccgcccag tgctcgttct ctggcgtcct gctggcgtca gcgttgggcg tctcgcgctc
     5401 gcggtaggcg tgcttgagac tggccgccac gttgcccatt ttcgccagct tcttgcatcg
     5461 catgatcgcg tatgccgcca tgcctgcccc tcccttttgg tgtccaaccg gctcgacggg
     5521 ggcagcgcaa ggcggtgcct ccggcgggcc actcaatgct tgagtatact cactagactt
     5581 tgcttcgcaa agtcgtgacc gcctacggcg gctgcggcgc cctacgggct tgctctccgg
     5641 gcttcgccct gcgcggtcgc tgcgctccct tgccagcccg tggatatgtg gacgatggcc
     5701 gcgagcggcc accggctggc tcgcttcgct cggcccgtgg acaaccctgc tggacaagct
     5761 gatggacagg ctgcgcctgc ccacgagctt gaccacaggg attgcccacc ggctacccag
     5821 ccttcgacca catacccacc ggctccaact gcgcggcctg cggccttgcc ccatcaattt
     5881 ttttaatttt ctctggggaa aagcctccgg cctgcggcct gcgcgcttcg cttgccggtt
     5941 ggacaccaag tggaaggcgg gtcaaggctc gcgcagcgac cgcgcagcgg cttggccttg
     6001 acgcgcctgg aacgacccaa gcctatgcga gtgggggcag tcgaaggcga agcccgcccg
     6061 cctgcccccc gagcctcacg gcggcgagtg cgggggttcc aagggggcag cgccaccttg
     6121 ggcaaggccg aaggccgcgc agtcgatcaa caagccccgg aggggccact ttttgccgga
     6181 gggggagccg cgccgaaggc gtgggggaac cccgcagggg tgcccttctt tgggcaccaa
     6241 agaactagat atagggcgaa atgcgaaaga cttaaaaatc aacaacttaa aaaagggggg
     6301 tacgcaacag ctcattgcgg caccccccgc aatagctcat tgcgtaggtt aaagaaaatc
     6361 tgtaattgac tgccactttt acgcaacgca taattgttgt cgcgctgccg aaaagttgca
     6421 gctgattgcg catggtgccg caaccgtgcg gcaccctacc gcatggagat aagcatggcc
     6481 acgcagtcca gagaaatcgg cattcaagcc aagaacaagc ccggtcactg ggtgcaaacg
     6541 gaacgcaaag cgcatgaggc gtgggccggg cttattgcga ggaaacccac ggcggcaatg
     6601 ctgctgcatc acctcgtggc gcagatgggc caccagaacg ccgtggtggt cagccagaag
     6661 acactttcca agctcatcgg acgttctttg cggacggtcc aatacgcagt caaggacttg
     6721 gtggccgagc gctggatctc cgtcgtgaag ctcaacggcc ccggcaccgt gtcggcctac
     6781 gtggtcaatg accgcgtggc gtggggccag ccccgcgacc agttgcgcct gtcggtgttc
     6841 agtgccgccg tggtggttga tcacgacgac caggacgaat cgctgttggg gcatggcgac
     6901 ctgcgccgca tcccgaccct gtatccgggc gagcagcaac taccgaccgg ccccggcgag
     6961 gagccgccca gccagcccgg cattccgggc atggaaccag acctgccagc cttgaccgaa
     7021 acggaggaat gggaacggcg cgggcagcag cgcctgccga tgcccgatga gccgtgtttt
     7081 ctggacgatg gcgagccgtt ggagccgccg acacgggtca cgctgccgcg ccggtagcac
     7141 ttgggttgcg cagcaacccg taagtgcgct gttccagact atcggctgta gccgcctcgc
     7201 cgccctatac cttgtctgcc tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc
     7261 gacctgaatg gaagccggcg gcacctcgct aacggattca ccgtttttat caggctctgg
     7321 gaggcagaat aaatgatcat atcgtcaatt attacctcca cggggagagc ctgagcaaac
     7381 tggcctcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     7441 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg
     7501 aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcacc
     7561 aggcgtttaa gtcgaccaat aac
//
