LOCUS       40924_31095        7565 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pMLBAD_GlycoLoopGFP, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7565)
  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 7565)
  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 7565)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7565)
  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..7565
                     /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             2872..2895
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     terminator      3132..3218
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3310..3337
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     promoter        complement(4192..4294)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     CDS             complement(4444..5463)
                     /codon_start=1
                     /gene="mob"
                     /product="Mob"
                     /function="required for plasmid mobilization"
                     /label=mob
                     /protein_id="ATN32199.1"
                     /translation="MAAYAIMRCKKLAKMGNVAASLKHAYRERETPNADASRTPENEHW
                     AASSTDEAMGRLRELLPEKRRKDAVLAVEYVMTASPEWWKSASQEQQAAFFEKAHKWLA
                     DKYGADRIVTASIHRDETSPHMTAFVVPLTQDGRLSAKEFIGNKAQMTRDQTTFAAAVA
                     DLGLQRGIEGSKARHTRIQAFYEALERPPVGHVTISPQAVEPRAYAPQGLAEKLGISKR
                     VETPEAVADRLTKAVRQGYEPALQAAAGAREMRKKADQAQETARDLRERLKPVLDALGP
                     LNRDMQAKAAAIIKAVGEKLLTEQREVQRQKQAQRQQERGRAHFPEKCHLTSKKPLLS"
     gene            complement(4444..5463)
                     /gene="mob"
                     /label=mob
     rep_origin      5687..6456
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             6457..7116
                     /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 tcttaagcgg ctccggcgct aacgcgaccg cgaccggttc tggtctgccg
     2761 gacaatcact acttatccac gcagtcggtg ctgagcaaag acccgaatga gaagcgtgac
     2821 cacatggtcc tgctggagtt cgtgacggcg gcgggcatca ccaaaactag ttggtcccat
     2881 cctcaatttg agaaataact ctagagtcga cctgcaggca tgcaagcttg gctgttttgg
     2941 cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat
     3001 aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc
     3061 agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa
     3121 ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     3181 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     3241 ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     3301 aaattaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttccac
     3361 tcactacagc agagccattt aaacaacatc ccctccccct ttccaccgcg tcagacgccc
     3421 gtagcagccc gctacgggct ttttcatgcc ctgccctagc gtccaagcct cacggccgcg
     3481 ctcggcctct ctggcggcct tctggcgctg aggtctgcct cgtgaagaag gtgttgctga
     3541 ctcataccag gcctgaatcg ccccatcatc cagccagaaa gtgagggagc cacggttgat
     3601 gagagctttg ttgtaggtgg accagttggt gattttgaac ttttgctttg ccacggaacg
     3661 gtctgcgttg tcgggaagat gcgtgatctg atccttcaac tcagcaaaag ttcgatttat
     3721 tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta caaccaatta
     3781 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     3841 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     3901 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     3961 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagcga gaaatcacca
     4021 tgagtgacga ctgaatccgg tgagaatggc aaaagctaaa aaggccgtaa tatccagctg
     4081 aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg
     4141 atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc
     4201 cttagctcct gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
     4261 atggtgaaag ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc
     4321 ccagggcttc ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc
     4381 cgtcacaggt atttattcga agacgaaagg gcctcgtgat acgcctattt ttataggtta
     4441 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     4501 cccgcgttcc tgctggcgct gggcctgttt ctggcgctgg acttcccgct gttccgtcag
     4561 cagcttttcg cccacggcct tgatgatcgc ggcggccttg gcctgcatat cccgattcaa
     4621 cggccccagg gcgtccagaa cgggcttcag gcgctcccga aggtctcggg ccgtctcttg
     4681 ggcttgatcg gccttcttgc gcatctcacg cgctcctgcg gcggcctgta gggcaggctc
     4741 atacccctgc cgaaccgctt ttgtcagccg gtcggccacg gcttccggcg tctcaacgcg
     4801 ctttgagatt cccagctttt cggccaatcc ctgcggtgca taggcgcgtg gctcgaccgc
     4861 ttgcgggctg atggtgacgt ggcccactgg tggccgctcc agggcctcgt agaacgcctg
     4921 aatgcgcgtg tgacgtgcct tgctgccctc gatgccccgt tgcagcccta gatcggccac
     4981 agcggccgca aacgtggtct ggtcgcgggt catctgcgct ttgttgccga tgaactcctt
     5041 ggccgacagc ctgccgtcct gcgtcagcgg caccacgaac gcggtcatgt gcgggctggt
     5101 ttcgtcacgg tggatgctgg ccgtcacgat gcgatccgcc ccgtacttgt ccgccagcca
     5161 cttgtgcgcc ttctcgaaga acgccgcctg ctgttcttgg ctggccgact tccaccattc
     5221 cgggctggcc gtcatgacgt actcgaccgc caacacagcg tccttgcgcc gcttctctgg
     5281 cagcaactcg cgcagtcggc ccatcgcttc atcggtgctg ctggccgccc agtgctcgtt
     5341 ctctggcgtc ctgctggcgt cagcgttggg cgtctcgcgc tcgcggtagg cgtgcttgag
     5401 actggccgcc acgttgccca ttttcgccag cttcttgcat cgcatgatcg cgtatgccgc
     5461 catgcctgcc cctccctttt ggtgtccaac cggctcgacg ggggcagcgc aaggcggtgc
     5521 ctccggcggg ccactcaatg cttgagtata ctcactagac tttgcttcgc aaagtcgtga
     5581 ccgcctacgg cggctgcggc gccctacggg cttgctctcc gggcttcgcc ctgcgcggtc
     5641 gctgcgctcc cttgccagcc cgtggatatg tggacgatgg ccgcgagcgg ccaccggctg
     5701 gctcgcttcg ctcggcccgt ggacaaccct gctggacaag ctgatggaca ggctgcgcct
     5761 gcccacgagc ttgaccacag ggattgccca ccggctaccc agccttcgac cacataccca
     5821 ccggctccaa ctgcgcggcc tgcggccttg ccccatcaat ttttttaatt ttctctgggg
     5881 aaaagcctcc ggcctgcggc ctgcgcgctt cgcttgccgg ttggacacca agtggaaggc
     5941 gggtcaaggc tcgcgcagcg accgcgcagc ggcttggcct tgacgcgcct ggaacgaccc
     6001 aagcctatgc gagtgggggc agtcgaaggc gaagcccgcc cgcctgcccc ccgagcctca
     6061 cggcggcgag tgcgggggtt ccaagggggc agcgccacct tgggcaaggc cgaaggccgc
     6121 gcagtcgatc aacaagcccc ggaggggcca ctttttgccg gagggggagc cgcgccgaag
     6181 gcgtggggga accccgcagg ggtgcccttc tttgggcacc aaagaactag atatagggcg
     6241 aaatgcgaaa gacttaaaaa tcaacaactt aaaaaagggg ggtacgcaac agctcattgc
     6301 ggcacccccc gcaatagctc attgcgtagg ttaaagaaaa tctgtaattg actgccactt
     6361 ttacgcaacg cataattgtt gtcgcgctgc cgaaaagttg cagctgattg cgcatggtgc
     6421 cgcaaccgtg cggcacccta ccgcatggag ataagcatgg ccacgcagtc cagagaaatc
     6481 ggcattcaag ccaagaacaa gcccggtcac tgggtgcaaa cggaacgcaa agcgcatgag
     6541 gcgtgggccg ggcttattgc gaggaaaccc acggcggcaa tgctgctgca tcacctcgtg
     6601 gcgcagatgg gccaccagaa cgccgtggtg gtcagccaga agacactttc caagctcatc
     6661 ggacgttctt tgcggacggt ccaatacgca gtcaaggact tggtggccga gcgctggatc
     6721 tccgtcgtga agctcaacgg ccccggcacc gtgtcggcct acgtggtcaa tgaccgcgtg
     6781 gcgtggggcc agccccgcga ccagttgcgc ctgtcggtgt tcagtgccgc cgtggtggtt
     6841 gatcacgacg accaggacga atcgctgttg gggcatggcg acctgcgccg catcccgacc
     6901 ctgtatccgg gcgagcagca actaccgacc ggccccggcg aggagccgcc cagccagccc
     6961 ggcattccgg gcatggaacc agacctgcca gccttgaccg aaacggagga atgggaacgg
     7021 cgcgggcagc agcgcctgcc gatgcccgat gagccgtgtt ttctggacga tggcgagccg
     7081 ttggagccgc cgacacgggt cacgctgccg cgccggtagc acttgggttg cgcagcaacc
     7141 cgtaagtgcg ctgttccaga ctatcggctg tagccgcctc gccgccctat accttgtctg
     7201 cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg
     7261 cggcacctcg ctaacggatt caccgttttt atcaggctct gggaggcaga ataaatgatc
     7321 atatcgtcaa ttattacctc cacggggaga gcctgagcaa actggcctca ggcatttgag
     7381 aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca atagacataa
     7441 gcggctattt aacgaccctg ccctgaaccg acgaccgggt cgaatttgct ttcgaatttc
     7501 tgccattcat ccgcttatta tcacttattc aggcgtagca ccaggcgttt aagtcgacca
     7561 ataac
//
