LOCUS       40924_9441        4905 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCDF_polyST-F116-F460T, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4905)
  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 4905)
  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 4905)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 4905)
  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..4905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..126
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA
                     polymerase, with embedded lac operator"
     RBS             169..191
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             207..230
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     terminator      1698..1745
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1919..2707)
                     /codon_start=1
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2708..2799)
                     /label=AmpR promoter
     rep_origin      complement(2847..3585)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     protein_bind    complement(3761..3782)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(3798..4877)
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(join(4878..4905,1..50))
                     /label=lacI promoter
ORIGIN
        1 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
       61 acgctctccc ttatgcgact atcataaaaa atttatttgc tttgtgagcg gataacaatt
      121 ataatagatt caattgtgag cggataacaa ttcccctgta gaaataattt tgtttaactt
      181 taataaggag atataccatg gatagctgga gccacccgca gttcgaaaaa ggcgccctgg
      241 atatcggatc cccgtcctac ctctccccgc ctcgtaataa cttgtttgtg atctccaatt
      301 tgggtcaact gaaccaggtg caatctttga tcaagatcca gaaactgacc gataacctgt
      361 tagtgatcct gtacaccagc caaaatctga aaatgccgaa gttggttcat caatccgcga
      421 acaagaatct gtttgagagc atctacctgt tcgaattgcc gcgttccccg aacattatta
      481 ccccgaagaa actgttgtac atctatcact cctacaagaa aatcctgaat atcattcagc
      541 cggcacacct gtatatgttg tcctttaccg gtcactattc ctacctgatt tcaatcgcga
      601 agaagaaaaa catcactacc cacctgatcg acgagggtac cggtacgtat gctccgttgc
      661 tggagagctt ctcttatcac ccgacgaagc tggaacgtta cctgatcggc aataacctga
      721 acatcaaagg ctacattgac cattttgata ttctgcacgt tccgttccca gagtatgcta
      781 aaaagatttt caacgcgaag aaatataacc gcttctttgc gcatgctggc ggcatttctg
      841 tcaataacaa tattgcgaat ctgcaaaaga aataccagat ttccaaaaat gactacatct
      901 ttgttagcca gcgttatccg atctccgacg atctgtatta caaatccatt gtggagattt
      961 tgaactccat ttccttacaa atcaaaggta aaatctttat caaattgcat ccgaaggaaa
     1021 tgggcaataa ctatgtgatg tctttgttcc tcaacatggt tgagattaat ccgcgtctgg
     1081 ttgtgattaa cgaacctccg tttctgatcg agccgttgat ctatttgacc aatcctaaag
     1141 gcattatcgg tctggcgtcc agctccttga tctacactcc gctgctctct ccgtccacgc
     1201 aatgcctgag cattggtgag ctgattatca acttgattca aaaatactct atggttgaaa
     1261 acaccgagat gattcaggtc catctggaga ttatcaagaa gtttaatttc gtcaacatct
     1321 taaacgactt gaacggtgtt aactccaccc cgttgtacaa gaccgaggaa accttcgaaa
     1381 ccctcttgaa atccgcggaa tttgcttaca taagcaagaa ctatttccag gcgatctttt
     1441 actggcaatt ggcgtccaag aataatatca ccctgttagg tcataaggcg ttgtggtact
     1501 acaatgccct gtataacgtt aaacaaatct acaaaatgga gtattccgac atcacataca
     1561 ttgataatat ctccgttgac ttccatagca aagataagct gacctgggaa aagatcaagc
     1621 actattacta tagcgcagac aaccgtatcg gccgtgatcg ctaattaacc taggctgctg
     1681 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1741 ttttgctgaa acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata
     1801 aaccggtaaa ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga
     1861 ccgggtcatc gtggccggat cttgcggccc ctcggcttga acgaattgtt agacattatt
     1921 tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg
     1981 cgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga
     2041 cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg
     2101 cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct
     2161 catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa
     2221 atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa
     2281 cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct
     2341 cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag
     2401 ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga
     2461 gaatctcgct ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc
     2521 gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct
     2581 tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat
     2641 ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt
     2701 ccctcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2761 gcggatacat atttgaatgt atttagaaaa ataaacaaat agctagctca ctcggtcgct
     2821 acgctccggg cgtgagactg cggcgggcgc tgcggacaca tacaaagtta cccacagatt
     2881 ccgtggataa gcaggggact aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt
     2941 tttccatagg ctccgccctc ctgccagagt tcacataaac agacgctttt ccggtgcatc
     3001 tgtgggagcc gtgaggctca accatgaatc tgacagtacg ggcgaaaccc gacaggactt
     3061 aaagatcccc accgtttccg gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc
     3121 cgtttaccgg atacctgttc cgcctttctc ccttacggga agtgtggcgc tttctcatag
     3181 ctcacacact ggtatctcgg ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa
     3241 gaactccccg ttcagcccga ctgctgcgcc ttatccggta actgttcact tgagtccaac
     3301 ccggaaaagc acggtaaaac gccactggca gcagccattg gtaactggga gttcgcagag
     3361 gatttgttta gctaaacacg cggttgctct tgaagtgtgc gccaaagtcc ggctacactg
     3421 gaaggacaga tttggttgct gtgctctgcg aaagccagtt accacggtta agcagttccc
     3481 caactgactt aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa
     3541 aagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgaaccg
     3601 ctctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga
     3661 tataagttgt aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt
     3721 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     3781 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3841 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     3901 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     3961 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     4021 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc
     4081 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     4141 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     4201 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     4261 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     4321 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     4381 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     4441 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     4501 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     4561 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     4621 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     4681 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     4741 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     4801 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     4861 aacgttactg gtttcacatt caccaccctg aattgactct cttcc
//
