LOCUS       Exported                4905 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pCDF_polyST-F116-F460T, complete sequence.
ACCESSION   KY212118
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..4905
                     /organism="Expression vector pCDF_polyST-F116-F460T"
                     /mol_type="other DNA"
                     /label=PMA1059
                     /note="PMA1059"
                     /db_xref="taxon:2045055"
     promoter        82..126
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     regulatory      98..104
                     /regulatory_class="minus_35_signal"
     misc_feature    102..120
                     /label=lacO1 site
                     /note="lacO1 site"
     protein_bind    102..118
                     /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)."
     regulatory      120..125
                     /regulatory_class="minus_10_signal"
     misc_feature    132..156
                     /label=lacO
                     /note="lacO"
     protein_bind    134..150
                     /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)."
     CDS             198..1664
                     /codon_start=1
                     /product="strep-polyST(F116-F460T)"
                     /label=strep-polyST
                     /protein_id="ATN32202.1"
                     /translation="MDSWSHPQFEKGALDIGSPSYLSPPRNNLFVISNLGQLNQVQSLI
                     KIQKLTDNLLVILYTSQNLKMPKLVHQSANKNLFESIYLFELPRSPNIITPKKLLYIYH
                     SYKKILNIIQPAHLYMLSFTGHYSYLISIAKKKNITTHLIDEGTGTYAPLLESFSYHPT
                     KLERYLIGNNLNIKGYIDHFDILHVPFPEYAKKIFNAKKYNRFFAHAGGISVNNNIANL
                     QKKYQISKNDYIFVSQRYPISDDLYYKSIVEILNSISLQIKGKIFIKLHPKEMGNNYVM
                     SLFLNMVEINPRLVVINEPPFLIEPLIYLTNPKGIIGLASSSLIYTPLLSPSTQCLSIG
                     ELIINLIQKYSMVENTEMIQVHLEIIKKFNFVNILNDLNGVNSTPLYKTEETFETLLKS
                     AEFAYISKNYFQAIFYWQLASKNNITLLGHKALWYYNALYNVKQIYKMEYSDITYIDNI
                     SVDFHSKDKLTWEKIKHYYYSADNRIGRDR"
     misc_feature    207..230
                     /label=Strep tag II
                     /note="Strep tag II"
     CDS             207..230
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     terminator      1698..1745
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     gene            complement(1916..2707)
                     /gene="spectR (variant)"
                     /label=spectR (variant)
     CDS             complement(1916..2707)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(2708..2799)
                     /gene="bla"
                     /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."
     misc_feature    complement(3795..4886)
                     /label=lacI
                     /note="lacI"
     CDS             complement(3795..4877)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(4878..50)
                     /gene="lacI"
                     /label=lacI promoter