LOCUS       Exported                7583 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector pMLBAD_GlycoTagGFP, complete sequence.
ACCESSION   KY212116
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7583
                     /organism="Expression vector pMLBAD_GlycoTagGFP"
                     /mol_type="other DNA"
                     /label=PMA1045
                     /note="PMA1045"
                     /db_xref="taxon:2045057"
     promoter        55..83
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             410..646
                     /codon_start=1
                     /gene="dhfrII"
                     /product="dihydrofolate reductase"
                     /label=dhfrII
                     /note="trimethoprim resistance determinant"
                     /protein_id="ATN32193.1"
                     /translation="MGQSSDEANAPVAGQFALPLSATFGLGDRVRKKSGAAWQGQVVGW
                     YCTKLTPEGYAVESESHPGSVQIYPVAALERVA"
     gene            410..646
                     /gene="dhfrII"
                     /label=dhfrII
     CDS             complement(869..1798)
                     /codon_start=1
                     /gene="araC"
                     /product="AraC"
                     /label=araC
                     /note="transcriptional regulator"
                     /protein_id="ATN32194.1"
                     /translation="MQYGQLVSSLNGGSMKSMAEAQNDPLLPGYSFNAHLVAGLTPIEA
                     NGYLDFFIDRPLGMKGYILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPE
                     AREWYHQWVYFRPRAYWHEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGR
                     YSELLAINLLEQLLLRRMEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVC
                     LSPSRLSHLFRQQLGISVLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRV
                     FKKCTGASPSEFRAGCEEKVNDVAVKLS"
     gene            complement(869..1798)
                     /gene="araC"
                     /label=araC
     CDS             complement(869..1747)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     regulatory      2023..2050
                     /regulatory_class="promoter"
                     /note="P-BAD"
     CDS             2092..2916
                     /codon_start=1
                     /product="GlycoTagGFP"
                     /label=GlycoTagGFP
                     /protein_id="ATN32195.1"
                     /translation="MGSHHHHHHHHHHGSSKGEELFTGVVPILVELDGDVNGHKFSVRG
                     EGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPE
                     GYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHN
                     VYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLS
                     KDPNEKRDHMVLLEFVTAAGITKTSAHATASGAHATAGSANATAWSHPQFEK"
     CDS             2101..2130
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     misc_feature    2833..2889
                     /label=NAT-tag (C-terminal glycosylation-tag)
                     /note="NAT-tag (C-terminal glycosylation-tag)"
     misc_feature    2890..2913
                     /label=Strep-tag II
                     /note="Strep-tag II"
     CDS             2890..2913
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     regulatory      2948..3373
                     /regulatory_class="terminator"
                     /note="rrnB"
     terminator      3150..3236
                     /gene="Escherichia coli rrnB"
                     /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..7137
                     /codon_start=1
                     /gene="rep"
                     /product="Rep"
                     /function="required for plasmid replication"
                     /label=rep
                     /protein_id="ATN32192.1"
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     gene            6475..7137
                     /gene="rep"
                     /label=rep