pMKEx2 vector (V004707)

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LOCUS       40924_31045        8061 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle-expression vector pMKEx2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8061)
  AUTHORS   Kortmann M, Kuhl V, Klaffl S, Bott M.
  TITLE     A chromosomally encoded T7 RNA polymerase-dependent gene expression 
            system for Corynebacterium glutamicum: construction and comparative 
            evaluation at the single-cell level
  JOURNAL   Microb Biotechnol (2014) In press
  PUBMED    25488698
REFERENCE   2  (bases 1 to 8061)
  AUTHORS   Kortmann M, Kuhl V, Klaffl S, Bott M.
  TITLE     Direct Submission
  JOURNAL   Submitted (25-SEP-2014) IBG-1: Biotechnology, Forschungszentrum 
            Juelich GmbH, Wilhelm-Johnen-Strasse, Juelich 52425, Germany
REFERENCE   3  (bases 1 to 8061)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8061)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Microb 
            Biotechnol (2014) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (25-SEP-2014) IBG-1: Biotechnology, Forschungszentrum Juelich GmbH, 
            Wilhelm-Johnen-Strasse, Juelich 52425, Germany"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(73..120)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(160..186)
                     /codon_start=1
                     /label=9xHis
                     /note="9xHis affinity tag"
                     /translation="HHHHHHHHH"
     CDS             complement(193..210)
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     CDS             complement(256..279)
                     /codon_start=1
                     /label=HRV 3C site
                     /note="recognition and cleavage site for human rhinovirus
                     3C and PreScission proteases"
                     /translation="LEVLFQGP"
     CDS             complement(286..309)
                     /codon_start=1
                     /label=Strep-Tag II
                     /note="peptide that binds Strep-Tactin(R), an engineered 
                     form
                     of streptavidin"
                     /translation="WSHPQFEK"
     RBS             complement(325..347)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(362..386)
                     /label=lac operator
                     /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)."
     promoter        complement(387..405)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        718..795
                     /label=lacI promoter
     CDS             796..1875
                     /codon_start=1
                     /label=lacI
                     /note="lac repressor"
                     /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     protein_bind    1891..1912
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      2451..2996
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     CDS             3591..4403
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5380..6837)
                     /codon_start=1
                     /gene="repA"
                     /product="replicase"
                     /label=repA
                     /protein_id="AIZ68604.1"
                     /translation="MTLADSRDIDTPAWKFSADLFDTHPELALRSRGWTSEDRREFLAH
                     LGRENFQGSKTRDFASAWIKDPDTGETQPKLYRVGSKSLARCQYVALTHAQHAAVLVLD
                     IDVPSHQAGGKIEHVNPEVYAILERWARLEKAPAWIGVNPLSGKCQLIWLIDPVYAAAG
                     MSSPNMRLLAATTEEMTRVFGADQAFSHRLSRWPLHVSDDPTAYRWHAQHNRVDRLADL
                     MEVARMISGTEKPKKRYEQEFSSGRARIEAARKATAEAKALATLEASLPSAAEASGELI
                     DGVRVLWTAPGRAARDETAFRHALTVGYQLKAAGERLKDTKIIDAYERAYTVAQAVGAD
                     GREPDLPPMRDRQTMARRVRGYVAKGQPVVPARQTETQSSRGRKALATMGRRGGKKAAE
                     RWKDPNSEYARAQREKLAKSSQRQARKAKGNRLTIAGWFMTVEGETGSWPTINEAMSEF
                     SVSRQTVNRALKSAGIELPRGRRKASQ"
     gene            complement(5380..6837)
                     /gene="repA"
                     /label=repA