LOCUS       Exported                8061 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Shuttle-expression vector pMKEx2, complete sequence.
ACCESSION   KM658503
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8061
                     /organism="Shuttle-expression vector pMKEx2"
                     /lab_host="Corynebacterium glutamicum"
                     /mol_type="other DNA"
                     /note="Escherichia coli/Corynebacterium glutamicum 
                     shuttle-expression vector; contains a T7 promoter and lac 
                     repressor-gene"
                     /db_xref="taxon:1582277"
     terminator      73..120
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(157..186)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             complement(193..210)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(256..279)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(286..309)
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     RBS             325..347
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    362..386
                     /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)."
     promoter        complement(387..405)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      complement(388..405)
                     /regulatory_class="promoter"
                     /note="T7 promoter"
     promoter        718..795
                     /gene="lacI"
                     /label=lacI promoter
     CDS             796..1878
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /protein_id="AIZ68603.1"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     gene            796..1878
                     /gene="lacI"
                     /label=lacI
     rep_origin      2451..2996
                     /direction=RIGHT
                     /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..4406
                     /codon_start=1
                     /gene="aph"
                     /product="aminoglycoside 3-N-acetyltransferase"
                     /label=aph
                     /note="kanamycin resistance"
                     /protein_id="AIZ68602.1"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     gene            3591..4406
                     /gene="aph"
                     /label=aph
     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