Basic Vector Information
- Vector Name:
- pMKEx2
- Antibiotic Resistance:
- Kanamycin
- Length:
- 8061 bp
- Type:
- Shuttle-expression vector
- Replication origin:
- p15A ori
- Source/Author:
- Kortmann M, Kuhl V, Klaffl S, Bott M.
pMKEx2 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
pMKEx2 vector Sequence
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
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