Basic Vector Information
- Vector Name:
- pMCSG8
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5340 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID.
- Copy Number:
- High copy number
pMCSG8 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 ℃.
pMCSG8 vector Sequence
LOCUS pMCSG8. 5340 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector with a 6xHis-S-loop-TEV leader for high-throughput
purification of toxic recombinant proteins.
ACCESSION .
VERSION .
KEYWORDS pMCSG8.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5340)
AUTHORS Eschenfeldt WH, Lucy S, Millard CS, Joachimiak A, Mark ID.
TITLE A family of LIC vectors for high-throughput cloning and purification
of proteins.
JOURNAL Methods Mol. Biol. 2009;498:105-15.
PUBMED 18988021
REFERENCE 2 (bases 1 to 5340)
AUTHORS Midwest Center for Structural Genomics
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5340)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Methods
Mol. Biol."; date: "2009"; volume: "498"; pages: "105-15"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT For ligation-independent cloning (LIC), linearize with SspI and
treat with T4 DNA polymerase plus dGTP.
FEATURES Location/Qualifiers
source 1..5340
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(26..73)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(140..157)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(223..243)
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
CDS complement(250..303)
/label=S loop
/note="GroES chaperone mobile loop that interacts with
GroEL"
CDS complement(322..339)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(340..342)
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
RBS complement(350..372)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(387..411)
/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(412..430)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 743..820
/label=lacI promoter
CDS 821..1900
/label=lacI
/note="lac repressor"
protein_bind 1916..1937
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 2712..2900
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 3005..3147
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(3333..3921)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4095..4952)
/label=AmpR
/note="beta-lactamase"
promoter complement(4953..5056)
/label=AmpR promoter
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