Basic Vector Information
- Vector Name:
- pMCSG30
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8314 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Midwest Center for Structural Genomics
- Copy Number:
- High copy number
- Promoter:
- sacB
pMCSG30 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 ℃.
pMCSG30 vector Sequence
LOCUS pMCSG30. 8314 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial expression vector with a 6xHis-TEV-MBP leader and a SacB
negative selection marker, for high-throughput purification of
recombinant proteins.
ACCESSION .
VERSION .
KEYWORDS pMCSG30.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8314)
AUTHORS Midwest Center for Structural Genomics
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8314)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT For ligation-independent cloning (LIC), linearize with SfiI to
remove the SacB cassette, and treat with T4 DNA polymerase plus
dTTP.
FEATURES Location/Qualifiers
source 1..8314
/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(258..1676)
/label=SacB
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"
promoter complement(1677..2122)
/label=sacB promoter
/note="sacB promoter and control region"
CDS complement(2147..3244)
/label=MBP
/note="maltose binding protein from E. coli"
CDS complement(3251..3271)
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
CDS complement(3296..3313)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(3314..3316)
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
RBS complement(3324..3346)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(3361..3385)
/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(3386..3404)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 3717..3794
/label=lacI promoter
CDS 3795..4874
/label=lacI
/note="lac repressor"
protein_bind 4890..4911
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 5686..5874
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 5979..6121
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(6307..6895)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7069..7926)
/label=AmpR
/note="beta-lactamase"
promoter complement(7927..8030)
/label=AmpR promoter
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