Basic Vector Information
- Vector Name:
- pMCSG75
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5876 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI,
- Copy Number:
- High copy number
pMCSG75 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 ℃.
pMCSG75 vector Sequence
LOCUS pMCSG75. 5876 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector encoding tRNA genes for rare Arg and Ile codons,
plus a C-terminal TEV-AviTag(TM)-6xHis cassette, plus the BirA
biotin ligase.
ACCESSION .
VERSION .
KEYWORDS pMCSG75.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5876)
AUTHORS Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI,
Jedrzejczak R, Joachimiak A.
TITLE New LIC vectors for production of proteins from genes containing
rare codons.
JOURNAL J. Struct. Funct. Genomics 2013;14:135-44.
PUBMED 24057978
REFERENCE 2 (bases 1 to 5876)
AUTHORS Midwest Center for Structural Genomics
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5876)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct.
Funct. Genomics"; date: "2013"; volume: "14"; pages: "135-44"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT For ligation-independent cloning (LIC), linearize with SmaI and
treat with T4 DNA polymerase plus dATP.
FEATURES Location/Qualifiers
source 1..5876
/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(244..1206)
/label=BirA
/note="E. coli biotin protein ligase"
RBS 1215..1220
/note="ribosome binding site"
CDS complement(1258..1275)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(1282..1326)
/label=AviTag(TM)
/note="peptide tag that allows for enzymatic biotinylation"
CDS complement(1333..1353)
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
RBS complement(1365..1385)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(1401..1425)
/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(1426..1444)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
tRNA complement(1719..1795)
/label=argU
tRNA complement(2065..2140)
/label=ileX
promoter 2280..2357
/label=lacI promoter
CDS 2358..3437
/label=lacI
/note="lac repressor"
protein_bind 3453..3474
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3869..4457)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4631..5488)
/label=AmpR
/note="beta-lactamase"
promoter complement(5489..5592)
/label=AmpR promoter
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