Basic Vector Information
- Vector Name:
- pMCSG65
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5369 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Eschenfeldt WH, Makowska-Grzyska M, Stols L, Donnelly MI,
- Copy Number:
- High copy number
pMCSG65 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 ℃.
pMCSG65 vector Sequence
LOCUS pMCSG65. 5369 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector for expressing a protein with a 6xHis-HA-TEV leader
plus a second untagged protein.
ACCESSION .
VERSION .
KEYWORDS pMCSG65.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5369)
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 5369)
AUTHORS Midwest Center for Structural Genomics
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5369)
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 There are two sites for ligation-independent cloning (LIC).
To express a 6xHis-HA-TEV-tagged protein, linearize with SspI and
treat with T4 DNA polymerase plus dGTP.
To express a second untagged protein, linearize with SmaI and treat
with T4 DNA polymerase plus dATP.
FEATURES Location/Qualifiers
source 1..5369
/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"
RBS 243..248
/note="ribosome binding site"
CDS complement(279..299)
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
CDS complement(306..332)
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
CDS complement(351..368)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(369..371)
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
RBS complement(379..401)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(416..440)
/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(441..459)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 772..849
/label=lacI promoter
CDS 850..1929
/label=lacI
/note="lac repressor"
protein_bind 1945..1966
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 2741..2929
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 3034..3176
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(3362..3950)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4124..4981)
/label=AmpR
/note="beta-lactamase"
promoter complement(4982..5085)
/label=AmpR promoter
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