Basic Vector Information
- Vector Name:
- pNYCOMPS-LIC-FH10T+ (N term)
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 6858 bp
- Type:
- Structural Genomics Vectors
- Replication origin:
- ori
- Source/Author:
- Mancia F, Love J.
- Copy Number:
- High copy number
- Promoter:
- lac UV5
pNYCOMPS-LIC-FH10T+ (N term) 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 ℃.
pNYCOMPS-LIC-FH10T+ (N term) vector Sequence
LOCUS pNYCOMPS-LIC-FH1 6858 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector for high-throughput expression and purification of
membrane proteins with N-terminal FLAG(R) and 10xHis tags.
ACCESSION .
VERSION .
KEYWORDS pNYCOMPS-LIC-FH10T+ (N term).
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6858)
AUTHORS Mancia F, Love J.
TITLE High-throughput expression and purification of membrane proteins.
JOURNAL J. Struct. Biol. 2010;172:85-93.
PUBMED 20394823
REFERENCE 2 (bases 1 to 6858)
AUTHORS New York Structural Biology Center
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6858)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J. Struct.
Biol."; date: "2010"; volume: "172"; pages: "85-93"
COMMENT SGRef: number: 2; type: "Journal Article"
COMMENT For ligation-independent cloning (LIC), linearize with SnaBI and
treat with T4 DNA polymerase plus dGTP.
FEATURES Location/Qualifiers
source 1..6858
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 6..23
/label=6xHis
/note="6xHis affinity tag"
terminator 90..137
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 174..629
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(725..1537)
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin 1659..2247
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(2433..2575)
/label=bom
/note="basis of mobility region from pBR322"
CDS complement(2680..2868)
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(3643..3664)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3680..4759)
/label=lacI
/note="lac repressor"
promoter complement(4760..4837)
/label=lacI promoter
promoter 5146..5164
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 5165..5189
/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)."
RBS 5204..5226
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 5233..5235
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 5236..5259
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 5260..5286
/label=9xHis
/note="9xHis affinity tag"
CDS 5290..5310
/label=TEV site
/note="tobacco etch virus (TEV) protease recognition and
cleavage site"
CDS complement(5393..5695)
/label=ccdB
/note="CcdB, a bacterial toxin that poisons DNA gyrase"
CDS complement(6040..6696)
/label=CmR
/note="chloramphenicol acetyltransferase"
promoter complement(6750..6780)
/label=lac UV5 promoter
/note="E. coli lac promoter with an 'up' mutation"
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