Basic Vector Information
- Vector Name:
- pET-32 Xa/LIC
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5926 bp
- Type:
- pET & Duet Vectors (Novagen)
- Replication origin:
- ori
- Source/Author:
- Novagen (EMD Millipore)
- Copy Number:
- High copy number
pET-32 Xa/LIC 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 ℃.
pET-32 Xa/LIC vector Sequence
LOCUS pET-32_Xa_LIC. 5926 bp DNA circular SYN 01-JAN-1980
DEFINITION Bacterial vector for ligation-independent cloning (LIC) to express
thioredoxin-tagged proteins with a Factor Xa site.
ACCESSION .
VERSION .
KEYWORDS pET-32 Xa_LIC.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5926)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5926)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT For LIC, linearize with BseRI and treat with T4 DNA polymerase plus
dCTP.
FEATURES Location/Qualifiers
source 1..5926
/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"
misc_feature 158..216
/label=MCS
/note="MCS"
/note="multiple cloning site"
CDS complement(233..244)
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
CDS complement(275..319)
/label=S-Tag
/note="affinity and epitope tag derived from pancreatic
ribonuclease A"
CDS complement(326..343)
/label=thrombin site
/note="thrombin recognition and cleavage site"
CDS complement(353..370)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(392..718)
/label=TrxA
/note="E. coli thioredoxin"
RBS complement(726..748)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(763..787)
/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(788..806)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 1119..1196
/label=lacI promoter
CDS 1197..2276
/label=lacI
/note="lac repressor"
protein_bind 2292..2313
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 3088..3276
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 3381..3523
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(3709..4297)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4471..5328)
/label=AmpR
/note="beta-lactamase"
promoter complement(5329..5433)
/label=AmpR promoter
rep_origin complement(5460..5915)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
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