Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V014016 | pCWLIC-2×Tac-CYP8B1(25-504aa) | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pCWLIC-2×Tac-CYP8B1(25-504aa)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8326 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- E. coli
- Promoter:
- sacB
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
pCWLIC-2×Tac-CYP8B1(25-504aa) vector Vector Map
Plasmid 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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
pCWLIC-2×Tac-CYP8B1(25-504aa) vector Sequence
LOCUS V014016 8326 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V014016
VERSION V014016
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8326)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8326
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin 12..467
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 494..598
/label="AmpR promoter"
CDS 599..1456
/label="AmpR"
/note="beta-lactamase"
rep_origin 1630..2218
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
misc_feature complement(2404..2546)
/label="bom"
/note="basis of mobility region from pBR322"
CDS complement(2955..3128)
/label="lacZ-alpha"
/note="LacZ-alpha fragment of beta-galactosidase"
protein_bind complement(3148..3164)
/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(3172..3202)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(3217..3238)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3254..4333)
/label="lacI"
/note="lac repressor"
promoter complement(4334..4411)
/label="lacI promoter"
promoter 4529..4557
/label="tac promoter"
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 4565..4581
/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)."
CDS 4727..6157
/gene="CYP8B1"
/label="7-alpha-hydroxycholest-4-en-3-one
12-alpha-hydroxylase"
/note="7-alpha-hydroxycholest-4-en-3-one
12-alpha-hydroxylase from Homo sapiens. Accession#: Q9UNU6"
CDS 6158..6175
/label="6xHis"
/note="6xHis affinity tag"
protein_bind complement(6188..6221)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
promoter 6236..6681
/label="sacB promoter"
/note="sacB promoter and control region"
CDS 6682..8100
/label="SacB"
/note="secreted levansucrase that renders bacterial growth
sensitive to sucrose"