Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012931 | pCold-SUMO-10×His-3C-Strep II-FLAG | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pCold-SUMO-10×His-3C-Strep II-FLAG is for cold shock-induced expression in E. coli of a protein with a translation enhancing element followed by SUMO-10×His-3C-Strep II-FLAG tags.
- Vector Name:
- pCold-SUMO-10×His-3C-Strep II-FLAG
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4930 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- E. coli
- Promoter:
- cspA
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pCold-SUMO-10×His-3C-Strep II-FLAG 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.
References
- Jerlström-Hultqvist J, Einarsson E, Svärd SG. Stable transfection of the diplomonad parasite Spironucleus salmonicida. Eukaryot Cell. 2012;11(11):1353-1361. doi:10.1128/EC.00179-12
pCold-SUMO-10×His-3C-Strep II-FLAG vector Sequence
LOCUS 62056_7845 4930 bp DNA circular SYN 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4930)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4930
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 15..81
/label=cspA promoter
/note="promoter of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
protein_bind 84..100
/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)."
5'UTR 122..255
/label=cspA 5'UTR
/note="5'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
CDS 256..270
/codon_start=1
/label=TEE
/note="translation enhancing element for E. coli (Qing et
al., 2004)"
/translation="MNHKV"
CDS 280..303
/codon_start=1
/label=Strep-Tag II
/note="peptide that binds Strep-Tactin(R), an engineered
form
of streptavidin"
/translation="WSHPQFEK"
CDS 325..342
/codon_start=1
/label=6xHis
/note="6xHis affinity tag"
/translation="HHHHHH"
CDS 364..654
/codon_start=1
/label=SUMO
/note="cleavable ubiquitin-like protein tag"
/translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
CDS 676..702
/codon_start=1
/label=9xHis
/note="9xHis affinity tag"
/translation="HHHHHHHHH"
CDS 706..729
/codon_start=1
/label=HRV 3C site
/note="recognition and cleavage site for human rhinovirus
3C and PreScission proteases"
/translation="LEVLFQGP"
CDS 775..798
/codon_start=1
/label=Strep-Tag II
/note="peptide that binds Strep-Tactin(R), an engineered
form
of streptavidin"
/translation="WSHPQFEK"
CDS 850..873
/codon_start=1
/label=FLAG
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/translation="DYKDDDDK"
3'UTR 890..1034
/label=cspA 3'UTR
/note="3'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
rep_origin complement(1234..1689)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1789..1893
/label=AmpR promoter
CDS 1894..2751
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 2925..3513
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind complement(3651..3672)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3688..4767)
/codon_start=1
/label=lacI
/note="lac repressor"
/translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
promoter complement(4768..4845)
/label=lacI promoter