Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001456 | pcDNA5/FRT | 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
pcDNA5/FRT vector is a 5.1 kb expression vector designed for use with the Flp-In™ System. This vector features: 1. CMV promoter for high level expression in mammalian cells; 2. ten unique restriction site for easy cloning; 3. FLP Recombination Target (FRT) site for Flp recombinase-mediated integration of the vector into the Flp-In™ host cell line and 4. Hygromycin resistance gene for selection of stable cell lines.
- Vector Name:
- pcDNA5/FRT
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5070 bp
- Type:
- Mammalian Expression Vectors
- Replication origin:
- ori
- Source/Author:
- Invitrogen
- Selection Marker:
- Hygromycin
- Copy Number:
- High copy number
- Promoter:
- CMV
- Cloning Method:
- Enzyme Cut
- 5' Primer:
- T7 promoter
- 3' Primer:
- BGH Reverse primer
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
- Expression Method:
- Constiutive, Stable / Transient
pcDNA5/FRT 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.
References
- Tamraz B, Fukushima H, Wolfe AR, et al. OATP1B1-related drug-drug and drug-gene interactions as potential risk factors for cerivastatin-induced rhabdomyolysis. Pharmacogenet Genomics. 2013;23(7):355-364. doi:10.1097/FPC.0b013e3283620c3b
pcDNA5/FRT vector Sequence
LOCUS 40924_10256 5070 bp DNA circular SYN 20-AUG-2020
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5070)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5070)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5070
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 235..614
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 615..818
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 863..881
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
polyA_signal 1028..1252
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
protein_bind 1536..1583
/label=FRT
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
CDS 1591..2610
/codon_start=1
/label=HygR
/note="aminoglycoside phosphotransferase from E. coli"
/translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
"
polyA_signal 2743..2876
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2913..2929)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(2937..2953)
/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(2961..2991)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3006..3027)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3315..3903)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4077..4934)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4935..5039)
/label=AmpR promoter