Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012186 | pCLBW cox8 EGFP mCherry | 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:
- pCLBW cox8 EGFP mCherry
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8923 bp
- Type:
- Mammalian Expression, Retroviral
- Replication origin:
- ori
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- CMV
pCLBW cox8 EGFP mCherry 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.
pCLBW cox8 EGFP mCherry vector Sequence
LOCUS 40924_10976 8923 bp DNA circular SYN 06-JAN-2022
DEFINITION Fluorescent mitophagy reporter.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8923)
AUTHORS Rojansky R, Cha MY, Chan DC
TITLE Elimination of paternal mitochondria in mouse embryos occurs through
autophagic degradation dependent on PARKIN and MUL1.
JOURNAL Elife. 2016 Nov 17;5. pii: e17896. doi: 10.7554/eLife.17896.
PUBMED 27852436
REFERENCE 2 (bases 1 to 8923)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 8923)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Elife.";
date: "2016-11-17"; pages: "
10.7554/eLife.17896"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8923
/mol_type="other DNA"
/organism="synthetic DNA construct"
intron 175..1191
/label=chimeric intron
/note="chimera between introns from chicken beta-actin and
rabbit beta-globin"
primer_bind 1199..1218
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
protein_bind 1279..1303
/label=attB1
/note="recombination site for the Gateway(R) BP reaction"
CDS 1310..1384
/codon_start=1
/product="mitochondrial presequence of human cytochrome c
oxidase subunit VIII (Rizutto et al., 1989)"
/label=COX8 presequence
/translation="MSVLTPLLLRGLTGSARRLPVPRAK"
CDS 1406..2119
/codon_start=1
/label=EGFP
/note="enhanced GFP"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
CDS 2135..2842
/codon_start=1
/label=mCherry
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
protein_bind complement(2846..2870)
/label=attB2
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 2915..3503
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3506..3522)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
LTR 3718..4309
/label=LTR
/note="long terminal repeat from Moloney murine leukemia
virus"
primer_bind complement(4439..4456)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4610..5198)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5361..6218)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
enhancer 6370..6749
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 6750..6953
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 7192..7549
/label=MMLV Psi
/note="packaging signal of Moloney murine leukemia virus
(MMLV)"
CDS 7614..8030
/codon_start=1
/label=gag (truncated)
/note="truncated Moloney murine leukemia virus (MMLV) gag
gene lacking the start codon"
/translation="GQTVTTPLSLTLGHWKDVERIAHNQSVDVKKRRWVTFCSAEWPTF
NVGWPRDGTFNRDLITQVKIKVFSPGPHGHPDQVPYIVTWEALAFDPPPWVKPFVHPKP
PPPLPPSAPSLPLEPPRSTPPRSSLYPALTPSLGA"
misc_feature 8040..8414
/label=pol region
/note="Moloney murine leukemia virus (MMLV) pol region
containing the splice acceptor site"
promoter 8453..8923
/label=CAG
/note="CMV early enhancer fused to modified chicken
beta-actin promoter"