Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001062 | pCMV CEPIA4mt | 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:
- pCMV CEPIA4mt
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6404 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Selection Marker:
- Neomycin (select with G418)
- Copy Number:
- High Copy
- Promoter:
- CMV
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- cgcaaatgggcggtaggcgtg
- 3' Primer:
- tagaaggcacagtcgagg-
pCMV CEPIA4mt 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.
pCMV CEPIA4mt vector Sequence
LOCUS 40924_12425 6404 bp DNA circular SYN 13-MAY-2021
DEFINITION Green fluorescent indicator for calcium imaging in the mitochondria
(low affinity variant).
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6404)
AUTHORS Suzuki J, Kanemaru K, Ishii K, Ohkura M, Okubo Y, Iino M
TITLE Imaging intraorganellar Ca(2+) at subcellular resolution using
CEPIA.
JOURNAL Nat Commun. 2014 Jun 13;5:4153. doi: 10.1038/ncomms5153.
PUBMED 24923787
REFERENCE 2 (bases 1 to 6404)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6404)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat
Commun."; date: "2014-06-13"; pages: "
10.1038/ncomms5153"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6404
/mol_type="other DNA"
/organism="synthetic DNA construct"
rep_origin complement(37..625)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(799..1656)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(1657..1761)
/label=AmpR promoter
enhancer 1825..2202
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 2204..2407
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 2450..2524
/codon_start=1
/product="mitochondrial presequence of human cytochrome c
oxidase subunit VIII (Rizutto et al., 1989)"
/label=COX8 presequence
/translation="MSVLTPLLLRGLTGSARRLPVPRAK"
CDS 2546..2620
/codon_start=1
/product="mitochondrial presequence of human cytochrome c
oxidase subunit VIII (Rizutto et al., 1989)"
/label=COX8 presequence
/translation="MSVLTPLLLRGLTGSARRLPVPRAK"
CDS 2654..4000
/codon_start=1
/label=GCaMP6f
/note="improved fluorescent protein-based calcium sensor
(Chen et al., 2013)"
/translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNKT
GHAVRAIGRLSSLENVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPV
LLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEEL
FTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTY
GVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGYYKTRAEVKFEGDTLVNRIVL
KGIDFKEDGNILGHKLEYNTRDQLTEEQIAEFKEAFSLFDKDGDGTITTKDLGTVLRSL
GQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVWDKDGNG
YISAAELRHVMTNLGEKLTDEEVDEMIREADIDGEGQVNYEEFVQMMTAK"
CDS 4010..4039
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
polyA_signal 4081..4305
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 4351..4779
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4793..5122
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 5148..5939
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MVEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 6116..6249
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6270..6287)
/label=L4440
/note="L4440 vector, forward primer"