Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012409 | pECFP-Nuc | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pECFP-Nuc vector encodes an enhanced cyan fluorescent protein (ECFP) targeted to the nucleus of mammalian cells via three copies of a nuclear localization signal (NLS), ensuring efficient nuclear translocation. This ECFP variant features optimized excitation (major peak at 433 nm) and emission (major peak at 475 nm) spectra, along with mutations that enhance its brightness and solubility . The vector contains a neomycin resistance gene for selection in eukaryotic cells using G418 and an SV40 origin for replication. It is primarily used to visualize the nucleus in living or fixed cells via fluorescence microscopy and is suitable for dual-labeling experiments with yellow fluorescent proteins like EYFP.
- Vector Name:
- pECFP-Nuc
- Antibiotic Resistance:
- Kanamycin
- Length:
- 4764 bp
- Type:
- Fluorescent Protein Reporter Vectors
- Replication origin:
- ori
- Selection Marker:
- Neomycin
- Promoter:
- CMV
- Fusion Tag:
- ECFP
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pECFP-Nuc 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
- Arango-Rodriguez ML, Navarro-Quiroga I, Gonzalez-Barrios JA, Martinez-Arguelles DB, Bannon MJ, Kouri J, Forgez P, Rostene W, Garcia-Villegas R, Jimenez I, Martinez-Fong D. Biophysical characteristics of neurotensin polyplex for in vitro and in vivo gene transfection. Biochim Biophys Acta. 2006 Jul;1760(7):1009-20. doi: 10.1016/j.bbagen.2006.02.021. Epub 2006 Apr 3. PMID: 16730907.
pECFP-Nuc vector Sequence
LOCUS 40924_16745 4764 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4764)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4764)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4764
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 613..1329
/codon_start=1
/label=ECFP
/note="enhanced CFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 1354..1374
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 1378..1398
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
CDS 1402..1422
/codon_start=1
/label=SV40 NLS
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/translation="PKKKRKV"
polyA_signal 1552..1673
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1680..2135)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2162..2266
/label=AmpR promoter
promoter 2268..2625
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2660..3451
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3686..3733
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4062..4650
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"