Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001112 | pEYFP-Mito | 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
pEYFP-Mito encodes a fusion of EYFP and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (1, 2). The EYFP gene (enhanced yellow fluorescent protein) contains four amino acid substitutions previously published as GFP-10C (3), which shift the emission of the chromophore from green to yellow-green. The fluorescence excitation maximum of EYFP is 513 nm, and the emission spectrum has a peak at 527 nm. In addition to the four chromophore mutations, the coding sequence of the EYFP gene contains more than 190 silent base changes corresponding to human codon-usage preferences (4), which increase the translational efficiency of the EYFP transcript.SV40 polyadenylation signals downstream of the EYFP-Mito fusion direct proper processing of the 3' end of the EYFP-Mito mRNA. The vector backbone also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-antigen. A neomycin resistance cassette (Neo r ), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HSV-TK) gene, allow stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E. coli . The pEYFP-Mito backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.The pEYFP-Mito Vector is designed to be used for the fluorescent labeling of mitochondria (5). Fluorescence can be observed in living or fixed cells by microscopy, fluorometry, or flow cytometry. pEYFP-Mito can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).
- Vector Name:
- pEYFP-Mito
- Antibiotic Resistance:
- Kanamycin
- Length:
- 4759 bp
- Type:
- Fluorescent Protein Reporter Vectors
- Replication origin:
- ori
- Selection Marker:
- Neomycin
- Promoter:
- CMV
- Cloning Method:
- Enzyme digestion and ligation
- Fusion Tag:
- EYFP
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
pEYFP-Mito 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.
pEYFP-Mito vector Sequence
LOCUS 40924_18956 4759 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 4759)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4759)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4759
/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 705..1421
/codon_start=1
/label=EYFP
/note="enhanced YFP"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1547..1668
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1675..2130)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2157..2261
/label=AmpR promoter
promoter 2263..2620
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
CDS 2655..3446
/codon_start=1
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3681..3728
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4057..4645
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"