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).
| 载体基本信息 | |||
| 载体名称 | pEYFP-Mito | 载体抗性 | Kanamycin |
|---|---|---|---|
| 载体长度 | 4759 bp | 载体类型 | Fluorescent Protein Reporter Vectors |
| 筛选标记 | Neomycin | 启动子 | CMV |
| 克隆方法 | Enzyme digestion and ligation | 载体标签 | EYFP |
pEYFP-Mito 载体载体图谱
pEYFP-Mito 载体载体序列
LOCUS Exported 4759 bp ds-DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4759)
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..4759
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 705..1424
/codon_start=1
/product="enhanced YFP"
/note="EYFP"
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 1547..1668
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1675..2130)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 2157..2261
/gene="bla"
/note="AmpR promoter"
promoter 2263..2620
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 2471..2606
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2655..3449
/codon_start=1
/gene="aph(3)-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/note="NeoR/KanR"
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
polyA_signal 3681..3728
/note="HSV TK poly(A) signal"
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4057..4645
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
