MITO-YFP plasmid vector線粒體定位黃色熒光表達(dá)質(zhì)粒載體

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 YFP-Mito fusion direct proper processing of the 3' end of the YFP-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 (Neor), 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 YFP-Mito backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

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