The vector sequence has been compiled using the information from sequence databases, published literature, and other sources, together with partial sequences obtained by Evrogen. This vector has not been completely sequenced.
pPhi-Yellow-mito is a mammalian expression vector intended for yellow fluorescent labeling of mitochondria in living cells. The vector encodes yellow fluorescent protein PhiYFP (see reporter description) fused to mitochondrial targeting sequence (MTS) derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995]. MTS is fused to the PhiYFP N-terminus.
Stably transfected PtK rat kangaroo cells expressing mitochondria-targeted PhiYFP.
Image was kindly provided by Dr. Christian Petzelt (Marinpharm).
PhiYFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].
pPhi-Yellow-mito vector can be used as a source of PhiYFP-MTS hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.
Note: The plasmid DNA was isolated from dam+-methylated
The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in
SV40 early promoter (PSV40) provides neomycin resistance gene (Neor) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr) in
Expression in mammalian cells
pPhi-Yellow-mito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the PhiYFP-MTS fusion in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].
Suitable host strains for propagation in
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Start codon (ATG): 597-599
Mitochondrial targeting sequence (MTS): 597-683
Start of PhiYFP coding sequence (ATG): 705-707
Stop codon: 1407-1409
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1623-1628 & 1652-1657
mRNA 3' ends: 1661 & 1673
f1 single-strand DNA origin: 1720-2175
Bacterial promoter for expression of Kanr gene
-35 region: 2237-2242
-10 region: 2260-2265
Transcription start point: 2272
SV40 origin of replication: 2516-2651
SV40 early promoter
Enhancer (72-bp tandem repeats): 2349-2420 & 2421-2492
21-bp repeats: 2496-2516, 2517-2537 & 2539-2559
Early promoter element: 2572-2578
Major transcription start points: 2568, 2606, 2612 & 2617
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2700-2702
Stop codon: 3492-3494
G->A mutation to remove Pst I site: 2882
C->A (Arg to Ser) mutation to remove BssH II site: 3228
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3730-3735 & 3743-3748
pUC plasmid replication origin: 4079-4722
PhiYFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,951,923; European Pat. 03779067; and other Evrogen Patents and/or Patent applications pending. By use of these Products, you accept the terms and conditions of the applicable Limited Use Label License.
The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839, and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.
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