pTagRFP-mito is a mammalian expression vector intended for red (orange) fluorescent labeling of mitochondria in living cells. The vector encodes red (orange) fluorescent protein TagRFP (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 TagRFP N-terminus.
Transiently transfected HeLa cells expressing mitochondria-targeted TagRFP.
Image was kindly provided by Michael W. Davidson (Florida State University).
TagRFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].
pTagRFP-mito vector can be used as a source of TagRFP-MTS hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.
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
pTagRFP-mito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagRFP-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 TagRFP coding sequence (ATG): 705-707
Stop codon: 1416-1418
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1571-1576 & 1600-1605
mRNA 3' ends: 1609 & 1621
f1 single-strand DNA origin: 1668-2123
Bacterial promoter for expression of Kanr gene
-35 region: 2185-2190
-10 region: 2208-2213
Transcription start point: 2220
SV40 origin of replication: 2464-2599
SV40 early promoter
Enhancer (72-bp tandem repeats): 2297-2368 & 2369-2440
21-bp repeats: 2444-2464, 2465-2485 & 2487-2507
Early promoter element: 2520-2526
Major transcription start points: 2516, 2554, 2560 & 2565
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2648-2650
Stop codon: 3440-3442
G->A mutation to remove Pst I site: 2830
C->A (Arg to Ser) mutation to remove BssH II site: 3176
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3678-3683 & 3691-3696
pUC plasmid replication origin: 4027-4670
TagRFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,638,615; European Pat. 1994149; 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.
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