pTagRFP-mito vector

cat.# FP147

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.

Download vector information: Data sheet (pdf) Sequence (txt) Restriction map (pdf) Restriction table (pdf)

Product Cat.# Size Price pTagRFP-mito FP147 20 μg € 400 / 200* *50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer. The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. Vector type mammalian expression vector Reporter TagRFP Reporter codon usage mammalian Promoter for TagRFP PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use red (orange) fluorescent labeling of mitochondria

Vector description

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 (P_{CMV IE}) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A) direct proper processing of the 3'-end of the reporter mRNA.

SV40 early promoter (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

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].

Propagation in E. coli

Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 μg/ml) to E. coli hosts. Copy number in E. coli is about 500.

Location of features

P_{CMV IE}: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
TagRFP-mito fusion
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 Kan^r 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