pTagGFP2-mito vector

cat.# FP197

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 pTagGFP2-mito FP197 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 TagGFP2 Reporter codon usage mammalian Promoter for TagGFP2 PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use green fluorescent labeling of mitochondria

Vector description

pTagGFP2-mito is a mammalian expression vector intended for green fluorescent labeling of mitochondria in living cells. The vector encodes green fluorescent protein TagGFP2 (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 TagGFP2 N-terminus.

Transiently transfected REF-52 cells expressing mitochondria-targeted TagGFP2.

TagGFP2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996].

pTagGFP2-mito vector can be used as a source of TagGFP2-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

pTagGFP2-mito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagGFP2-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
TagGFP2-mito fusion
Start codon (ATG): 597-599
Mitochondrial targeting sequence (MTS): 597-683
Start of TagGFP2 coding sequence (ATG): 705-707
Stop codon: 1419-1421
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1574-1579 & 1603-1608
mRNA 3' ends: 1612 & 1624
f1 single-strand DNA origin: 1671-2126
Bacterial promoter for expression of Kan^r gene
-35 region: 2188-2193
-10 region: 2211-2216
Transcription start point: 2223
SV40 origin of replication: 2467-2602
SV40 early promoter
Enhancer (72-bp tandem repeats): 2300-2371 & 2372-2443
21-bp repeats: 2447-2467, 2468-2488 & 2490-2510
Early promoter element: 2523-2529
Major transcription start points: 2519, 2557, 2563 & 2568
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2651-2653
Stop codon: 3443-3445
G->A mutation to remove Pst I site: 2833
C->A (Arg to Ser) mutation to remove BssH II site: 3179
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3681-3686 & 3694-3699
pUC plasmid replication origin: 4030-4673