pTurboRFP-mito

pTurboRFP-mito vector

cat.# FP237

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.


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pTurboRFP-mitoFP23720 μg€ 320 / 160*
*50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer.
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Vector typemammalian expression vector
ReporterTurboRFP
Reporter codon usagemammalian
Promoter for TurboRFPPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use red (orange) fluorescent labeling of mitochondria

Vector description

pTurboRFP-mito is a mammalian expression vector intended for red (orange) fluorescent labeling of mitochondria in living cells. The vector encodes red (orange) fluorescent protein TurboRFP (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 TurboRFP N-terminus.

Transiently transfected HeLa cells expressing mitochondria-targeted TurboRFP.

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

pTurboRFP-mito vector can be used as a source of TurboRFP-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 E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.

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 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 (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 E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.


Expression in mammalian cells

pTurboRFP-mito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TurboRFP-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

PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
TurboRFP-mito fusion
Start codon (ATG): 597-599
Mitochondrial targeting sequence (MTS): 597-683
Start of TurboRFP coding sequence (ATG): 705-707
Stop codon (TGA): 1398
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1554-1559 & 1583-1588
mRNA 3' ends: 1592 & 1604
f1 single-strand DNA origin: 1651-2106
Bacterial promoter for expression of Kanr gene
-35 region: 2168-2173
-10 region: 2191-2196
Transcription start point: 2203
SV40 origin of replication: 2447-2582
SV40 early promoter
Enhancer (72-bp tandem repeats): 2280-2351 & 2352-2423
21-bp repeats: 2427-2447, 2448-2468 & 2470-2490
Early promoter element: 2503-2509
Major transcription start points: 2499, 2537, 2543 & 2548
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2631-2633
Stop codon: 3423-3425
G->A mutation to remove Pst I site: 2813
C->A (Arg to Ser) mutation to remove BssH II site: 3159
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3661-3666 & 3674-3679
pUC plasmid replication origin: 4010-4653


References:

  • Gorman C. High efficiency gene transfer into mammalian cells. In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.). 1985; 143-90.
  • Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996; 6 (3):315-24. / pmid: 8805248
  • Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol. 1995; 5 (6):635-42. / pmid: 7552174
  • Rizzuto R, Nakase H, Darras B, Francke U, Fabrizi GM, Mengel T, Walsh F, Kadenbach B, DiMauro S, Schon EA. A gene specifying subunit VIII of human cytochrome c oxidase is localized to chromosome 11 and is expressed in both muscle and non-muscle tissues. J Biol Chem. 1989; 264 (18):10595-600. / pmid: 2543673

Notice to Purchaser:

TurboRFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 8,138,320; 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.

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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