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    pKillerRed-dMito

pKillerRed-dMito vector

cat.# FP964

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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vector information:
ProductCat.#SizePrice
pKillerRed-dMitoFP96420 μg€ 400 / 200*
*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
ReporterKillerRed
Reporter codon usagemammalian
Promoter for KillerRedPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use Expression of mitochondria-targeted KillerRed in mammalian cells under the control of CMV promoter; source of mitochondria-targeted KillerRed coding sequence

Vector description

pKillerRed-dMito is a mammalian expression vector encoding mitochondria-targeted KillerRed (see reporter description). KillerRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Duplicated mitochondrial targeting sequence (MTS) is fused to the KillerRed N-terminus. MTS was derived from the subunit VIII of human cytochrome C oxidase [Rizzuto et al., 1989; Rizzuto et al., 1995].

pKillerRed-dMito vector can be used as a source of dMTS-KillerRed hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. Alternatively, dMTS-KillerRed coding sequence can be amplified by PCR.

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

pKillerRed-dMito vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mitochondria-targeted KillerRed in eukaryotic cells. If required, stable transformants can be selected using G418 [Gorman, 1985].

Note: KillerRed shows no cell toxic effects before light activation. Upon green light irradiation KillerRed generates reactive oxygen species (ROS) that damage the neighboring molecules.


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
KillerRed-dMito fusion
Start codon (ATG): 597-599
Mitochondrial localization signal 1 (MLS-1): 597-689
Mitochondrial localization signal 2 (MLS-2): 690-782
Start of KillerRed coding sequence (ATG): 798-800
Stop codon: 1515-1517
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1671-1676 & 1700-1705
mRNA 3' ends: 1709 & 1721
f1 single-strand DNA origin: 1768-2223
Bacterial promoter for expression of Kanr gene
-35 region: 2285-2290
-10 region: 2308-2313
Transcription start point: 2320
SV40 origin of replication: 2564-2699
SV40 early promoter
Enhancer (72-bp tandem repeats): 2397-2468 & 2469-2540
21-bp repeats: 2544-2564, 2565-2585 & 2587-2607
Early promoter element: 2620-2626
Major transcription start points: 2616, 2654, 2660 & 2665
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2748-2750
Stop codon: 3540-3542
G->A mutation to remove Pst I site: 2930
C->A (Arg to Ser) mutation to remove BssH II site: 3276
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3778-3783 & 3791-3796
pUC plasmid replication origin: 4127-4770


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:

KillerRed-related materials (also referred to as "Products") are intended for research use only.

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