pFusionRed-ER

pFusionRed-ER vector

cat.# FP420

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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pFusionRed-ERFP42020 μg€ 480 / 240*
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Vector typemammalian expression vector
ReporterFusionRed
Reporter codon usagemammalian
Promoter for FusionRedPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use red fluorescent labeling of the lumen of the endoplasmic reticulum

Vector description

pFusionRed-ER is a mammalian expression vector intended for red fluorescent labeling of the lumen of the endoplasmic reticulum (ER) [Roderick et al., 1997]. The vector encodes red fluorescent protein FusionRed (see reporter description) containing ER targeting signal (calreticulin signal sequence [Fliegel et al., 1989]) fused to the FusionRed N-terminus and ER retention signal (KDEL sequence [Munro and Pelham, 1987]) fused to the FusionRed C-terminus.

HeLa cell expressing fusion of human calreticulin ER targeting signal and KDEL peptide with FusionRed.

The fusion protein is localized in the lumen of the endoplasmic reticulum.
Scale bar represents 10 μm. Data from Shemiakina et al., 2012

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

pFusionRed-ER vector can be used as a source of FusionRed-ER 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

pFusionRed-ER vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-ER 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
FusionRed-ER fusion: 603-1397
Start codon (ATG): 603-605
Calreticulin signal sequence: 603-653
FusionRed: 669-1364
ER retention sequence (KDEL): 1383-1394
Stop codon: 1395-1397
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1589-1594 & 1618-1623
mRNA 3' ends: 1627 & 1639
f1 single-strand DNA origin: 1686-2141
Bacterial promoter for expression of Kanr gene
-35 region: 2203-2208
-10 region: 2226-2231
Transcription start point: 2238
SV40 origin of replication: 2482-2617
SV40 early promoter
Enhancer (72-bp tandem repeats): 2315-2386 & 2387-2458
21-bp repeats: 2462-2482, 2483-2503 & 2505-2525
Early promoter element: 2538-2544
Major transcription start points: 2534, 2572, 2578 & 2583
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2666-2668
Stop codon: 3458-3460
G->A mutation to remove Pst I site: 2848
C->A (Arg to Ser) mutation to remove BssH II site: 3194
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3696-3701 & 3709-3714
pUC plasmid replication origin: 4045-4688


References:

  • Fliegel L, Burns K, MacLennan DH, Reithmeier RA, Michalak M. Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1989; 264 (36):21522-8. / pmid: 2600080
  • 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
  • Munro S, Pelham HR. A C-terminal signal prevents secretion of luminal ER proteins. Cell. 1987; 48 (5):899-907. / pmid: 3545499
  • Roderick HL, Campbell AK, Llewellyn DH. Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors. FEBS Lett. 1997; 405 (2):181-5. / pmid: 9089287
  • Shemiakina II, Ermakova GV, Cranfill PJ, Baird MA, Evans RA, Souslova EA, Staroverov DB, Gorokhovatsky AY, Putintseva EV, Gorodnicheva TV, Chepurnykh TV, Strukova L, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM, Shcherbo D. A monomeric red fluorescent protein with low cytotoxicity. Nat Commun. 2012; 3 :1204. doi: 10.1038/ncomms2208 / pmid: 23149748

Notice to Purchaser:

FusionRed-related materials (also referred to as "Products") are intended for research use only. The Products are covered by 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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