pFusionRed-endo

pFusionRed-endo vector

cat.# FP427

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
pFusionRed-endoFP42720 μg€ 480 / 240*
*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
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 vesicles of the endocytic pathway

Vector description

pFusionRed-endo is a mammalian expression vector intended for red fluorescent labeling of vesicles of the endocytic pathway [Adamson et al., 1992], allowing the monitoring of intracellular membrane traffic during endocytosis in living cells. The vector encodes red fluorescent protein FusionRed (see reporter description) targeted to endosomes by human RhoB GTPase fused to the FusionRed C-terminus. The fusion also contains c-Myc epitope tag.

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(A) – Human endosomes in normal African green monkey kidney fibroblast cells expressing . The compartments are seen shuttling back and forth on their membrane transport pathway between the plasma membrane and lysosomes. Movie from Shemiakina et al., 2012.
(B) – HeLa cell expressing fusion of human RhoB GTPase and c-Myc epitope tag with FusionRed. The fusion protein is localized in the endosomes. 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-endo vector can be used as a source of FusionRed-RhoB 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-endo vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-RhoB 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-Human RhoB GTPase fusion
Start codon (ATG): 613-615
Start of FusionRed coding sequence (ATG): 613-615
Last amino acid in FusionRed: 1306-1308
c-Myc epitope: 1363-1395
Human RhoB GTPase: 1399-1977
Stop codon: 1975-1977
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2169-2174 & 2198-2203
mRNA 3' ends: 2207 & 2219
f1 single-strand DNA origin: 2266-2721
Bacterial promoter for expression of Kanr gene
-35 region: 2783-2788
-10 region: 2806-2811
Transcription start point: 2818
SV40 origin of replication: 3062-3197
SV40 early promoter
Enhancer (72-bp tandem repeats): 2895-2966 & 2967-3038
21-bp repeats: 3042-3062, 3063-3083 & 3085-3105
Early promoter element: 3118-3124
Major transcription start points: 3114, 3152, 3158 & 3163
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3246-3248
Stop codon: 4038-4040
G->A mutation to remove Pst I site: 3428
C->A (Arg to Ser) mutation to remove BssH II site: 3774
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4276-4281 & 4289-4294
pUC plasmid replication origin: 4625-5268


References:

  • Adamson P, Paterson HF, Hall A. Intracellular localization of the P21rho proteins. J Cell Biol. 1992; 119 (3):617-27. / pmid: 1383236
  • 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
  • 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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