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    pFusionRed-f-mem

pFusionRed-f-mem vector

cat.# FP418

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-f-memFP41820 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 plasma membrane

Vector description

pFusionRed-f-mem is a mammalian expression vector intended for red fluorescent labeling of plasma membrane in living cells. The vector encodes red fluorescent protein FusionRed (see reporter description) targeted to plasma membrane by 20 amino acid farnesylation signal from c-Ha-Ras [Aronheim et al., 1994; Hancock et al., 1991]. The farnesylation signal is fused to the FusionRed C-terminus.

Two African green monkey kidney fibroblast cells expressing FusionRed targeted to plasma membrane by 20-amino acid farnesylation signal from c-Ha-Ras.

FusionRed codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the FusionRed-f-mem coding sequence [Kozak, 1987].

pFusionRed-f-mem vector can be used as a source of FusionRed-f-mem 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-f-mem vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-f-mem 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
Kozak consensus translation initiation site: 606-616
FusionRed
Start codon (ATG): 613-615
Last amino acid in FusionRed: 1306-1308
Farnesylation signal: 1336-1398
Stop codon: 1396-1398
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1590-1595 & 1306-1624
mRNA 3' ends: 1628 & 1640
f1 single-strand DNA origin: 1687-2142
Bacterial promoter for expression of Kanr gene
-35 region: 2204-2209
-10 region: 2227-2232
Transcription start point: 2239
SV40 origin of replication: 2483-2618
SV40 early promoter
Enhancer (72-bp tandem repeats): 2316-2387 & 2388-2459
21-bp repeats: 2463-2483, 2484-2504 & 2506-2526
Early promoter element: 2539-2545
Major transcription start points: 2535, 2573, 2579 & 2584
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2667-2669
Stop codon: 3459-3461
G->A mutation to remove Pst I site: 2849
C->A (Arg to Ser) mutation to remove BssH II site: 3195
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3697-3702 & 3710-3715
pUC plasmid replication origin: 4046-4689


References:

  • Aronheim A, Engelberg D, Li N, al-Alawi N, Schlessinger J, Karin M. Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway. Cell. 1994; 78 (6):949-61. / pmid: 7923364
  • 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
  • Hancock JF, Cadwallader K, Marshall CJ. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991; 10 (3):641-6. / pmid: 2001678
  • Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987; 15 (20):8125-48. / pmid: 3313277

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.

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