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.
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
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
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].
Suitable host strains for propagation in
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
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
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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