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.

Download vector information: Data sheet (pdf) Sequence (txt) Restriction map (pdf) Restriction table (pdf)

Product Cat.# Size Price pFusionRed-f-mem FP418 20 μg € 480 / 240* *50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer. The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. Vector type mammalian expression vector Reporter FusionRed Reporter codon usage mammalian Promoter for FusionRed PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – 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.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV 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 (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r 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

P_{CMV 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 Kan^r 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