pFusionRed-Rab5a vector

cat.# FP431

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-Rab5a FP431 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 Rab-5A protein

Vector description

pFusionRed-Rab5a is a mammalian expression vector encoding FusionRed-Rab5a fusion protein (see reporter description). The vector can be used for fluorescent labeling of Rab-5A protein in living cells.

HeLa cell expressing fusion of canine Rab5a 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]. Human Ras-related protein Rab-5A is fused to the FusionRed C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the FusionRed-Rab5a coding sequence [Kozak, 1987].

pFusionRed-Rab5a vector can be used as a source of FusionRed-Rab5a 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-Rab5a vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-Rab5a 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

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
Ras-related protein Rab-5A: 1342-1989
Stop codon: 1987-1989
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2150-2155 & 2179-2184
mRNA 3' ends: 2188 & 2200
f1 single-strand DNA origin: 2247-2702
Bacterial promoter for expression of Kan^r gene
-35 region: 2764-2769
-10 region: 2787-2792
Transcription start point: 2799
SV40 origin of replication: 3043-3178
SV40 early promoter
Enhancer (72-bp tandem repeats): 2876-2947 & 2948-3019
21-bp repeats: 3023-3043, 3044-3064 & 3066-3086
Early promoter element: 3099-3105
Major transcription start points: 3095, 3133, 3139 & 3144
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3227-3229
Stop codon: 4019-4021
G->A mutation to remove Pst I site: 3409
C->A (Arg to Ser) mutation to remove BssH II site: 3755
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4257-4262 & 4270-4275
pUC plasmid replication origin: 4606-5249