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.
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.
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.
Note: The plasmid DNA was isolated from dam+-methylated
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-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].
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
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 Kanr 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
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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