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.
pTagRFP-vinculin is a mammalian expression vector encoding TagRFP-vinculin fusion protein (see reporter description). The vector can be used for fluorescent labeling of vinculin in living cells.
Transiently transfected HeLa cells expressing TagRFP-vinculin fusion.
Image was kindly provided by Michael W. Davidson (Florida State University).
TagRFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human vinculin is fused to the TagRFP C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TagRFP-vinculin coding sequence [Kozak, 1987].
pTagRFP-vinculin vector can be used as a source of TagRFP-vinculin 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
pTagRFP-vinculin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagRFP-vinculin 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 TagRFP: 1321-1323
Stop codon: 4591-4593
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 4746-4751 & 4775-4780
mRNA 3' ends: 4784 & 4796
f1 single-strand DNA origin: 4843-5298
Bacterial promoter for expression of Kanr gene
-35 region: 5360-5365
-10 region: 5383-5388
Transcription start point: 5395
SV40 origin of replication: 5639-5774
SV40 early promoter
Enhancer (72-bp tandem repeats): 5472-5543 & 5544-5615
21-bp repeats: 5619-5639, 5640-5660 & 5662-5682
Early promoter element: 5695-5701
Major transcription start points: 5691, 5729, 5735 & 5740
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 5823-5825
Stop codon: 6615-6617
G->A mutation to remove Pst I site: 6005
C->A (Arg to Ser) mutation to remove BssH II site: 6351
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 6853-6858 & 6866-6871
pUC plasmid replication origin: 7202-7845
TagRFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,638,615; 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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