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-Golgi is a mammalian expression vector intended for red (orange) fluorescent labeling of Golgi apparatus in living cells. The vector encodes red (orange) fluorescent protein TagRFP (see reporter description) fused to Golgi targeting sequence (GTS), the fragment of human β-1,4-galactosyltransferase. GTS is fused to the TagRFP N-terminus.
Transiently transfected HeLa cells expressing TagRFP targeted to the Golgi apparatus.
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].
pTagRFP-Golgi vector can be used as a source of TagRFP-GTS 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-Golgi vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagRFP-GTS 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
Golgi targeting sequence (GTS), fragment of human beta 1,4- galactosyltransferase: 597-842
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1730-1735 & 1759-1764
mRNA 3' ends: 1768 & 1780
f1 single-strand DNA origin: 1827-2282
Bacterial promoter for expression of Kanr gene
-35 region: 2344-2349
-10 region: 2367-2372
Transcription start point: 2379
SV40 origin of replication: 2623-2758
SV40 early promoter
Enhancer (72-bp tandem repeats): 2456-2527 & 2528-2599
21-bp repeats: 2603-2623, 2624-2644 & 2646-2666
Early promoter element: 2679-2685
Major transcription start points: 2675, 2713, 2719 & 2724
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2807-2809
Stop codon: 3599-3601
G->A mutation to remove Pst I site: 2989
C->A (Arg to Ser) mutation to remove BssH II site: 3335
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3837-3842 & 3850-3855
pUC plasmid replication origin: 4186-4829
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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