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.
pTagFP635-Cx26 is a mammalian expression vector encoding TagFP635-Cx26 fusion protein (see reporter description). The vector can be used for fluorescent labeling of connexin 26 in living cells.
Transiently transfected HeLa cells expressing TagFP635 fusion with connexin 26.
Image was kindly provided by Michael W. Davidson (Florida State University).
TagFP635 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Rat connexin 26 is fused to the TagFP635 N-terminus.
pTagFP635-Cx26 vector can be used as a source of TagFP635-Cx26 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
pTagFP635-Cx26 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagFP635-Cx26 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
Connexin 26: 683-1360
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2248-2253 & 2277-2282
mRNA 3' ends: 2286 & 2298
f1 single-strand DNA origin: 2345-2800
Bacterial promoter for expression of Kanr gene
-35 region: 2862-2867
-10 region: 2885-2890
Transcription start point: 2897
SV40 origin of replication: 3141-3276
SV40 early promoter
Enhancer (72-bp tandem repeats): 2974-3045 & 3046-3117
21-bp repeats: 3121-3141, 3142-3162 & 3164-3184
Early promoter element: 3197-3203
Major transcription start points: 3193, 3231, 3237 & 3242
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3325-3327
Stop codon: 4117-4119
G->A mutation to remove Pst I site: 3507
C->A (Arg to Ser) mutation to remove BssH II site: 3853
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4355-4360 & 4368-4373
pUC plasmid replication origin: 4704-5347
TagFP635-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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