pTagRFP-profilin vector

cat.# FP371

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 pTagRFP-profilin FP371 20 μg € 400 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 TagRFP Reporter codon usage mammalian Promoter for TagRFP PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use red (orange) fluorescent labeling of profilin

Vector description

pTagRFP-profilin is a mammalian expression vector encoding TagRFP-profilin fusion protein (see reporter description). The vector can be used for fluorescent labeling of profilin in living cells.

Transiently transfected HeLa cells expressing TagRFP fusion with profilin. 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]. Mouse profilin is fused to the TagRFP C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TagRFP-profilin coding sequence [Kozak, 1987].

pTagRFP-profilin vector can be used as a source of TagRFP-profilin 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

pTagRFP-profilin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagRFP-profilin 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
TagRFP
Start codon (ATG): 613-615
Last amino acid in TagRFP: 1321-1323
Profilin: 1354-1776
Stop codon: 1774-1776
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1937-1942 & 1966-1971
mRNA 3' ends: 1975 & 1987
f1 single-strand DNA origin: 2034-2489
Bacterial promoter for expression of Kan^r gene
-35 region: 2551-2556
-10 region: 2574-2579
Transcription start point: 2586
SV40 origin of replication: 2830-2965
SV40 early promoter
Enhancer (72-bp tandem repeats): 2663-2734 & 2735-2806
21-bp repeats: 2810-2830, 2831-2851 & 2853-2873
Early promoter element: 2886-2892
Major transcription start points: 2882, 2920, 2926 & 2931
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3014-3016
Stop codon: 3806-3808
G->A mutation to remove Pst I site: 3196
C->A (Arg to Ser) mutation to remove BssH II site: 3542
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4044-4049 & 4057-4062
pUC plasmid replication origin: 4393-5036