pTagBFP-actin vector

cat.# FP174

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 pTagBFP-actin FP174 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 TagBFP Reporter codon usage mammalian Promoter for TagBFP PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use blue fluorescent labeling of β-actin filaments

Vector description

pTagBFP-actin is a mammalian expression vector encoding TagBFP-actin fusion protein (see reporter description). The vector can be used for fluorescent labeling of β-actin in living cells.

Transiently transfected HeLa cells expressing TagBFP-tagged β-actin.

TagBFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human cytoplasmic β-actin is fused to the TagBFP C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TagBFP-actin coding sequence [Kozak, 1987].

pTagBFP-actin vector can be used as a source of TagBFP-actin 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

pTagBFP-actin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagBFP-actin 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
TagBFP- beta-Actin fusion: 607-2454
Kozak consensus translation initiation site: 600-610
Start codon (ATG): 607-609
Last amino acid in TagBFP: 1303-1305
beta-Actin: 1327-2454
Stop codon: 2452-2454
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2615-2620 & 2644-2649
mRNA 3' ends: 2653 & 2665
f1 single-strand DNA origin: 2712-3167
Bacterial promoter for expression of Kan^r gene
-35 region: 3229-3234
-10 region: 3252-3257
Transcription start point: 3264
SV40 origin of replication: 3508-3643
SV40 early promoter
Enhancer (72-bp tandem repeats): 3341-3412 & 3413-3484
21-bp repeats: 3488-3508, 3509-3529 & 3531-3551
Early promoter element: 3564-3570
Major transcription start points: 3560, 3598, 3604 & 3609
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3692-3694
Stop codon: 4484-4486
G->A mutation to remove Pst I site: 3874
C->A (Arg to Ser) mutation to remove BssH II site: 4220
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4722-4727 & 4735-4740
pUC plasmid replication origin: 5071-5714