pTagCFP-tubulin vector

cat.# FP115

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 pTagCFP-tubulin FP115 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 TagCFP Reporter codon usage mammalian Promoter for TagCFP PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use cyan fluorescent labeling of α-tubulin filaments

Vector description

pTagCFP-tubulin is a mammalian expression vector encoding TagCFP-tubulin fusion protein (see reporter description). The vector can be used for fluorescent labeling of α-tubulin in living cells.

Transiently transfected 3T3 cells expressing TagCFP-tagged α-tubulin.

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

pTagCFP-tubulin vector can be used as a source of TagCFP-tubulin 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

pTagCFP-tubulin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagCFP-tubulin 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
TagCFP
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 2698-2700
Last amino acid in TagCFP: 1324-1326
Tubulin: 1345-2700
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2861-2866 & 2890-2895
mRNA 3' ends: 2899 & 2911
f1 single-strand DNA origin: 2958-3413
Bacterial promoter for expression of Kan^r gene
-35 region: 3475-3480
-10 region: 3498-3503
Transcription start point: 3510
SV40 origin of replication: 3754-3889
SV40 early promoter
Enhancer (72-bp tandem repeats): 3587-3658 & 3659-3730
21-bp repeats: 3734-3754, 3755-3775 & 3777-3797
Early promoter element: 3810-3816
Major transcription start points: 3806, 3844, 3850 & 3855
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3938-3940
Stop codon: 4730-4732
G->A mutation to remove Pst I site: 4120
C->A (Arg to Ser) mutation to remove BssH II site: 4466
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4968-4973 & 4981-4986
pUC plasmid replication origin: 5317-5960