pTagBFP-C vector

The vector sequence has been compiled using the informa-

tion from sequence databases, published literature, and other Product Cat.# Size
sources, together with partial sequences obtained by Evrogen.
This vector has not been completely sequenced.
pTagBFP-C vector FP171 20 µg
PCMV IE
pUC ori Vector type mammalian expression vector
TagBFP Reporter TagBFP
HSV TK poly A
Reporter codon usage mammalian
pTagBFP-C vector,
Promoter for TagBFP PCMV IE
4.7kb MCS
Host cells mammalian
Kanr /Neor SV40 poly A
Selection prokaryotic - kanamycin
fl ori
PSV40 eukaryotic - neomycin (G418)
SV40 ori P
Replication prokaryotic - pUC ori
eukaryotic - SV40 ori
For vector sequence, please visit our Web site at
http://www.evrogen.com/products/vectors.shtml Use TagBFP expression in mammalian cells; generation of
fusions to the TagBFP C-terminus
Multiple cloning site (MCS)
Bgl II Sac I EcoR I Sal I Sac II Sma I/Xma I Xba I# Bcl I#
TagBFP BspE I Xho I Hind III* Pst I Kpn I Apa I BamH I STOPs

... TCC.GGA.CTC.AGA.TCT.CGA.GCT.CAA.GCT.TCG.AAT.TCT.GCA.GTC.GAC.GGT.ACC.GCG.GGC.CCG.GGA.TCC.ACC.GGA.TCT.AGA.TAA.CTG.ATC.A ...

S G L R S R A Q A S N S A V D G T A G P G S T G S R * L I
*
− not unique sites.
# − sites are blocked by dam methylation. If you wish to digest the vector with these enzymes, you will need to transform the vector into a dam- host and make fresh DNA.

Location of features Vector description

PCMV IE : 1-589 pTagBFP-C is a mammalian expression vector encoding blue fluorescent protein TagBFP. The vector allows gen-
Enhancer region: 59-465 eration of fusions to the TagBFP C-terminus and expression of TagBFP fusions or TagBFP alone in eukaryotic
TATA box: 554-560
(mammalian) cells.
Transcription start point: 583
TagBFP TagBFP codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al. 1996]. To
Kozak consensus translation initiation site: 600-610 increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of
Start codon (ATG): 607-609; Stop codon: 1384-1386 the TagBFP coding sequence [Kozak 1987]. Multiple cloning site (MCS) is located between TagBFP coding
Last amino acid in TagRFP: 1303-1305 sequence and SV40 polyadenylation signal (SV40 polyA).
MCS: 1306-1383
SV40 early mRNA polyadenylation signal The vector backbone contains immediate early promoter of cytomegalovirus (PCMV IE ) for protein expression,
Polyadenylation signals: 1526-1531 & 1555-1560 SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propa-
mRNA 3’ ends: 1564 & 1576 gation in E. coli, and f1 origin for single-stranded DNA production. SV40 polyadenylation signals (SV40 poly A)
f1 single-strand DNA origin: 1623-2078 direct proper processing of the 3’-end of the reporter mRNA.
Bacterial promoter for expression of Kanr gene
-35 region: 2140-2145; -10 region: 2163-2168 SV40 early promoter (PSV40 ) provides neomycin resistance gene (Neor ) expression to select stably transfected
Transcription start point: 2175 eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kanr )
SV40 origin of replication: 2419-2554 in E. coli. Kanr /Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation
SV40 early promoter
signals.
Enhancer (72-bp tandem repeats): 2252-2323 & 2324-
2395
Generation of TagBFP fusion proteins
21-bp repeats: 2399-2419, 2420-2440 & 2442-2462
Early promoter element: 2475-2481 A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a
Major transcription start points: 2471, 2509, 2515 & fusion to the TagBFP C-terminus when inserted in the same reading frame as TagBFP and no in-frame stop
2520 codons are present. TagBFP-tagged fusions retain fluorescent properties of the native protein allowing fusion
Kanamycin/neomycin resistance gene
localization in vivo. Unmodified vector will express TagBFP when transfected into eukaryotic (mammalian)
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2603-2605; Stop codon: 3395-3397 cells.
G->A mutation to remove Pst I site: 2785 Note: The plasmid DNA was isolated from dam+ -methylated E.coli. Therefore some restriction sites are blocked by methylation. If you wish to
C->A (Arg to Ser) mutation to remove BssH II site: 3131 digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.
Herpes simplex virus (HSV) thymidine kinase (TK)
polyadenylation signal
Expression in mammalian cells
Polyadenylation signals: 3633-3638 & 3646-3651 pTagBFP-C vector can be transfected into mammalian cells by any known transfection method. CMV pro-
pUC plasmid replication origin: 3982-4625 moter provides strong, constitutive expression of TagBFP or its fusions in eukaryotic cells. If required, stable
transformants can be selected using G418 [Gorman 1985].

Propagation in E. coli
References Suitable host strains for propagation in E. coli include DH5alpha, HB101, XL1-Blue, and other general purpose
Gorman, C. (1985). “High efficiency gene transfer into strains. Plasmid incompatibility group is pMB1/ColE1. The vector confers resistance to kanamycin (30 µg/ml)
mammalian cells.” In: DNA cloning: A Practical Ap- to E. coli hosts. Copy number in E. coli is about 500.
proach, Vol. II. Ed. by Glover. (IRL Press, Oxford, U.K.)
Pp. 143–190.
Haas, J. et al. (1996) “Codon usage limitation in the
expression of HIV-1 envelope glycoprotein.” Curr Biol,
6 (3): 315–324 / pmid: 8805248
Kozak, M. (1987) “An analysis of 5’-noncoding se-
quences from 699 vertebrate messenger RNAs.” Nu-
cleic Acids Res, 15 (20): 8125–8148 / pmid: 3313277

Notice to Purchaser:
TagBFP-related materials (also referred to as "Products") are intended for research use only.
The Products are covered by 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 #001: http://www.evrogen.com/products/Evrogen-FP-license.shtml.
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.
MSDS information is available at http://www.evrogen.com/MSDS.shtml