pTagRFP-actin

pTagRFP-actin vector

cat.# FP144

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.


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pTagRFP-actinFP14420 μg€ 400 / 200*
*50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer.
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Vector typemammalian expression vector
ReporterTagRFP
Reporter codon usagemammalian
Promoter for TagRFPPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use red (orange) fluorescent labeling of β-actin filaments

Vector description

pTagRFP-actin is a mammalian expression vector encoding TagRFP-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 TagRFP fusion with β-actin.

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]. Human cytoplasmic β-actin is fused to the TagRFP C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the TagRFP-actin coding sequence [Kozak, 1987].

pTagRFP-actin vector can be used as a source of TagRFP-actin 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 E. coli. Therefore some restriction sites are blocked by methylation. If you wish to digest the vector using such sites you will need to transform the vector into a dam- host and make fresh DNA.

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 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 (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 E. coli. Kanr/Neor gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.


Expression in mammalian cells

pTagRFP-actin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagRFP-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

PCMV 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
Beta-Actin: 1345-2472
Stop codon: 2470-2472
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2633-2638 & 2662-2667
mRNA 3' ends: 2671 & 2683
f1 single-strand DNA origin: 2730-3185
Bacterial promoter for expression of Kanr gene
-35 region: 3247-3252
-10 region: 3270-3275
Transcription start point: 3282
SV40 origin of replication: 3526-3661
SV40 early promoter
Enhancer (72-bp tandem repeats): 3359-3430 & 3431-3502
21-bp repeats: 3506-3526, 3527-3547 & 3549-3569
Early promoter element: 3582-3588
Major transcription start points: 3578, 3616, 3622 & 3627
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3710-3712
Stop codon: 4502-4504
G->A mutation to remove Pst I site: 3892
C->A (Arg to Ser) mutation to remove BssH II site: 4238
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4740-4745 & 4753-4758
pUC plasmid replication origin: 5089-5732


References:

  • Gorman C. High efficiency gene transfer into mammalian cells. In DNA cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.). 1985; 143-90.
  • Haas J, Park EC, Seed B. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol. 1996; 6 (3):315-24. / pmid: 8805248
  • Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987; 15 (20):8125-48. / pmid: 3313277

Notice to Purchaser:

TagRFP-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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