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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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Vector description
pTagRFP-actin is a mammalian expression vector encoding TagRFP-actin fusion protein (see TagRFP description). The vector can be used for fluorescent labeling of β-actin in living cells.
TagRFP codon usage is optimized for high expression in mammalian cells, i.e. 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 TagRFP-actin coding sequence [Kozak, 1987].
pTagRFP-actin can be used as a source of TagRFP-actin hybrid sequence. The vector backbone contains unique restriction sites that permit it 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 also 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 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
Eukaryotic 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:
Evrogen FP-related products are intended for research use only and covered by 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.
MATERIAL SAFETY DATA SHEET INFORMATION
To the best of our knowledge, these products do not require a Material Safety Data Sheet. However, all the properties of these products (and, if applicable, each of their components) have not been thoroughly investigated. Therefore, we recommend that you use gloves and eye protection, and wear a laboratory coat when working with these products.
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Copyright 2002-2008 Evrogen. All rights reserved. Evrogen JSC, 16/10 Miklukho-Maklaya str., Moscow, Russia, +7(495)4298020, e-mail:evrogen@evrogen.com |