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    pTagYFP-tubulin

pTagYFP-tubulin vector

cat.# FP135

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 information:
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pTagYFP-tubulinFP13520 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
ReporterTagYFP
Reporter codon usagemammalian
Promoter for TagYFPPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use yellow fluorescent labeling of α-tubulin filaments

Vector description

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

Stably transfected MDCK canine kidney epithelial cells expressing TagYFP-tagged α-tubulin.

Image was kindly provided by Dr. Christian Petzelt (Marinpharm).

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

pTagYFP-tubulin vector can be used as a source of TagYFP-tubulin 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

pTagYFP-tubulin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the TagYFP-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

PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
TagYFP
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 2701-2703
Last amino acid in TagYFP: 1327-1329
Tubulin: 1348-2703
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2864-2869 & 2893-2898
mRNA 3' ends: 2902 & 2914
f1 single-strand DNA origin: 2961-3416
Bacterial promoter for expression of Kanr gene
-35 region: 3478-3483
-10 region: 3501-3506
Transcription start point: 3513
SV40 origin of replication: 3757-3892
SV40 early promoter
Enhancer (72-bp tandem repeats): 3590-3661 & 3662-3733
21-bp repeats: 3737-3757, 3758-3778 & 3780-3800
Early promoter element: 3813-3819
Major transcription start points: 3809, 3847, 3853 & 3858
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3941-3943
Stop codon: 4733-4735
G->A mutation to remove Pst I site: 4123
C->A (Arg to Ser) mutation to remove BssH II site: 4469
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4971-4976 & 4984-4989
pUC plasmid replication origin: 5320-5963


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:

TagYFP-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,888,113; European Pat. 06809023; 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.

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