pmKate2-C

pmKate2-C vector

cat.# FP181

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:
ProductCat.#SizePrice
pmKate2-CFP18120 μg€ 400 / 200*
*50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer.
The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information.

Vector typemammalian expression vector
ReportermKate2
Reporter codon usagemammalian
Promoter for mKate2PCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use mKate2 expression in mammalian cells; generation of fusions to the mKate2 C-terminus
Multiple cloning site (MCS)
mKate2 Bgl II Sac I EcoR I Sal I Kpn I Apa I BamH I STOPs
BspE I Xho I Hind III Pst I Sac II Sma I/Xma I Xba I# Bcl I#
        ... GGT.GGA.GGA.GGT. TCC.GGA .CTC. AGA. T CT. C GA.G CT.C AA.GCT.T C G.AAT.T C T.GCA. G TC.GAC. GGT.A CC .GC G.G G C.CC G. GG A.TCC .ACC.GGA. TC T.AG A. TAA .C TG.A TC.A

# – 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.

Vector description

pmKate2-C is a mammalian expression vector encoding far-red fluorescent protein mKate2 (see reporter description). The vector allows generation of fusions to the mKate2 C-terminus and expression of mKate2 fusions or mKate2 alone in eukaryotic (mammalian) cells.

mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2 coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between mKate2 coding sequence and SV40 polyadenylation signal (SV40 polyA).

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.


Generation of mKate2 fusion proteins

A localization signal or a gene of interest can be cloned into MCS of the vector. It will be expressed as a fusion to the mKate2 C-terminus when inserted in the same reading frame as mKate2 and no in-frame stop codons are present. mKate2-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express mKate2 when transfected into eukaryotic (mammalian) cells.

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.


Expression in mammalian cells

pmKate2-C vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mKate2 or its fusions 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
mKate2
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 1399-1401
Last amino acid in mKate2: 1306-1308
MCS: 1321-1398
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1541-1546 & 1570-1575
mRNA 3' ends: 1579 & 1591
f1 single-strand DNA origin: 1638-2093
Bacterial promoter for expression of Kanr gene
-35 region: 2155-2160
-10 region: 2178-2183
Transcription start point: 2190
SV40 origin of replication: 2434-2569
SV40 early promoter
Enhancer (72-bp tandem repeats): 2267-2338 & 2339-2410
21-bp repeats: 2414-2434, 2435-2455 & 2457-2477
Early promoter element: 2490-2496
Major transcription start points: 2486, 2524, 2530 & 2535
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2618-2620
Stop codon: 3410-3412
G->A mutation to remove Pst I site: 2800
C->A (Arg to Ser) mutation to remove BssH II site: 3146
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3648-3653 & 3661-3666
pUC plasmid replication origin: 3997-4640


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:

mKate2-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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