pmKate2-annexin

pmKate2-annexin vector

cat.# FP321

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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pmKate2-annexinFP32120 μg€ 400 / 200*
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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 far-red fluorescent labeling of annexin A4

Vector description

pmKate2-annexin is a mammalian expression vector encoding mKate2-annexin fusion protein (see reporter description). The vector can be used for fluorescent labeling of annexin A4 in living cells.

Translocation of mKate2 fusion with human annexin A4 from the cytoplasm to the plasma and nuclear membranes upon induction with ionomycin.

Human cervical adenocarcinoma cells (HeLa).
Scale bar represents 10 μm. Image from Shcherbo et al., 2009.

mKate2 codon usage is optimized for high expression in mammalian cells (humanized) [Haas et al., 1996]. Human annexin A4 is fused to the mKate2 C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2-annexin coding sequence [Kozak, 1987].

pmKate2-annexin vector can be used as a source of mKate2-annexin 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

pmKate2-annexin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2-annexin 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
mKate2-annexin fusion: 603-2313
mKate2: 603-1314
Start codon (ATG): 603-605
Last amino acid in mKate2: 1312-1314
Annexin A4: 1351-2313
Stop codon: 2314-2316
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2477-2482 & 2506-2511
mRNA 3' ends: 2515 & 2527
f1 single-strand DNA origin: 2574-3029
Bacterial promoter for expression of Kanr gene
-35 region: 3091-3096
-10 region: 3114-3119
Transcription start point: 3126
SV40 origin of replication: 3370-3505
SV40 early promoter
Enhancer (72-bp tandem repeats): 3203-3274 & 3275-3346
21-bp repeats: 3350-3370, 3371-3391 & 3393-3413
Early promoter element: 3426-3432
Major transcription start points: 3422, 3460, 3466 & 3471
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3554-3556
Stop codon: 4346-4348
G->A mutation to remove Pst I site: 3736
C->A (Arg to Ser) mutation to remove BssH II site: 4082
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4584-4589 & 4597-4602
pUC plasmid replication origin: 4933-5576


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
  • Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM. Far-red fluorescent tags for protein imaging in living tissues. Biochem J. 2009; 418 (3):567-74. doi: 10.1042/BJ20081949 / pmid: 19143658

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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