pFusionRed-annexin

pFusionRed-annexin vector

cat.# FP414

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
pFusionRed-annexinFP41420 μg€ 480 / 240*
*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
ReporterFusionRed
Reporter codon usagemammalian
Promoter for FusionRedPCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use red fluorescent labeling of annexin A4

Vector description

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

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(A) – FusionRed fused to human annexin A4 in human bone osteosarcoma cells during translocation from the cytoplasm to the plasma and nuclear membranes upon induction with ionomycin. Movie from Shemiakina et al., 2012.
(B) – Translocation of FusionRed fusion with human annexin A4 from the cytoplasm to the plasma and nuclear membranes upon induction with ionomycin. HeLa cells. Scale bar represents 10 μm. Data from Shemiakina et al., 2012

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

pFusionRed-annexin vector can be used as a source of FusionRed-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

pFusionRed-annexin vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the FusionRed-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
FusionRed-annexin fusion: 613-2322
FusionRed: 613-1308
Start codon (ATG): 613-615
Last amino acid in FusionRed: 1306-1308
Annexin A4: 1357-2322
Stop codon: 2320-2322
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2483-2488 & 2512-2517
mRNA 3' ends: 2521 & 2533
f1 single-strand DNA origin: 2580-3035
Bacterial promoter for expression of Kanr gene
-35 region: 3097-3102
-10 region: 3120-3125
Transcription start point: 3132
SV40 origin of replication: 3376-3511
SV40 early promoter
Enhancer (72-bp tandem repeats): 3209-3280 & 3281-3352
21-bp repeats: 3356-3376, 3377-3397 & 3399-3419
Early promoter element: 3432-3438
Major transcription start points: 3428, 3466, 3472 & 3477
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3560-3562
Stop codon: 4352-4354
G->A mutation to remove Pst I site: 3742
C->A (Arg to Ser) mutation to remove BssH II site: 4088
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4590-4595 & 4603-4608
pUC plasmid replication origin: 4939-5582


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
  • Shemiakina II, Ermakova GV, Cranfill PJ, Baird MA, Evans RA, Souslova EA, Staroverov DB, Gorokhovatsky AY, Putintseva EV, Gorodnicheva TV, Chepurnykh TV, Strukova L, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM, Shcherbo D. A monomeric red fluorescent protein with low cytotoxicity. Nat Commun. 2012; 3 :1204. doi: 10.1038/ncomms2208 / pmid: 23149748

Notice to Purchaser:

FusionRed-related materials (also referred to as "Products") are intended for research use only. The Products are covered by 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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