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    pPS-CFP2-N

pPS-CFP2-N vector

cat.# FP802

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
pPS-CFP2-NFP80220 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
ReporterPS-CFP2
Reporter codon usagemammalian
Promoter for PS-CFP2PCMV IE
Host cellsmammalian
Selectionprokaryotic – kanamycin
eukaryotic – neomycin (G418)
Replicationprokaryotic – pUC ori
eukaryotic – SV40 ori
Use PS-CFP2 expression in mammalian cells; generation of fusions to the PS-CFP2 N-terminus
Multiple cloning site (MCS)
Nhe I Bgl II Sac I Hind III EcoR I Sal I Kpn I Apa I BamH I Age I PS-CFP2
Afe I Xho I* Pst I Sac II Sma I/Xma I
GCT A.GC G.CT A.CCG.GAC.TC A.GAT. CT C. GAG. CTC. AAG.CTT. C GA.ATT. C TG.CA G. TCG.AC G.GTA. CC G.C GG. G CC.C G G.G AT.CC A.CCG.GT C.GCC.ACC. ATG.AGC.AAG

* – not unique site.

Vector description

pPS-CFP2-N is a mammalian expression vector encoding cyan-to-green fluorescent protein PS-CFP2 (see reporter description). The vector allows generation of fusions to the PS-CFP2 N-terminus and expression of PS-CFP2 fusions or PS-CFP2 alone in eukaryotic (mammalian) cells.

PS-CFP2 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 PS-CFP2 coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between PCMV IE and PS-CFP2 coding sequence.

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 PS-CFP2 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 PS-CFP2 N-terminus when inserted in the same reading frame as PS-CFP2 and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. PS-CFP2-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express PS-CFP2 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

pPS-CFP2-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of PS-CFP2 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
MCS: 591-671
PS-CFP2
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1393-1395
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1549-1554 & 1578-1583
mRNA 3' ends: 1587 & 1599
f1 single-strand DNA origin: 1646-2101
Bacterial promoter for expression of Kanr gene
-35 region: 2163-2168
-10 region: 2186-2191
Transcription start point: 2198
SV40 origin of replication: 2442-2577
SV40 early promoter
Enhancer (72-bp tandem repeats): 2275-2346 & 2347-2418
21-bp repeats: 2422-2442, 2443-2463 & 2465-2485
Early promoter element: 2498-2504
Major transcription start points: 2494, 2532, 2538 & 2543
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2626-2628
Stop codon: 3418-3420
G->A mutation to remove Pst I site: 2808
C->A (Arg to Ser) mutation to remove BssH II site: 3154
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3656-3661 & 3669-3674
pUC plasmid replication origin: 4005-4648


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:

PS-CFP2-related materials (also referred to as "Products") are intended for research use only. The Products are covered by U.S. Pat. 7,897,726; 7,879,988; 7,667,016; European Pat. 1485481; 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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