RNA-i test vector, p2FP-RNAi

cat.# FP981

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.

Download vector information: Data sheet (pdf) Sequence (txt) Restriction map (pdf) Restriction table (pdf)

Product Cat.# Size Price p2FP-RNAi vector FP981 20 μg € 400 Price does not include delivery. Please contact your local distributor for exact prices and delivery information. Vector type mammalian expression vector Reporters TurboGFP / JRed-Neor fusion Reporter codon usage mammalian Promoters for TurboGFP and JRed-Neor PCMV IE Host cells mammalian Selection prokaryotic - kanamycin / eukaryotic - neomycin (G418) Replication pUC ori Use RNAi test vector

Multiple cloning site

TurboGFP			Bgl II			Sac I				Sal I			Sma I/Xma I
	Stop			Xho I*				Hind III			Kpn I
GT.GAA.GAA.	TGA	.TCC.CGG.A	AG.AT	C.T	C	G.AG	C.TC	A.AGC.TT	C	G.TCG.AC	G.GTA.CC	G.	CCC.GGG.	ACT.

Description

p2FP-RNAi vector is a mammalian expression vector encoding two fluorescent proteins: TurboGFP and JRed-neomycin phosphotransferase II protein fusion (JRed-Neo^r). Both proteins are under the control of immediate early promoter of cytomegalovirus (P_{CMV IE}): the first regulates TurboGFP expression, while the second provides JRed-Neo^r expression. Codon usages of both reporters are optimized for high expression in mammalian cells (Haas et al., 1996). To increase TurboGFP mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of TurboGFP coding sequence (Kozak, 1987).

Multiple cloning site (MCS) is located between the TurboGFP stop codon and SV40 polyadenylation signals (SV40 poly A) allowing to clone a DNA fragment of interest into the TurboGFP 3' untranslated region (3'UTR). SV40 poly A direct proper processing of the 3' end of TurboGFP mRNA.

Note: The plasmid DNA was isolated from dam⁺-methylated E. coli. Therefore some restriction sites are bloked 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 also contains pUC origin of replication for propagation in E. coli, and f1 origin for single-stranded DNA production. Bacterial promoter (PT5/LacO) allows the expression of kanamycin resistance gene (fusion with JRed) in E. coli. Kan^r/Neo^r-JRed fusion gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Expression in mammalian cells

The vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (Gorman, 1985). When transfected into eukaryotic cells in the absence of a functional siRNA, the vector expresses both JRed and TurboGFP proteins. In this case intensity of green fluorescence prevails significantly over the red signal.

TurboGFP expression is knocked down in the presence of siRNA(s) directed against the cloned DNA fragment. JRed expression at that remains unmodified or (in some experimental systems) increases because of translational competition.

Note: Because of lower phosphotransferase activity of the JRed-Neor fusion protein in comparison with neomycin phosphotransferase II alone, working concentration of the neomycin for cultivation of mammalian cells comprising p2FP-RNAi vector must be 2-4 fold lower than standard one for cells with neomycin resistance.

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 (10 mg/ml) to E. coli hosts. Copy number in E. coli is about 500.

Note: The working concentration of the kanamycin should be 2-4 fold lower that standard one for vectors with Kan^r/Neo^r resistance.

Location of features

P_{CMV IE}: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
TurboGFP Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 1309-1311
MCS: 1312-1360
Polyadenylation signals: 1505-1510 & 1534-1539
mRNA 3' ends: 1543 & 1555
f1 single-strand DNA origin: 1602-2057 (Packages the noncoding strand of TurboGFP.)
P_{CMV IE}: 2231-2812
Enhancer region: 2282-2688
TATA box: 2777-2783
Transcription start point: 2806
T5 promoter/lac operator element: 2894-2974
T5 transcription start: 2948
JRed-Neo^r
Start codon (ATG): 3002-3004
JRed: 3038-3760
Kanamycin/neomycin resistance gene: 3785-4576
Stop codon: 4574-4576
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4812-4817 & 4825-4830
pUC plasmid replication origin: 5161-5804

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