pPhi-Yellow-PRL vector

cat.# FP604

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 pPhi-Yellow-PRL FP604 20 μg € 400.00 The price does not include delivery. The price varies in different countries. Please contact your local distributor for exact prices and delivery information. Vector type promoterless expression vector Reporter PhiYFP Reporter codon usage mammalian Promoter for PhiYFP NO Host cells mammalian, prokaryotic Selection prokaryotic - kanamycin eukaryotic - neomycin (G418) Replication prokaryotic - pUC ori eukaryotic - SV40 ori Use Monitoring transcription from different promoters and promoter/enhancer combinations

Multiple cloning site (MCS)

			Bgl II*			Sac I		Hind III		EcoR I			Sal I		Kpn I			Apa I				BamH I		Age I		PhiYFP
	Afe I			Xho I							Pst I					Sac II*				Sma I/Xma I
...T	A.GCG.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...

Vector description

pPhi-Yellow-PRL is a promoterless vector encoding yellow fluorescent protein, PhiYFP (see protein description), which can be used as in vivo reporter of gene expression. PhiYFP 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 PhiYFP coding sequence [Kozak, 1987].

Multiple cloning site (MCS) is located upstream of the upstream of the Kozak consensus translation initiation site and can be used to clone a promoter or a promoter/enchancer combination of interest. Without the addition of a functional promoter, this vector will not express PhiYFP.

The vector backbone contains 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 (P_SV40) provides neomycin resistance gene (Neo^r) expression to select stably transfected eukaryotic cells using G418. Bacterial promoter (P) provides kanamycin resistance gene expression (Kan^r) in E. coli. Kan^r/Neo^r gene is linked with herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signals.

Expression in mammalian cells

pPhi-Yellow-PRL can be transfected into mammalian cells by any known transfection method. 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

MCS: 12-89
PhiYFP
Kozak consensus translation initiation site: 90-100
Start codon (ATG): 97-99
Stop codon: 799-801
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1015-1020 & 1044-1049
mRNA 3' ends: 1053 & 1065
f1 single-strand DNA origin: 1112-1567
Eukaryotic promoter for expression of Kan^r gene
-35 region: 1629-1634
-10 region: 1652-1657
Transcription start point: 1664
SV40 origin of replication: 1908-2043
SV40 early promoter
Enhancer (72-bp tandem repeats): 1741-1812 & 1813-1884
21-bp repeats: 1888-1908, 1909-1929 & 1931-1951
Early promoter element: 1964-1970
Major transcription start points: 1960, 1998, 2004 & 2009
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2092-2094
Stop codon: 2884-2886
G->A mutation to remove Pst I site: 2274
C->A (Arg to Ser) mutation to remove BssH II site: 2620
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3122-3127 & 3135-3140
pUC plasmid replication origin: 3471-4114