pPhi-Yellow-PRL-dest1 vector

cat.# FP605

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-dest1 FP605 20 μg € 400 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-m Reporter codon usage mammalian Promoter for PhiYFP-m NO Host cells mammalian, prokaryotic Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use Monitoring of activity of 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-m
	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-dest1 is a promoterless vector encoding destabilized variant of the yellow fluorescent protein PhiYFP-m, which can be used as in vivo reporter of promoter activity (see reporter description). To generate PhiYFP-m-dest1 variant, residues 422-461 of mouse ornithine decarboxylase (MODC) were fused to the PhiYFP-m C-terminus. This MODC region contains a PEST amino acid sequence that targets the protein for degradation and provides for rapid protein turnover [Li et al., 1998]. PhiYFP-m-dest1 retains fluorescent properties of the native protein and has a half-life of approximately 1-1.5 hours, as measured by fluorescence intensity of cells treated with the protein synthesis inhibitor, cycloheximide. Rapid PhiYFP-m-dest1 turnover allows accurate analysis of changes in gene regulation.

PhiYFP-m-dest1 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 PhiYFP-m-dest1 coding sequence [Kozak, 1987].

Multiple cloning site (MCS) is located 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-m-dest1.

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.

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-m-dest1
Kozak consensus translation initiation site: 90-100
Start codon (ATG): 97-99
Last amino acid in XGFP: 796-798
Stop codon: 934-936
MODC PEST sequence: 814-936
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1091-1096 & 1120-1125
mRNA 3' ends: 1129 & 1141
f1 single-strand DNA origin: 1188-1643
Bacterial promoter for expression of Kan^r gene
-35 region: 1705-1710
-10 region: 1728-1733
Transcription start point: 1740
SV40 origin of replication: 1984-2119
SV40 early promoter
Enhancer (72-bp tandem repeats): 1817-1888 & 1889-1960
21-bp repeats: 1964-1984, 1985-2005 & 2007-2027
Early promoter element: 2040-2046
Major transcription start points: 2036, 2074, 2080 & 2085
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2168-2170
Stop codon: 2960-2962
G->A mutation to remove Pst I site: 2350
C->A (Arg to Ser) mutation to remove BssH II site: 2696
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3198-3203 & 3211-3216
pUC plasmid replication origin: 3547-4190