pPhi-Yellow-dest1 vector

cat.# FP608

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-dest1 FP608 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 mammalian expression vector Reporter PhiYFP-m Reporter codon usage mammalian Promoter for PhiYFP-m PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use PhiYFP-m expression in mammalian cells; generation of fusions to the PhiYFP-m-dest1 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		PhiYFP-m
	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.AGC

Vector description

pPhi-Yellow-dest1 is a mammalian expression vector encoding destabilized yellow fluorescent protein PhiYFP-m-dest1 (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.

pPhi-Yellow-dest1 carries synthetic version of the PhiYFP-m-dest1 gene which 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].

pPhi-Yellow-dest1 vector can be used to express PhiYFP-m-dest1 in eukaryotic (mammalian) cells. For example it can be used as a positive control with a pPhi-Yellow-PRL-dest1 promoterless vector (Cat.# FP605). The vector can be also used to generate destabilized PhiYFP-m-tagged fusion proteins. Multiple cloning site (MCS) is located upstream of PhiYFP-m-dest1 coding sequence.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV 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 (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.

Generation of PhiYFP-m-dest1-tagged fusions

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 PhiYFP-m-dest1 N-terminus when inserted in the same reading frame as PhiYFP-m and no in-frame stop codons are present. PhiYFP-m-dest1-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express PhiYFP-m-dest1 when transfected into eukaryotic (mammalian) cells.

Expression in mammalian cells

pPhi-Yellow-dest1 vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of PhiYFP-m-dest1 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

P_{CMV IE}: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
MCS: 591-671
PhiYFP-m
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Last amino acid in PhiYFP-m: 1378-1380
Stop codon: 1516-1518
MODC PEST sequence: 1396-1518
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1673-1678 & 1702-1707
mRNA 3' ends: 1711 & 1723
f1 single-strand DNA origin: 1770-2225
Bacterial promoter for expression of Kan^r gene
-35 region: 2287-2292
-10 region: 2310-2315
Transcription start point: 2322
SV40 origin of replication: 2566-2701
SV40 early promoter
Enhancer (72-bp tandem repeats): 2399-2470 & 2471-2542
21-bp repeats: 2546-2566, 2567-2587 & 2589-2609
Early promoter element: 2622-2628
Major transcription start points: 2618, 2656, 2662 & 2667
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2750-2752
Stop codon: 3542-3544
G->A mutation to remove Pst I site: 2932
C->A (Arg to Ser) mutation to remove BssH II site: 3278
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3780-3785 & 3793-3798
pUC plasmid replication origin: 4129-4772