pTurboRFP-dest1 vector

cat.# FP239

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 pTurboRFP-dest1 FP239 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 TurboRFP Reporter codon usage mammalian Promoter for TurboRFP PCMV IE Host cells mammalian Selection prokaryotic - kanamycin eukaryotic - neomycin (G418) Replication prokaryotic - pUC ori eukaryotic - SV40 ori Use TurboRFP expression in mammalian cells; generation of fusions to the TurboRFP-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		TurboRFP
	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.GAG

Vector description

pTurboRFP-dest1 is a mammalian expression vector encoding destabilized red (orange) fluorescent protein TurboRFP-dest1 (see reporter description). To generate TurboRFP-dest1 variant, residues 422-461 of mouse ornithine decarboxylase (MODC) were fused to the TurboRFP 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]. TurboRFP-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.

pTurboRFP-dest1 carries synthetic version of the TurboRFP-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 TurboRFP-dest1 coding sequence [Kozak, 1987].

pTurboRFP-dest1 vector can be used to express TurboRFP-dest1 in eukaryotic (mammalian) cells. For example it can be used as a positive control with a pTurboRFP-PRL-dest1 promoterless vector (Cat.# FP238). The vector can be also used to generate destabilized TurboRFP-tagged fusion proteins. Multiple cloning site (MCS) is located upstream of TurboRFP-dest1 coding sequence.

The vector backbone contains immediate early promoter of cytomegalovirus (P_{CMV IE}) for reporter 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 TurboRFP-dest1-tagged fusions

A localization signal or a gene of interest should be cloned into MCS of the vector. It will be expressed as a fusion to the TurboRFP-dest1 N-terminus when inserted in the same reading frame as TurboRFP and no in-frame stop codons are present. TurboRFP-dest1-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified pTurboRFP-dest1 vector will express TurboRFP-dest1 when transfected into eukaryotic (mammalian) cells.

Expression in mammalian cells

pTurboRFP-dest1 can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of TurboRFP-dest1 or its fusions in many cell types. 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
TurboRFP
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Last amino acid in TurboRFP: 1405-1407
Stop codon: 1519-1521
MODC PEST sequence: 1399-1518
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1676-1681 & 1705-1710
mRNA 3' ends: 1714 & 1726
f1 single-strand DNA origin: 1773-2228
Eukaryotic promoter for expression of Kan^r gene
-35 region: 2290-2295
-10 region: 2313-2318
Transcription start point: 2325
SV40 origin of replication: 2569-2704
SV40 early promoter
Enhancer (72-bp tandem repeats): 2402-2473 & 2474-2545
21-bp repeats: 2549-2569, 2570-2590 & 2592-2612
Early promoter element: 2625-2631
Major transcription start points: 2621, 2659, 2665 & 2670
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2753-2755
Stop codon: 3545-3547
G->A mutation to remove Pst I site: 2935
C->A (Arg to Ser) mutation to remove BssH II site: 3281
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3783-3788 & 3796-3801
pUC plasmid replication origin: 4132-4775