pTurboRFP-PRL vector

cat.# FP235

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-PRL FP235 20 μg € 320 / 160* *50% discount on the second and subsequent vectors encoding same fluorescent protein or sensor ordered by the same customer. 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 TurboRFP Reporter codon usage mammalian Promoter for TurboRFP 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		TurboRFP
	Afe I				Xho I							Pst I					Sac II				Sma I/Xma I
ACT	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-PRL is a promoterless vector encoding red (orange) fluorescent protein TurboRFP, which can be used as in vivo reporter of promoter activity (see reporter description). TurboRFP 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 TurboRFP 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 TurboRFP.

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: 12-96
TurboRFP
Kozak consensus translation initiation site: 90-100
Start codon (ATG): 97-99
Stop codon: 790-792
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 946-951 & 975-980
mRNA 3' ends: 984 & 996
f1 single-strand DNA origin: 1043-1498
Bacterial promoter for expression of Kan^r gene
-35 region: 1560-1565
-10 region: 1583-1588
Transcription start point: 1595
SV40 origin of replication: 1839-1974
SV40 early promoter
Enhancer (72-bp tandem repeats): 1672-1743 & 1744-1815
21-bp repeats: 1819-1839, 1840-1860 & 1862-1882
Early promoter element: 1895-1901
Major transcription start points: 1891, 1929, 1935 & 1940
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2023-2025
Stop codon: 2815-2817
G->A mutation to remove Pst I site: 2205
C->A (Arg to Ser) mutation to remove BssH II site: 2551
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3053-3058 & 3066-3071
pUC plasmid replication origin: 3402-4045