pTurboGFP-PRL vector

cat.# FP515

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 pTurboGFP-PRL FP515 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 TurboGFP Reporter codon usage mammalian Promoter for TurboGFP 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			TurboGFP
	Afe I				Xho I							Pst I*					Sac II				Sma I/Xma I						Nco 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.A	CC.	ATG.G	AG.AGC

Vector description

pTurboGFP-PRL is a promoterless vector encoding green fluorescent protein, TurboGFP (see protein description), which can be used as in vivo reporter of gene expression. TurboGFP 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 TurboGFP 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 TurboGFP.

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

pTurboGFP-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
TurboGFP
Kozak consensus translation initiation site: 90-100
Start codon (ATG): 97-99
Stop codon: 793-795
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 949-954 & 978-983
mRNA 3' ends: 987 & 999
f1 single-strand DNA origin: 1046-1501
Eukaryotic promoter for expression of Kan^r gene
-35 region: 1563-1568
-10 region: 1586-1591
Transcription start point: 1598
SV40 origin of replication: 1842-1977
SV40 early promoter
Enhancer (72-bp tandem repeats): 1675-1746 & 1747-1818
21-bp repeats: 1822-1842, 1843-1863 & 1865-1885
Early promoter element: 1898-1904
Major transcription start points: 1894, 1932, 1938 & 1943
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2026-2028
Stop codon: 2818-2820
G->A mutation to remove Pst I site: 2208
C->A (Arg to Ser) mutation to remove BssH II site: 2554
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3056-3061 & 3069-3074
pUC plasmid replication origin: 3405-4048