pJRed-C vector

cat.# FP701

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 pJRed-C FP701 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 mammalian expression vector Reporter JRed Reporter codon usage mammalian Promoter for JRed PCMV IE Host cells mammalian Selection prokaryotic - kanamycin eukaryotic - neomycin (G418) Replication prokaryotic - pUC ori eukaryotic - SV40 ori Use JRed expression in mammalian cells; generation of fusions to the JRed C-terminus

Multiple cloning site (MCS)

JRed			Bgl II			Sac I				EcoR I			Sal I		Kpn I			Apa I				BamH I				Stops
	BspE I			Xho I				Hind III			Pst I					Sac II				Sma I/Xma I					Xba I#					Bcl I#
GAG.GAT.	TCC.GGA.	CTC.	AGA.T	CT	.C	GA.G	CT.C	AA.GCT.T	C	G.AAT.T	C	T.GCA.	G	TC.GAC	.GGT.A	CC.	GC	G.G	G	C.CC	G.	GG	A.TCC.	ACC.GGA.	TC	T.AG	A.	TAA.	C	TG.A	TC.A

Vector description

pJRed-C is a mammalian expression vector encoding true-red fluorescent protein JRed (see protein description). The vector allows generation of fusions to the JRed C-terminus and expression of JRed fusions or JRed alone in eukaryotic (mammalian) cells.

JRed 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 JRed sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between JRed coding sequence and SV40 polyadenylation signal (SV40 polyA).

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 polyA 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 JRed-fusion proteins

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

Expression in mammalian cells

pJRed-C can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of JRed 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
JRed
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 1417-1419
Last amino acid in JRed: 1336-1338
MCS: 1339-1424
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1559-1564 & 1588-1593
mRNA 3' ends: 1597 & 1609
f1 single-strand DNA origin: 1656-2111
Eukaryotic promoter for expression of Kan^r gene
-35 region: 2173-2178
-10 region: 2196-2201
Transcription start point: 2208
SV40 origin of replication: 2452-2587
SV40 early promoter
Enhancer (72-bp tandem repeats): 2285-2356 & 2357-2428
21-bp repeats: 2432-2452, 2453-2473 & 2475-2495
Early promoter element: 2508-2514
Major transcription start points: 2504, 2542, 2548 & 2553
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2636-2638
Stop codon: 3428-3430
G->A mutation to remove Pst I site: 2818
C->A (Arg to Ser) mutation to remove BssH II site: 3164
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3666-3671 & 3679-3684
pUC plasmid replication origin: 4015-4658