pKindling-Red-N vector

cat.# FP301

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 pKindling-Red-N FP301 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 mammalian expression vector Reporter KFP-Red Reporter codon usage mammalian Promoter for KFP-Red PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use KFP-Red expression in mammalian cells; generation of fusions to the KFP-Red 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			KFP-Red
	Afe I				Xho I							Pst I					Sac II				Sma I/Xma I						Nco 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.A	CC.	ATG.G	CC

Vector description

pKindling-Red-N is a mammalian expression vector encoding kindling red fluorescent protein KFP-Red (see reporter description). The vector allows generation of fusions to the KFP-Red N-terminus and expression of KFP-Red fusions or KFP-Red alone in eukaryotic (mammalian) cells.

KFP-Red 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 KFP-Red coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between P_{CMV IE} and KFP-Red 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 KFP-Red fusion proteins

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 KFP-Red N-terminus when inserted in the same reading frame as KFP-Red and no in-frame stop codons are present. The inserted sequence should contain an initiating ATG codon. KFP-Red-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express KFP-Red when transfected into eukaryotic (mammalian) cells.

Expression in mammalian cells

pKindling-Red-N vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of KFP-Red 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
KFP-Red
Kozak consensus translation initiation site: 672-682
Start codon (ATG): 679-681
Stop codon: 1375-1377
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1530-1535 & 1559-1564
mRNA 3' ends: 1568 & 1580
f1 single-strand DNA origin: 1627-2082
Bacterial promoter for expression of Kan^r gene
-35 region: 2144-2149
-10 region: 2167-2172
Transcription start point: 2179
SV40 origin of replication: 2423-2558
SV40 early promoter
Enhancer (72-bp tandem repeats): 2256-2327 & 2328-2399
21-bp repeats: 2403-2423, 2424-2444 & 2446-2466
Early promoter element: 2479-2485
Major transcription start points: 2475, 2513, 2519 & 2524
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2607-2609
Stop codon: 3399-3401
G->A mutation to remove Pst I site: 2789
C->A (Arg to Ser) mutation to remove BssH II site: 3135
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3637-3642 & 3650-3655
pUC plasmid replication origin: 3986-4629