pmKate2-C vector

cat.# FP181

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 pmKate2-C FP181 20 μg € 400 / 200* *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 mKate2 Reporter codon usage mammalian Promoter for mKate2 PCMV IE Host cells mammalian Selection prokaryotic – kanamycin eukaryotic – neomycin (G418) Replication prokaryotic – pUC ori eukaryotic – SV40 ori Use mKate2 expression in mammalian cells; generation of fusions to the mKate2 C-terminus

Multiple cloning site (MCS)

mKate2				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#
...	GGT.GGA.GGA.GGT.	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

pmKate2-C is a mammalian expression vector encoding far-red fluorescent protein mKate2 (see reporter description). The vector allows generation of fusions to the mKate2 C-terminus and expression of mKate2 fusions or mKate2 alone in eukaryotic (mammalian) cells.

mKate2 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 mKate2 coding sequence [Kozak, 1987]. Multiple cloning site (MCS) is located between mKate2 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 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 mKate2 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 mKate2 C-terminus when inserted in the same reading frame as mKate2 and no in-frame stop codons are present. mKate2-tagged fusions retain fluorescent properties of the native protein allowing fusion localization in vivo. Unmodified vector will express mKate2 when transfected into eukaryotic (mammalian) cells.

Expression in mammalian cells

pmKate2-C vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of mKate2 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
mKate2
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 1399-1401
Last amino acid in mKate2: 1306-1308
MCS: 1321-1398
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 1541-1546 & 1570-1575
mRNA 3' ends: 1579 & 1591
f1 single-strand DNA origin: 1638-2093
Bacterial promoter for expression of Kan^r gene
-35 region: 2155-2160
-10 region: 2178-2183
Transcription start point: 2190
SV40 origin of replication: 2434-2569
SV40 early promoter
Enhancer (72-bp tandem repeats): 2267-2338 & 2339-2410
21-bp repeats: 2414-2434, 2435-2455 & 2457-2477
Early promoter element: 2490-2496
Major transcription start points: 2486, 2524, 2530 & 2535
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 2618-2620
Stop codon: 3410-3412
G->A mutation to remove Pst I site: 2800
C->A (Arg to Ser) mutation to remove BssH II site: 3146
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 3648-3653 & 3661-3666
pUC plasmid replication origin: 3997-4640