The mKate2 codon usage is optimized for high expression in mammalian cells, i.e. humanized. Human α-tubulin is fused to the mKate2 C-terminus. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of the mKate2-tubulin coding sequence. The pmKate2-tubulin vector can be used as a source of mKate2-tubulin hybrid sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice.
|Application Notes:||Expression in mammalian cells |
The vector can be transfected into mammalian cells by any known transfection method. CMV promoter provides strong, constitutive expression of the mKate2 fusion in eukaryotic cells. If required, stable transformants can be selected using G418.
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 (30μg/ml) to E. coli hosts. Copy number in E. coli is about 500.
|Long Term Storage:||-20°C|
|Background / Technical Information:|
Far-red fluorescent protein mKate2 (excitation/emission maxima at 588 and 633 nm, respectively) is almost 3-fold brighter than its parental version TagFP635, and is 10-fold brighter than mPlum at the physiological pH=7.5. Within the optical window optimal for light penetration in living tissues, the calculated brightness of mKate2 is at least 2-fold higher compared to any monomeric fluorescent protein reported to date. Also, mKate2 is more photostable under both widefield and confocal illumination than other monomeric far-red FPs. The exceptional brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues.
Location of features
PCMV IE: 1-589
Enhancer region: 59-465
TATA box: 554-560
Transcription start point: 583
Kozak consensus translation initiation site: 606-616
Start codon (ATG): 613-615
Stop codon: 2692-2694
Last amino acid in mKate2: 1306-1308
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2855-2860 & 2884-2889
mRNA 3` ends: 2893 & 2905
f1 single-strand DNA origin: 2952-3407
Bacterial promoter for expression of Kanr gene
-35 region: 3469-3474
-10 region: 3492-3497
Transcription start point: 3504
SV40 origin of replication: 3748-3883
SV40 early promoter
Enhancer (72-bp tandem repeats): 3581-3652 & 3653-3724
21-bp repeats: 3728-3748, 3749-3769 & 3771-3791
Early promoter element: 3804-3810
Major transcription start points: 3800, 3838, 3844 & 3849
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3932-3934
Stop codon: 4724-4726
G->A mutation to remove Pst I site: 4114
C->A (Arg to Ser) mutation to remove BssH II site: 4460
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4962-4967 & 4975-4980
pUC plasmid replication origin: 5311-5954
For the Original Manufacturer’s Data Sheet please click here.
Our product description may differ slightly from the Original Manufacturer’s Data Sheet.