mKate2 codon usage is optimized for high expression in mammalian cells, i.e. humanized. Chicken paxillin is fused to the mKate2 N-terminus. pmKate2-paxillin vector can be used as a source of mKate2-paxillin hybrid sequence. To increase mRNA translation efficiency, Kozak consensus translation initiation site is generated upstream of mKate2-paxillin coding sequence. The vector backbone contains unique restriction sites that permit its excision and further insertion into expression vector of choice. The vector backbone also contains immediate early promoter of cytomegalovirus (PCMV IE) for protein expression, SV40 origin for replication in mammalian cells expressing SV40 T-antigen, pUC origin of replication for propagation in E.
|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-paxillin 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 (30ìg/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
mKate2-clathrin fusion: 613-1992
Start codon (ATG): 613-615
Last amino acid in mKate2: 1312-1314
Clathrin LCB: 1360-1992
Stop codon: 1993-1995
SV40 early mRNA polyadenylation signal
Polyadenylation signals: 2156-2161 & 2185-2190
mRNA 3’ ends: 2194 & 2206
f1 single-strand DNA origin: 2253-2708
Bacterial promoter for expression of Kanr gene
-35 region: 2770-2775
-10 region: 2793-2798
Transcription start point: 2805
SV40 origin of replication: 3049-3184
SV40 early promoter
Enhancer (72-bp tandem repeats): 2882-2953 & 2954-3025
21-bp repeats: 3029-3049, 3050-3070 & 3072-3092
Early promoter element: 3105-3111
Major transcription start points: 3101, 3139, 3145 & 3150
Kanamycin/neomycin resistance gene
Neomycin phosphotransferase coding sequences:
Start codon (ATG): 3233-3235
Stop codon: 4025-4027
G->A mutation to remove Pst I site: 3415
C->A (Arg to Ser) mutation to remove BssH II site: 3761
Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
Polyadenylation signals: 4263-4268 & 4276-4281
pUC plasmid replication origin: 4612-5255
For the Original Manufacturer’s Data Sheet please click here.
Our product description may differ slightly from the Original Manufacturer’s Data Sheet.