pCaspase3-sensor质粒载体图谱序列抗性说明书价格Biovector公司

pCaspase3-Sensor Vector can be used to detect the onset of caspase-3 activity in mammalian cells.This vector encodes the enhanced yellow-green variant (EYFP) of the Aequorea victoria greenfluorescent protein (GFP) fused at the 3' end to three copies of the nuclear localization signal (NLS)of the simian virus 40 large T-antigen (1,2). At the 5' end the gene contains a sequence encodingthe nuclear export signal (NES) of the Map Kinase Kinase (MAPKK;3). The NES is separated fromthe EYFP coding region by a 36-nucleotide sequence encoding the region of Poly (ADP-ribose)polymerase (PARP) cleaved by caspase-3. The complete coding sequence for this fusion proteinis human codon-optimized (4).Because the NES of MAPKK dominates the NLS, the full-length fluorescent fusion protein distributesto the cytosol. If caspase-3 is activated, the NES will be cleaved from the fusion protein and thetruncated EYFP-NLS fusion will translocate to the nucleus via the NLS. The translocation of thefluorescent protein from the cytosol to the nucleus indicates caspase-3 activity at a cellular level.The fluorescence excitation maximum of EYFP is 513 nm; the emission spectrum has a peak at 527nm (in the yellow-green region). The Em of EYFP at 513 nm is 36,500 cm–1M–1 and its quantum yieldis 0.63 (5), resulting in a bright fluorescent signal. The fluorescence intensity is roughly equivalentto that of EGFP.The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene forselection (using G418) in eukaryotic cells. A bacterial promoter (P) upstream of Neor expresseskanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication forpropagation in E. coli and an f1 origin for single-stranded DNA production. The pCaspase3-SensorVector can be introduced into mammalian cells using any standard transfection method. If desired,stable transfectants can be selected using G418 (6).