pCMV-MEK1载体使用信息: pCMV-MEK1 is designed for the constitutive expression of MAPK/ERK kinase 1 (MEK 1) in mammalian cells. It can also be used to study the effects of a given stimulus on the MAPK signaling pathway. In conjunction with Clontech's In Vivo Kinase Assay Kits, pCMV-MEK1 is an ideal positive control for studying a gene or molecule and its affect on kinase activation in the MAPK pathway. MEK1, a Raf isoform, phosphorylates and activates ERK-1 and ERK-2 (1–3). Studies show that ERK1/2 are activated by mitogens in most cell types, linking their involvement in mitogenic signaling (2). ERKs may also be linked to cell transformation, differentiation, and apoptosis in some cell types (ibid.).
pCMV-MEK1 contains the gene encoding MEK1 (human), which is controlled by the human cytomegalovirus (CMV) promoter. SV40 polyadenylation signals downstream of MEK1 direct proper processing of the 3' end of the mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor)—consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene—allows stably transfected eukaryotic cells to be selected using G418 (4). A bacterial promoter upstream of this cassette expresses kanamycin resistance in E. coli. The pCMV-MEK1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production. The recombinant vector can be transfected into mammalian cells using any standard method.
Propagation in E. coli:
Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM109 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.
E. coli replication origin: pUC
Copy number: ≈500
Plasmid incompatibility group: pMB1/ColE1
