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The pGEM-T is 3kb in length, and contains the amplicin resistance gene, conferring selection of the plasmid in E. coli, and the ori site which is the bacterial origin of replication. The plasmid has multiple cloning sites as shown below. The coding sequence was inserted by TA cloning. Many E. coli strains are suitable for the propagation of this vector including JM109, DH5α and TOP10.
The coding sequence can be easily obtained by digesting the vector with proper restriction enzyme(s). The coding sequence can also be amplified by PCR with M13 primers, or primer pair SP6 and T7.
MKI67 contains 1 FHA domain and plays a key role in cell proliferation. During interphase, the MKI67 antigen can be exclusively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. MKI67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), but is absent from resting cells. MKI67 is an excellent marker to determine the growth fraction of a given cell population. The fraction of MKI67-positive tumor cells is often correlated with the clinical course of cancer. It is also associated with ribosomal RNA transcription. Inactivation of antigen MKI67 leads to inhibition of ribosomal RNA synthesis.