The Carboxy Flexi Enzyme Blend (SgfI & EcoICRI) is required to cut acceptor Flexi Vectors containing a carboxy-terminal peptide or protein tag. The donor vector (i.e., the clone containing the protein coding region of interest) is cut with SgfI and PmeI in a separate reaction. The two restriction digests are heat-inactivated and then combined with ligase to transfer the protein coding region to the acceptor vector, thereby appending the carboxy-terminal tag to the protein of interest. The ligated product no longer contains the EcoICRI site of the acceptor vector or the PmeI site of protein coding region, as these two blunt-end cutter target sites are not recreated in the ligation. Since the acceptor Flexi Vector contains a lethal barnase gene, complete digestion with Carboxy Flexi Enzyme Blend is not required for good transfer rates of the protein coding region from the donor Flexi Vector.
