.Thismethodisbased on the restriction enzyme digestion of the recombinant to determine the correct orientation of the insert. The restriction enzyme XcmI (CCANNNNN/NNNNTGG), resulting in a one-base3′-protrudingend,wasusedforconstructingsuch vectors.Forsimilarpurposes,restrictionenzymesEam11105I (GACNNN/NNGTC), AspEI (GACNNN/NNGTC), and AhdI (GACNNN /NNGTC) were also available