Vectors with hidden cloning sites

A general strategy is described for using the cleavage site of restriction enzymes in vectors for cloning regardless of how many sites the given enzym es have in the vector. The application of this method allows one to open an y vector at its cloning site with protruding ends which can be compatible w ith almost every commercially available Class Ii restriction enzyme. By emp loying this method, the laborious construction of new vectors can be simpli fied considerably. This general strategy is based on the known ability of C lass HS restriction enzymes to cut any sequence located outside of their re cognition site; the introduction of a linker containing recognition site(s) for Class IIS restriction enzyme(s), not present originally in the vector, gives rise to the possibility of opening the vector so as to produce overh angs of arbitrary sequence. In particular, when a symmetrical short sequenc e representing the protruding end of any Class II enzyme is situated at the cutting position of the Class IIS enzyme, cleavage with the Class IIS enzy me exposes the hitherto hidden, "unique" cloning site. This technique is de monstrated by cloning the cDNA of the multidrug resistance protein to an ex pression vector, (C) 2000 Academic Press.