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 enzymes have in the vector. The application of this method allows one to open any vector at its cloning site with protruding ends which can be compatible with almost every commercially available Class II restriction enzyme. By employing this method, the laborious construction of new vectors can be simplified considerably. This general strategy is based on the known ability of Class IIS restriction enzymes to cut any sequence located outside of their recognition 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 overhangs of arbitrary sequence. In particular, when a symmetrical short sequence 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 enzyme exposes the hitherto hidden, "unique" cloning site. This technique is demonstrated by cloning the cDNA of the multidrug resistance protein to an expression vector. Copyright 2000 Academic Press.