A vector for facile PCR product cloning and modification generating any desired 4-base 5' overhang: pRPM.

A pair of plasmids were developed for cloning PCR products in order to facilitate the preparation of products with 5' overhangs consisting of any desired 4-nucleotide sequence. These vectors allow DNA to be cloned into a unique restriction site by blunt-end ligation or by AT-cloning. The cloned DNA is subsequently excised using class IIS restriction enzyme sites flanking the insert yielding a fragment that is entirely free of vector sequences. These enzymes recognize sequences in the vector but "reach-over" the junction to cut within the insert thereby generating a 4-base 5' overhang sequence determined by the 5' sequences in the insert. Thus, in cases where the insert was originally generated by PCR, the overhangs are specified by the primer. More generally, these vectors offer a unique capability for the reversible cloning of any blunt DNA fragment because excision and fill-in reactions precisely regenerate the original blunt fragment regardless of its sequence. These "reach-over" product modification vectors represent general and flexible tools for the generation of fragments for use in engineering DNA constructs.