A VERSATILE PROKARYOTIC CLONING VECTOR WITH 6 DUAL RESTRICTION ENZYMESITES IN THE POLYLINKER FACILITATES EFFICIENT SUBCLONING INTO VECTORSWITH UNIQUE CLONING SITES

In large and complex vectors a single restriction enzyme recognition s ite may be available for introduction of additional DNA requiring the development of linker fragments to create compatible insertion sites. This technology can he time consuming and costly. We describe the cons truction of a simple phagemid, pSFI, with a polylinker that contains s ix pairs of dual, rare-cutting, restriction enzyme recognition sites ( NotI, SpeI, EcoRV, PstI, SacII. EagI) with multiple unique sites betwe en each pair. This has permitted rapid subcloning of DNA with creation of single flanking restriction enzyme sites. pSFI was used to expedit e transfer of viral genes to a LacZ-inducible expression vector and to an adenovirus expression cassette for production of replication-defec tive virus. The use of this phagemid has facilitated complex vector ma nipulations and is a valuable adjunct to the family of multifunctional cloning vectors. (C) 1998 Academic Press.