A PLASMID VECTOR WITH POSITIVE SELECTION AND DIRECTIONAL CLONING BASED ON A CONDITIONALLY LETHAL GENE

A plasmid vector with a multiple cloning site (MCS) for positive selec tion of cloned inserts in Escherichia coli (Ec) has been devised, base d on the expression plasmid (pMT416) for the bacterial ribonuclease ba rnase (Barn). The host is protected from the lethal effect of moderate expression of barn by expression of the gene bars, encoding its inhib itor, barstar (Bars), placed on the same plasmid. Full expression, how ever, is lethal. Induction is also lethal with the derived plasmid, pM T440, which has the pUC19 MCS inserted into barn. Under inducing condi tions, transformation by the vector is lethal unless the product of th e modified barn is inactivated by insertion of cloned DNA fragments in to the MCS, Plasmid pMT440 is, therefore, a generally useful selective cloning vector not requiring any special strain of Ec.