A Positive Selection Vector for Cloning of Long Polymerase Chain Reaction Fragments Based on a Lethal Mutant of thecrpGene ofEscherichia coli

Abstract We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this “suicide” vector, pCAP s , is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene.