The pAC92 plasmid is a direct screening cloning vector which allows po
sitive selection of recombinant clones (re-clones). This new high-copy
-number plasmid vector encodes ampicillin resistance and carries the B
acillus subtilis alpha-amylase (alpha-Amy)-encoding gene (amy) contain
ing a multiple cloning site. The pAC92 plasmid confers to Escherichia
coil transformants an amylolytic phenotype easily detected by iodine v
apor staining. The re-clones are identified by insertional inactivatio
n of alpha-Amy activity. During pAC92 construction, a bacterial growth
defect was observed in host cells after some modifications of the pro
moter region that caused the increase in the amy expression. This suic
ide characteristic permitted the positive selection of re-clones. A se
cond transformation step was performed to enhance the rate of re-clone
s per plate.