Generation of Sfi I sites by cloning a target ORF only in correct direction. The top line is the cloning site of pCA24N. To clone a target ORF, blunt-ended PCR-amplified protein-coding region with three (GCC) and two (GG) additional nucleotides at the N- and C-terminal, respectively, was inserted into the Stu I site (filled triangle). Ligation of blunt-ended 5′-GCCXXX…XXXGG-3′ fragment (X indicates nucleotides of target gene) generates Sfi I sites at both boundaries of the fragment when the ligation was done in correct direction (left) but not in opposite direction (right). Open arrows show PCR-amplified fragments with arrowhead indicating direction of the ORF.