Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease

► Our sandwiched ZFN completely cleaved 40-fold excess of plasmid DNA within 1 h. ► The single sandwiched ZFN cleaved two heterogeneous sites in one plasmid. ► The sandwiched ZFN allowed cloning of a foreign gene in the directed orientation. ► DNA fragments generated by the sandwiched ZFN were religated with no mutation.