A plasmid substrate (left) was incubated with NdeI and dimeric FokI-EcoCascade complexes targeting one of 16 adjacent sites containing interspacers ranging from 25–45 bp. Agarose gel electrophoresis reveals the expected cleavage products in each case. Interspacer distances for each target are shown at the top. Target 3–7 digestions are identical to those in Fig. 1d. N, nicked; L, linear; SC, supercoiled; P, dropout product. Experiment was repeated two independent times with similar results; uncropped gels can be found in Source Data Fig. 2.