To determine the in vitro cleavage efficiency by the restriction enzyme I–SceI, we first linearized each reporter gene plasmid by a restriction enzyme digest, followed by enzyme inactivation, and extracted the DNA using a column purification protocol (NZYTech): The EX2 reporter construct was Xho I digested, and the PROP and EX2-AS reporter constructs were each Hind III digested (all enzymes are from Thermo Fisher Scientific). Incubation of the predigested EX2 plasmid with the I–SceI restriction enzyme (Thermo Fisher Scientific) generated 1.5– and 1.2–kilo-bp (kbp) fragments. Aliquots were removed at different time points (2.5, 5, 10, and 15 min after incubation with I–SceI). Predigested PROP and EX2-AS plasmids incubated for 15 min with I–SceI generated one additional fragment of 1.5 and 2.0 kbp, respectively, and all digests were analyzed on 1% agarose gels, together with non–I–SceI–digested controls. DNA was visualized by GelRed fluorescence (Biotium), and agarose gel images were acquired using the Bio-Rad gel imager. The bands were quantified using ImageJ software. The in vitro I–SceI cleavage efficiency was calculated by measuring the background-corrected integrated band intensity of the uncleaved 2.7 kbp and the total of the cleaved 1.5- + 1.2-kbp fragments at the indicated times of I–SceI incubation.

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