TALEN Construction Using Golden Gate Assembly

For human cell and frog experiments, a two-step Golden Gate assembly method using the Platinum Gate TALEN Kit (Addgene; cat#1000000043)15 (link) was used to construct Platinum TALEN plasmids containing the homodimer-type FokI nuclease domain. Briefly, single DNA-binding repeats were assembled into the intermediate array vectors. The assembled repeat arrays were subsequently inserted into the final destination vectors, ptCMV-153/47-VR. For the silkworm experiments, the Golden Gate TALEN and TAL Effector Kit 2.0 (Addgene; cat#1000000024)31 (link) were used to construct TALENs (BLTS-5A and BLTS-4B18 (link)), and the repeat arrays were inserted into the scaffold plasmid, pBlue-TAL18 (link).