Generation of TALE driver constructs

Assembled TALEs were subcloned into either pJC-TALE-VP64-SV40 (gift from David Stern, Addgene plasmid #46147)¹² or pJC-TALE-VP64-P10 (Supplementary Fig. ¹). pJC-TALE-VP64-P10 was generated from pJC-TALE-VP64-SV40 as follows: The TALE-VP64-SV40 sequence was removed from the pJC-TALE-VP64-SV40 plasmid by XhoI and EcoRI digest. The P10 terminator sequence was then amplified from plasmid pJFRC81 using primers P10_InFusion_Fwd and P10_InFusion_Rev (Supplementary Data ³), and the TALE-VP64 construct was amplified from plasmid pJC-TALE-VP64-SV40 using primers TALE-VP64_InFusion_Fwd and TALE-VP64_In-Fusion_Rev. Finally, the three components were re-assembled into the new pJC-TALE-VP64-P10 vector using the In-Fusion HD Cloning Kit (Clontech, 639648). To generate plasmid pJC-20×UAS-GAL4-SV40, the GAL4 sequence was amplified from plasmid pChs-GAL4 (gift from A. Bassett) with primers IF-pJC-GAL4-Fwd and IF-pJC-GAL4-Rev. The amplicon was then digested with XhoI and XbaI and inserted into pJC-TALE-VP64-SV40 replacing the TALE-VP64 sequence. pJC-20×UAS-GFP-SV40 was obtained through excision of the EGFP sequence from plasmid pJFRC81 with XhoI and XbaI, and insertion of the resulting fragment into pJC-TALE-VP64-SV40 replacing the TALE-VP64 sequence. pJC-10×UAS-GFP-SV40 was generated by removing 10×UAS from the plasmid by restriction enzyme cloning (HindIII, NheI) and inserting annealed oligonucleotides HindIII-20bp-NheI-Fwd and HindIII-20bp-NheI-Rev.