Q5 hotstart start high fidelity 2x master mix

All plasmids were cloned using Gibson enzymatic assembly (Gibson et al., 2009 (link)). DNA fragments were either amplified from available plasmids or synthesized gBlocks (Integrated DNA Technologies) using Q5 Hotstart Start High-Fidelity 2X Master Mix (New England Biolabs, Cat. #M0494S). The generated plasmids were transformed into Zymo JM109 chemically competent E. coli (Zymo Research, Cat. #T3005), amplified, isolated (Zymo Research, Zyppy plasmid miniprep kit, Cat. #D4036), and Sanger sequenced. Final selected plasmids were maxi-prepped (Zymo Research, ZymoPURE II Plasmid Maxiprep kit, Cat. #D4202) and sequenced thoroughly using Oxford Nanopore Sequencing at Primordium Labs (https://www.primordiumlabs.com). All plasmids and annotated DNA sequence maps are available at https://www.addgene.org/ under accession numbers: 200252 (gRNA^dsx, 1067B) 200251 (gRNA^ix, 1055J), 200253 (gRNA^dsx,ix,βTub, 1067L), and 164846 (Nup50-Cas9 or Cas9, 874PA).

All plasmids were cloned using Gibson enzymatic assembly^{44 (link)}. DNA fragments were either amplified from available plasmids or synthesized gBlocks™ (Integrated DNA Technologies) using Q5 Hotstart Start High-Fidelity 2X Master Mix (New England Biolabs, Cat. #M0494S). The generated plasmids were transformed into Zymo JM109 chemically competent E. coli (Zymo Research, Cat # T3005), amplified, isolated (Zymo Research, Zyppy plasmid miniprep kit, Cat. #D4036), and Sanger sequenced. Final selected plasmids were maxi-prepped (Zymo Research, ZymoPURE II Plasmid Maxiprep kit, Cat. #D4202) and sequenced thoroughly using Oxford Nanopore Sequencing at Primordium Labs (https://www.primordiumlabs.com). All plasmids and annotated DNA sequence maps are available at www.addgene.com under accession numbers: 200252 (gRNA^dsx, 1067B) 200251 (gRNA^ix, 1055J), 200253 (gRNA^dsx,ix,βTub, 1067L) and 164846 (Nup50-Cas9 or Cas9, 874PA).