Versatile Tol2-based Plasmid Construction

To generate pTol2-mpeg1:KalTA4, we first cloned the middle entry vector pME-KalTA4GI by a BP reaction of pTol2-sox10:KalTA4GI (kind gift of Tim Czopka [Almeida and Lyons, 2015 (link)]) with pDONR 221, using Gateway BP Clonase II Enzyme mix (Thermo Fisher Scientific). pME-KalTA4GI was then recombined with p5E-mpeg1 and p3E-polyA into pDestTol2CG2 (Kawakami, 2007 (link)), using LR Clonase II Plus enzyme (Thermo Fisher Scientific). pTol2-UAS:EGFP-Rab7 was generated by recombining p5E-UAS, pME-EGFP no stop and p3E-Rab7 (kind gift of Brian Link [Clark et al., 2011 (link)]) into pDestTol2-CG2 in an LR reaction, as described above. To obtain pTol2-UAS:MFG-E88-C1C2-EGFP, we first cloned pME-MFG-E8-C1C2-EGFP by PCR amplification of the mouse MFG-E8 C1 and C2 domain (insert) and of the middle entry vector backbone (vector), using Q5 polymerase (New England Biolabs Inc). Fragments were generated with the following primers (template plasmids indicated in parentheses): insert fragment (pD2523-mMFG-E8_C1C2-EGFP, kind gift of Jan Kranich [Kranich et al., 2020 (link)]): 5′- ATG​CAA​GTC​TCT​AGG​GTA​C-3′ (fwd) and 5′-CTT​ATA​AAG​TTC​ATC​CAT​GCC​A-3′ (rev), vector fragment (pME-KalTA4GI): 5′-GCA​TGG​ATG​AAC​TTT​ATA​AGT​AAA​CCC​AGC​TTT​CTT​G-3′ (fwd), and 5′- AGT​ACC​CTA​GAG​ACT​TGC​ATG​GTG​GCG​GCA​GCC​T-3′ (rev). This was followed by a 2-fragment Gibson assembly, using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, Inc.) according to the manufacturer’s protocol. pTol2-UAS:MFG-E8-C1C2-EGFP was then generated by recombining p5E-UAS, pME-MFG-E8-C1C2-EGFP and p3E-polyA into pDestTol2-CG2 in a Gateway LR reaction. All sequences were verified by Sanger sequencing.