atlas genomic rescue line

We constructed an HA-tagged atlas rescue line that contained the atlas gene flanked by 1345 bp of sequence upstream of the start codon (but excluding the coding sequence of upstream gene CG3124) and 3000 bp of sequence downstream of the stop codon (including the full 3’ UTR) as follows. Genomic sequences were PCR amplified using Q5 High fidelity Polymerase (NEB), purified Canton S genomic DNA (Gentra Puregene Tissue Kit, Qiagen, Germantown, MD), and the atlas rescue F1/R1 and atlas rescue F3/R3 primer sets (see S4 Table). The 3x-HA tag was likewise amplified from pTWH (DGRC 1100; T. Murphy) using atlas rescue F2/R2 primers. These DNA fragments were subsequently assembled into a XbaI/AscI-linearized w+attB plasmid (Addgene, Watertown, MA, plasmid 30326, deposited by J. Sekelsky). The assembled construct was then phiC31 integrated into the PBac{y⁺-attP-3B}VK00037 (Bloomington Drosophila Stock Center (BDSC) stock #24872) docking site (Rainbow Transgenics) and crossed into the atlas null background to assess rescue.