Figure 5
From: Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi
Timeline for constructing a KO plasmids using MS/GW strategy. The Multisite Gateway based method consists of three steps: 1) PCR with attB-containing primers to amplify 5' and 3' UTR from genomic DNA; 2) BP recombination of each PCR products with specific donor vectors to generate entry clones containing the UTRs; 3) LR recombination of the two entry clones made in step 2 and a third entry clone containing Neo/Hyg to create the final construct. (Kan, kanamycin-resistance gene; Amp, ampicillin-resistance gene; Ori, Origin of replication).