A two-step method to flexibly engineer the fly genome.

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A two-step method to flexibly engineer the fly genome. A two-step method to flexibly engineer the fly genome. Overview of the two-step procedure. In step 1, a donor vector consisting of two attP sites (P), a splice acceptor (SA), and STOP codons (yellow box, black asterisk) followed by an SV40 polyadenylation signal (pA) and a 3xP3dsRed marker are inserted on Cas9 cleavage with two sgRNAs. The orange coding exon is excised. In step 2, ΦC31-mediated RMCE inserts any DNA sequence between the two attB sites (B). Examples for various engineered exons are given, resulting in attR sites (R) in introns. F stands for FRT. Xu Zhang et al. G3 2014;4:2409-2418 ©2014 by Genetics Society of America