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. 2014 Oct 15;4(12):2409–2418. doi: 10.1534/g3.114.013979

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Copyright © 2014 Zhang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.