The LG cassette was PCR amplified from an α-crystallin:EGFP cassette (Kikuchi et al., 2011), and the PCR product was inserted between two FRT sites of pL451 (Liu et al., 2003) (provided by Dr. Stephan Creekmore, NCI-Frederick). The resulting vector was referred to as pL451-FRT-LG-FRT. Tandem LoxP-Lox5171 sites, the splice acceptor (SA) sequence of pFT1 (provided by Dr. Wenbiao Chen, Vanderbilt University) (Ni et al., 2012), and the P2A sequence (SA-P2A) were synthesized using Ultramer Oligo Synthesis by IDT Technologies. TagRFP cDNA was PCR-amplified from pTagRFP-C (Evrogen, Moscow, Russia), and 5× BGHpA repeats were derived from pFT1 via restriction enzyme digestion. The components were inserted into pL451-FRT-LG-FRT via restriction enzyme digestion, and the resulting construct was referred to as pZwitch. As this version was designed for +1 reading frame, it was designated pZwitch+1. To use different reading frames, we created pZwitch+2 and pZwitch+3 using SA-P2A oligonucleotides with the addition of one or five base pairs upstream of the P2A sequence (Figure 1—figure supplement 1B).