Control construct for use in screens using FOFO2.0 based constructs. Expression from the ubiquitous Act5C promoter is blocked by the presence of two STOP cassettes, flanked by FRT and mFRT71 sites respectively. The first STOP cassette is approximately 2.8kb and contains the Lam coding sequence followed by Hsp70Aa and Hsp27 polyA signals. The second STOP cassette is approximately 5.7kb and consists of four tandem SV40 polyA sequences. An intron flanked by splice donor and acceptor sites is present downstream of the stop cassettes; this contains short hairpin artificial microRNAs (miRs) targeted against both Rnor\Cd2 and the GAL4 driver. Downstream of the intron is the coding sequence for the EGFP fluorescent protein, tagged at the C-terminal end with the Tag:NLS(SV40-largeT) nuclear localization signal. Expression from the Act5C promoter should only occur after excision of both the STOP cassettes, which requires two different recombinases; FLP (targets FRT) and FLPm5 (targets mFRT71). After excision of the two cassettes, both the miRs and the fluorescent marker (represented by Avic\GFP^{EGFP.FOFO2.0.Act5C}) are expressed, in the overlap of expression between the FLP and FLPm5 recombinases; typically FLP is expressed using a specific enhancer (to give spatial control) and FLPm5 is expressed using heat shock (to give temporal control).

Short hairpin artificial microRNAs (miRs) that target Rnor\Cd2. Present in transgenic constructs made using a 'FOFO2.0' based plasmid that contains an Act5C promoter; the construct is designed so that expression of the miRs should only occur once both of the FRT and mFRT71 STOP cassettes located downstream of the Act5C promoter have been excised (requires both the FLP and FLPm5 recombinases, which target FRT and mFRT71 respectively).

Short hairpin artificial microRNAs (miRs) that target GAL4. Present in transgenic constructs made using a 'FOFO2.0' based plasmid that contains an Act5C promoter; the construct is designed so that expression of the miRs should only occur once both of the FRT and mFRT71 STOP cassettes located downstream of the Act5C promoter have been excised (requires both the FLP and FLPm5 recombinases, which target FRT and mFRT71 respectively).