Gblock

The enhancer of Per and Tim were synthesized as gBlocks (Integrated DNA Technologies) in 2× tandem multiplexes. Then two copies of the gBlocks were amplified by PCR and inserted into a reporter vector using In-Fusion HD Cloning (Clontech) to generate 4× Per and 4× Tim enhancers (Figure S4). The reporter vector contains fly heat shock mini promoter and an nlsGFP fused with PEST domain at the C terminus (destabilized GFP) as previously used for the construction of Notch reporter(Hunter et al., 2016 (link)).

Varying lengths of homology were made by reconstructing the P{wIw} construct used previously (Rong and Golic 2003 (link); Wei and Rong 2007 (link)). We started with w+attB (previously deposited into AddGene, plasmid # 30326), which has a fully functional white gene under control of a basal Hsp70Bb promoter (Hsp70Bb::white; referred to as mini-white). We added a full-length, nonfunctional mini-white gene with truncated exon 1 (3.5 kb) and a 3′ I-SceI site upstream of the functional mini-white in the form of two gBlocks (Integrated DNA Technologies). These incorporated restriction enzyme sites such that cleavage and relegation would delete various segments of the upstream mini-white gene sequence. Deleting between HindIII sites left 2 kb 5′ homology; between 2 NheI sites left 2 kb 3′ homology; between two AvrII left 500-bp 5′ homology; between 2 AgeI sites left 500-bp 3′ homology; and between two MluI sites left 50-bp 3′ homology. The two gBlocks were added to the w+attB vector by InFusion cloning (Clontech/Takara) into the HindIII site. Once the full-length, nonfunctional 5′ copy was inserted, varying homologies described were created by cutting with a particular enzyme and ligating with T4 DNA ligase (New England Biolabs). All vectors were checked via Sanger sequencing to confirm proper amount/arrangement of upstream homology.