The following information accompanied stocks donated to the Bloomington Stock Center by Stephanie Gamez and Omar Akbari, University of California at San Diego.
TI{vas-Cas9.T2A.GFP,attP} is a vasa-Cas9.T2A.GFP construct with an attP target site and marked with Disc\RFPtdTom.3xP3. These core elements are flanked on both sides by insulator sequences (su(Hw) protein-binding sites from the gypsy element on one side and CTCF protein-binding sites on the other side). The vasa-Cas9.T2A.GFP sequence is identical to that present in PBac{vas-Cas9.T2A.GFP}.
It has the general structure:
homology arm-su(Hw) binding sites-vasa regulatory sequences-Cas9-T2A-GFP-p10 3'UTR-3XP3-tdTomato-SV40 3'UTR-attP-CTCF binding sites-homology arm
The construct was inserted upon Cas9-mediated genomic cleavage at a position immediately 3' of kl-2 using the flanking homology arms directed to  Y:666 ,064..666,876 and  Y:666 ,990..667,710 to generate TI{vas-Cas9.T2A.GFP,attP}SGyA.
Despite the presence of the su(Hw) and CTCF binding site sequences, the 3XP3-tdTomato marker is not completely insulated from heterochromatic suppression. It shows variable expression and often appears completely suppressed. Nevertheless, Cas9 expression was sufficient to allow guide RNA-based cleavage.