Donor transgenic construct for use in the homology assisted CRISPR knock-in (HACK) method. Designed to convert a GAL4 line to a FLPD5 line in vivo. The construct contains the DNA of interest for incorporation into the genome (in this case a 2A-FLPD5 cassette) flanked by 5' and 3' homology arms to the targeted sequence which is to be replaced (in this case GAL4). It also contains a Equa\eqFP578^{mTagBFP2.Ubi-p63E.Tag:NLS(SV40-largeT)} marker and a CR7T-U63(2.1) dual-gRNA cassette that ubiquitously expresses 2 gRNAs targeting GAL4 coding sequences (one using a pol III promoter reported to be potent in both the soma and the germline (CR7T) and the other using the snRNA:U6:96Ac promoter); these gRNAs target the middle of GAL4 to increase CRISPR efficiency. The Equa\eqFP578^{mTagBFP2.Ubi-p63E.Tag:NLS(SV40-largeT)} marker and gRNA cassette are not included in the converted target, thus the marker can be selected against when screening for convertants. CRISPR/Cas9 is used to induce a double-strand DNA break in the target GAL4 sequence, which is then repaired via homology-directed repair using the donor as a template, resulting in insertion of the 2A-FLPD5 cassette in place of the targeted GAL4 sequence. The 'self-cleaving' 2A peptide will release FLPD5 and a truncated and nonfunctional GAL4 fragment as two separate proteins after translation of the transcript that is produced by the converted transgene.