The following information accompanied stocks donated to the Bloomington Drosophila Stock Center by Kate Koles, Brandeis University.
TI{T-STEP.TagRFP-T}CsasKI - A T-STEP cassette was knocked into Csas to tag the endogenous protein with TagRFP-T. In the presence of Rippase, TagRFP-T is replaced by GFP, allowing tissue/cell specific tagging of endogenous protein with GFP. Introduction of the T-STEP cassette leads to a Csas fusion protein tagged at its C-terminal amino acid, with the translated Rippase Recognition Site acting as a linker (LMKEYYVILSS) in front of TagRFPt (MVSKGEELIKE...), leading to the following amino acid sequence of the Csas-RRS-TagRFPt fusion protein at the C-terminal junction: ...ILSSETKTELAYALMKEYYVILSSSMVSKGEEL..., where ILSSETKTEL are the last 10 amino acids of CSAS, AYA are amino acids derived from the cloning procedure, LMKEYYVILSS is the translated Rippase Recognition Site, S is another cloning derived amino acid, and MVSKGEEL are the first eight amino acids of TagRFPt."
TI{T-STEP.EGFP}CsasKI-GFP – Germline Rippase conversion of progenitor TI{T-STEP.TagRFP-T}CsasKI has converted RFP-tagged Csas to GFP-tagged Csas. The C-terminally GFP-tagged CSAS fusion protein has the following sequence: ...ILSSETKTELAYALMKEYYVILSSSSGSSLEVLFQPGSGSGSMSKGEELFT..., where ILSSETKTEL are the last 10 amino acids of Csas, AYA are amino acids derived from the cloning procedure, LMKEYYVILSS is the translated Rippase Recognition Site, which is followed by five linker amino acids "SSGSS"; then a recognition sequence for the PreScission ProteaseTM "LEVLFQP", which is followed by six linker amino acids "GSGSGS," then the coding sequence for GFP (MSKGEELFT...)."