An ∼7.0-kb fragment (genomic region 19509164–19502181) encompassing the cyst gene was amplified from BACR27M12 (BACPAC) and recombined into pENTR221 (Invitrogen). To confer RNAi resistance on the resulting Entry Clone, silent mutations were introduced at two distinct sites corresponding to SH00146.N (TRiP). All other genomic constructs were derived from this Entry Clone. The GFP-tagged full-length construct was generated by inserting Drosophila codon-optimized superfolder GFP (Pédelacq et al., 2006 (link)) in between the ATG and the second codon of cyst. The GFP-tagged deletion constructs were generated by replacing the domains described in Fig. 6 A with superfolder GFP. In all constructs, an S(GGGGS)2 linker was introduced in between GFP and Cyst, and in CystΔN, the intron was left intact to preserve potential regulatory sequences. Finally, all Entry Clones were recombined into the Drosophila transformation vector pBID-G (Wang et al., 2012b (link)).
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Cloning and Tagging of Cyst Protein
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Corresponding Organization : Howard Hughes Medical Institute
Other organizations : RIKEN Center for Biosystems Dynamics Research
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- Inserting Drosophila codon-optimized superfolder GFP in between the ATG and the second codon of cyst
- Replacing the domains described in Fig. 6 A with superfolder GFP
- The GFP-tagged full-length construct
- The GFP-tagged deletion constructs
- The 7.0-kb fragment (genomic region 19509164–19502181) encompassing the cyst gene was amplified from BACR27M12 (BACPAC)
- Silent mutations were introduced at two distinct sites corresponding to SH00146.N (TRiP) to confer RNAi resistance on the resulting Entry Clone
- An S(GGGGS)2 linker was introduced in between GFP and Cyst
- In CystΔN, the intron was left intact to preserve potential regulatory sequences
- All Entry Clones were recombined into the Drosophila transformation vector pBID-G (Wang et al., 2012b)
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