Technical note: Fly KCR construct maps
We have uploaded the verified vector information for the publishedDrosophilaKCR constructs to Zenodo. Files can be assessed under:https://zenodo.org/records/15074206The file contains vector maps for the below constructs:pJFRC7_20xUAS_HcKCR1_AAA_YFPpJFRC7_20xUAS_HcKCR1_C29D_YFPpJFRC7_20xUAS_HcKCR1_ET_YFPpJFRC7_20xUAS_HcKCR1_GS_YFPpJFRC7_20xUAS_HcKCR2_AAA_YFPpJFRC7_20xUAS_HcKCR2_ET_YFPpJFRC7_20xUAS_HcKCR2_GS_YFPpJFRC7_20xUAS_WiChR_ET_YFPThesewere reported in:Ott, S., Xu, S., Lee, N.et al.Kalium channelrhodopsins effectively inhibit neurons.Nat Commun15, 3480 (2024). https://doi.org/10.1038/s41467-024-47203-wAlso:Kalium channelrhodopsins effectively inhibit neurons in the small model animalsStanislavOtt,SangyuXu,NicoleLee,Ivan Hee KeanHong,JonathanAnns,Danesha DeviniSuresh,ZhiyiZhang,XianyuanZhang,RaihanahHarion,WeiyingYe,VaishnaviChandramouli,SureshJesuthasan,YasunoriSaheki,AdamClaridge-ChangbioRxiv2024.01.14.575538;doi:https://doi.org/10.1101/2024.01.14.575538Fly constructs and geneticsUAS-KCR1-ET,UAS-KCR2-ET,UAS-KCR1-GSand UAS-WiChR transgenic lines were generated byde novosynthesis (Genscript) ofDrosophilacodon-optimized HcKCR insert sequences43(Genbank #MZ826861and #MZ826862) or the WiChR sequence[45](https://www.biorxiv.org/content/10.1101/2024.01.14.575538v1.full#ref-45)(Genbank #OP710241) as eYFP fusions. After Sanger sequencing verification (Genscript), the fragments were cloned into anpJFRC7-20XUAS-IVS-mCD8::GFPvector (Addgeneplasmid # 26220), replacing themCD8::GFPinsert via restriction enzyme digest (XhoI, Xba I). ForUAS-KCR1-GS, a 3×GGGGS sequence was used to link the opsin with the fluorophore. For the KCR-ET and WiChR constructs, an AAA linker sequence was used as the starting point, to which two modifications were made: (1) an FCYENEV motif was added to the C terminus of eYFP to boost protein export from the endoplasmic reticulum and prevent potential aggregate formation51; and (2) a KSRITSEGEYIPLDQIDINV trafficking signal from Kir 2.152was added to the linker at C terminus of the opsin to boost protein expression10. The KCR1-C29D variant45was obtained by site-directed mutagenesis of the KCR1-ET sequence, where the cysteine at position 29 was replaced by aspartic acid (Genscript). The synthesized constructs were injected into flies and targeted to attP1 or attP2 insertion sites on the second or third chromosomes respectively and the transgenic progeny were balanced either over CyO or TM6C (BestGene). Expression was verified by imaging of eYFP fluorescence with a Leica TCS SP8 STED confocal microscope. Opsin transgenic flies were crossed with relevant Gal4 driver lines to produce F1 offspring for use as test subjects. Driver Gal4 lines and UAS-opsin responder lines were each crossed with an otherwise wild-typew1118line and the F1 progeny (e.g.UAS-KCR1-ET/+orelav-Gal4/+) were used as control subjects.