Pnl1.3 secnluc plasmid

For studying intracellular BRET, the Nanoluciferase (Nluc,Promega, USA) was fused to the C terminus of npr-11. cDNA of npr-11 was amplified with XbaI_npr-11_f/npr-11_Nluc_r from pSP168 inserting an XbaI restriction site at the 5′ end and parts of a SGGGGS linker at the 3′ end. The Nluc was amplified from pNL1.3_secNluc plasmid (Promega, USA) using Nluc_Linker_npr-11_f/Nluc_XmaI_r to add a 5′ SGGGGS Linker and an XmaI restriction site at the 3′ end. Both fragments were fused together via overlap PCR and inserted into a modified pPD95.79 containing 3 kb upstream of npr-11 (generated as described above) using XbaI and XmaI restriction sites resulting in pSP185. Primer sequences are shown in Table S2.

For expressing a variant of the nanoluciferase (Nluc, Promega, USA), fused to the N terminus of npr-11, 3 kb upstream of npr-11 was amplified from fosmid WRM0616cB05 with primer pnpr-11_SbfI_f and pnpr-11_XbaI_r attaching SbfI and XbaI restriction sites. The fragment was cloned into two modified pPD95.79 (from A. Fire, Addgene plasmid #1496) expression vectors, one with and one without a GFP, using SbfI and XbaI. npr-11 cDNA was purchased from GenScript and amplified with npr-11_nluc_f and npr-11_XmaI_r/ npr-11_XmaI_GFP_r (for sequences see Table S2) generating an overlap to the Nluc at the 5′ end and an XmaI restriction site at the 3′ end. The Nluc with an additional SGGGGS linker at the 3′ end was amplified from pNL1.3_secNluc plasmid (Promega, USA) with primers Nluc_XbaI_f and Nluc_NPR-11_r generating an XbaI restriction site at 5′ and an overlap to npr-11 at the 3′ end. The Nluc and npr-11 fragments were fused together by overlap PCR and inserted into the modified pPD95.79 with pnpr-11 using XbaI and XmaI restriction sides (pSP167 with GFP, pSP168 without GFP). Primer sequences are shown in Table S2.