Azyx 1 gblock

For azyx-1 localization, 3,474 bp upstream of the azyx-1 stop codon were amplified by PCR from wild-type genomic DNA, along with 558 bp of azyx-1 3′ UTR and fused to 5′ and 3′ ends of mNeonGreen (minus its start-AUG) using HiFi DNA assembly (NEBuilder). The resultant linear transgene was purified (Wizard Genomic DNA purification kit, Promega) and confirmed by sequencing (oligos p001-p006, S3 Table), and injected into wild-type N2 to generate C. elegans strain LSC1959 (see “Transgenesis” and S1 Table). For overexpression and rescue strains (LSC1950, LSC1951, LSC1960, LSC1997, LSC1999, LSC2001, LSC2052, LSC2053, LSC2055; S1 Table), a 757 bp promoter region upstream of azyx-1 was amplified, as were 535 bp of its 3′ UTR. Next, these were fused to 5′ and 3′ ends of a 587 bp synthesized azyx-1 gBlock (Integrated DNA Technologies (IDT); containing all azyx-1 exons and its first intron, with the ATG at the zyx-1a start mutated to CTG) using HiFi DNA assembly (NEBuilder) and confirmed by sequencing (oligos p007-p010, S3 Table). To build the neuronal marker transgene, mCherry was fused to 1,800 bp of the unc-47 promoter region and 497 bp of the unc-47 3′ UTR using HiFi DNA assembly (NEBuilder) and confirmed by sequencing (oligos p011-p016, S3 Table).

For azyx-1 localization, 3,474 bp upstream of the azyx-1 stop codon were amplified by PCR from wild-type genomic DNA, along with 558 bp of azyx-1 3′ UTR and fused to 5′ and 3′ ends of mNeonGreen (minus its start-AUG) using HiFi DNA assembly (NEBuilder). The resultant linear transgene was purified (Wizard Genomic DNA purification kit, Promega) and confirmed by sequencing (oligos p001-p006, S3 Table), and injected into wild-type N2 to generate C. elegans strain LSC1959 (see “Transgenesis” and S1 Table). For overexpression and rescue strains (LSC1950, LSC1951, LSC1960, LSC1997, LSC1999, LSC2001, LSC2052, LSC2053, LSC2055; S1 Table), a 757 bp promoter region upstream of azyx-1 was amplified, as were 535 bp of its 3′ UTR. Next, these were fused to 5′ and 3′ ends of a 587 bp synthesized azyx-1 gBlock (Integrated DNA Technologies (IDT); containing all azyx-1 exons and its first intron, with the ATG at the zyx-1a start mutated to CTG) using HiFi DNA assembly (NEBuilder) and confirmed by sequencing (oligos p007-p010, S3 Table). To build the neuronal marker transgene, mCherry was fused to 1,800 bp of the unc-47 promoter region and 497 bp of the unc-47 3′ UTR using HiFi DNA assembly (NEBuilder) and confirmed by sequencing (oligos p011-p016, S3 Table).