Hifi dna assembly

pTriEx-mTFP1(cp175)-Luciferase-Venus(cp173) (called here MLucV) was created by using NEB Gibson assembly (NEBuilder HiFi DNA Assembly_E5520S) taking pTriEx-mTFP1(cp175)-Barnase-Venus(cp173) as template plasmid^{27 (link),28 (link)} in which Barnase was replaced by mutant Luciferase^{6 (link)}.
To construct plasmid with a low leaky MLucV expression in Escherichia coli, MLucV was cloned into the pSE380 plasmid. MLucV was PCR amplified from the mTFP1-Luciferase-Venus-pTriEx4 plasmid as DNA template, using primers 5′-AGGAAACAGAATGGGCGGCCACCACCGC-3′ and 5′-CGCCAAAACATTAGATGTTGTGGCGGATCTTGAAGTTGG-3′. The pSE380 vector was PCR amplified using primers 5′-CAACATCTAATGTTTTGGCGGATGAGAG-3′ and 5′-GGCCGCCCATTCTGTTTCCTGTGTGAAATTG-3′ and pSE380-MLucV was created using the NEB Gibson assembly kit (NEBuilder HiFi DNA Assembly_ E5520S). All constructs were confirmed by Sanger sequencing.
GrpE and DnaJ were cloned into pET28-Smt3 expression vector. GrpE and DnaJ were first PCR amplified using E. coli genomic DNA then assembled by Gibson assembly (NEBuilder HiFi DNA Assembly_E5520S). The complete maps and sequences of the plasmids described above are available at 10.6084/m9.figshare.20502495.

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).

Blasticidin S deaminase (BSD) selection marker and a destabilization domain (DD) were cloned into lentiCRISPRv2 (Addgene, 52961) using NEBuilder HiFi DNA Assembly in a stepwise manner. Cloning of BSD was performed by assembling three components: two PCR-amplified pieces of lentiCRISPRv2 and one PCR-amplified piece of pLX304 (Addgene, 25890) with 13 bp overlaps. The lentiCRISPRv2 primers were used to remove the puromycin N-acetyltransferase selection marker. Cloning of the DD was performed by assembling two components: PCR-amplified piece of lentiCRISPRv2 BSD and PCR-amplified piece of DD-Cas9 with filler sequence and Venus (Addgene, 90085) with 25 bp overlaps. Small PCR products were purified using a PCR purification kit (QIAGEN, 28104) and large PCR products (> 6 kb) were run on a 1% agarose gel. Fragments were extracted using a gel extraction kit (QIAGEN, 28706) and were assembled by HiFi DNA Assembly according to manufacturer instructions (NEB, E2621).

All the strains used in this study are listed in Table 1.
All the plasmids used in this study are listed in Table 2. All the plasmids based on the pSB1C3 backbone were constructed through digestion and ligation. The xylA promoter was obtained by amplification of 800 bp of the regulatory area of the gene upstream of its ATG from genomic DNA of the BW25113 strain. The xylA mutated promoter was designed as shown in Fig. S5 in the supplemental material and obtained as a synthetic gene (Eurofins). EcoRI and XbaI restriction sites at the 5′ end and SpeI restriction site at the 3′ end were used for cloning on the chassis plasmid. Primers (Fig. S6) were designed from the sequences in the Ecocyc database obtained from Eurofins (France). For the construction of the double fluorescence plasmid, the pSB1C3 P_ihfBA-mTagBFP was digested with EcoRI and SpeI to be ligated in the pSB1C3 P_xylA-mRFP1 linearized with EcoRI and XbaI digestion. The plasmids based on pBR322 were constructed by DNA HiFi assembly (New England Biolabs). Fragments were amplified from E. coli DNA or plasmid purification with 20 bp of homology between each fragment. For all constructs, transformants were selected on LB plates containing 40 mg/liter of chloramphenicol. The plasmids were checked by sequencing (Eurofins). Plasmids were then transformed into E. coli BW25113 strain.