NMp3610

synthetic codon optimized S. kuruvilia GAL4 DNA BD (aa 1 to 147 ) with N -terminal NLS and 1 intron

NMp3710

Synthetic codon optimized S. kuruvilia GAL4 DNA binding domain in a SapI GGT GCG assembly vector. NMp3697 was digested with SapI and a synthetic DNA fragment (3610, Table S3) encoding a C. elegans codon optimized version of the S. kudriavzevii GAL4 DNA binding domain was digested with SapI and inserted by ligation.

pCS410

pCS410 was created by modifying plasmid pRL475 (Lin, 2003). DNA encoding a codon-optimized S tag and tobacco etch virus (TEV) protease cleavage site was created using gene synthesis (Hoover and Lubkowski, 2002) and ligated into a NheI site created between OMA-1 and GFP coding sequence.

pNP77

"we cloned an amplicon encompassing the entire genomic ceh-17 coding sequence"; "To ectopically express ceh-17 in glr-1+ neurons, we cloned the same ceh-17 genomic fragment as above into the NheI-NcoI sites of the PV6 plasmid, containing the glr-1 promoter (a gift from S. Clark) to produce pglr-1::ceh-17 (pNP77)."

pCE2-2

To generate a repair template (pCE2-2), a 5'-lin-41 homology arm was generated by PCR using primers GA-B52 and GA-1R and a lin-41 fosmid (WRM064dG06) as template. To generate the 3'-lin-41 homology arm, nested PCR was performed using primers lin-41-1F and lin-41-1R, followed by GA-B3 and GA-3F. The gfp::tev::s insert was generated by PCR using primers GA-2862F and GA-2R with pIC26 as the template. A four-piece Gibson assembly (New England BioLabs) was used to stitch together the 5'-lin-41 homology arm, the gfp::tev::s insert, the 3'-lin-41 homology arm, and the pBluescript KS-vector, digested with SacI and KpnI. Site-directed mutagenesis was performed using a Q5 site-directed mutagenesis kit (New England BioLabs) and primers C13-PAMF and C13-PAMR to alter the PAM site from TTGG to CTTG without changing the coding sequence of LIN-41.

pJP#38

A fusion between the GFP gene of the vector pPD95.81 (A. Fire, S. Xu, J. Ahnn and G. Seydoux, personal communication) and the first six codons of cdh-3. A KpnI site was introduced into the fifth codon of cdh-3 by amplification of genomic DNA with primers CDH3-2 (5'-TACTTTCAAGGATCTCGACTG-3', located 270 bp upstream of the start codon) and CDH3-5 (5'GGTACCCGTATTGTCATCTATTCAG-3', which contains the first four codons of cdh-3). The amplification product was used to replace the corresponding region of cdh-3 and together with the 6 kb upstream presumptive promoter region contained in pJP#30, cloned into the KpnI site of pPD95.81.

pTG96

pTG96 was constructed for the purpose of assessing the pattern of expression of sur-5, a gene encoding a novel protein that was identified in a genetic screen for suppressors of a vulvaless phenotype associated with certain dominant-negative mutations in let-60Ras (Gu et al. 1998). The plasmid contains 7.3 kb of genomic DNA from the sur-5 locus and a vector, pPD95.70 (A. Fire, J. Ahnn, G. Seydoux and S. Xu, personal communication), whose expres- sion of GFP is dependent on the addition of heterologous DNA. The form of GFP encoded by the vector has a nuclear localization signal (A. Fire, personal communication) and an amino acid change (cysteine for serine at position 65) that increases the intensity of green fluorescence (Heim et al. 1995). Also, the DNA encoding GFP contains artificial introns for enhanced expression in transgenic C. elegans (A. Fire, personal communication). | pTG96 includes a 3.68-kb fragment of the 5-flanking sequence and the full-length sur-5 genomic sequence fused at its C terminus to GFP containing a potent, artificial nuclear localization signal (NLS) sequence.

WBCnstr00017906

S/T residues converted to A

WBCnstr00017907

S/T residues converted to A

WBCnstr00017903

S/T residues converted to A