{"text_format":"2","workbook":"xlsx","n_sheets":3,"sheets":["Oligos","Strains","Plasmids"],"not_rendered":{"charts":0,"images":0,"macros":0,"external_links":0,"defined_names":0,"comments":0,"pivot_tables":0},"unopened":[],"bounds":{"document_bytes":16777216,"sheet_cells":100000,"run_cost":"one","rows_first":2000,"rows_last":500,"rows_between":2000,"halvings":0,"cell_chars":16384,"cell_head_chars":4096},"cells_cut":0,"cells_raw":0,"read":"whole","date_system":"1900"}

{"sheet":"Oligos","index":0,"n_rows":39,"n_cols":3,"rows_kept":39,"cut":false,"hidden":false,"merged":[]}
["AF#","Sequence","Description"]
["AF-JA-138","TCTTG GGAAGAGGCAGAGGATATG","AF-JA-138/139 make gRNA overlapping Asp25 of pelo-1"]
["AF-JA-139","AAAC CATATCCTCTGCCTCTTCC C","AF-JA-138/139 make gRNA overlapping Asp25 of pelo-1"]
["AF-JA-143","TCTTG TGCGTCTTTCACAAATCCA","AF-JA-143/144 make gRNA overlapping RGF motif in pelo-1"]
["AF-JA-144","AAAC TGGATTTGTGAAAGACGCA C","AF-JA-143/144 make gRNA overlapping RGF motif in pelo-1"]
["AF-JA-147","TTTTCATAGGAAAGCTTTGCAAA","AF-JA-147/150 make 1520bp amplicon over pelo-1 locus"]
["AF-JA-150","GCCAGACGATAGTAAATGTCCCT","AF-JA-147/150 make 1520bp amplicon over pelo-1 locus"]
["AF-JA-152","ACTTCATCTTTTGCAGATTCGTAGTTTTAATGGCGGAAGAGGCAGAGGATAGATCTATTTGTGAAAGACGCATTTATGCAGCATTTAATAGCACATGCAGATGCAA","ultramer to delete region between gRNAs in pelo-1 (pJA60 and pJA61). Creates BglII site."]
["AF-JA-175","TCTTG ATGGTATAGGAGCACATGG","AF-JA-175/176 make gRNA #1 in skih-2"]
["AF-JA-176","AAAC CCATGTGCTCCTATACCAT C","AF-JA-175/176 make gRNA #1 in skih-2"]
["AF-JA-177","TCTTG TAGCTGCTCACACTTCTGC","AF-JA-177/178 make gRNA #2 in skih-2"]
["AF-JA-178","AAAC GCAGAAGTGTGAGCAGCTA C","AF-JA-177/178 make gRNA #2 in skih-2"]
["AF-JA-179","CGGAACGTTTGTTCCGTTTGGAATGTCTGGAAACTCTGAACTGGCCACCAAAUAAAAGATCTTGCTGGAAAAACTGTTGTTGCTGAGTATGCAATTGCACTTTGTCAAGCAC","donor oligo for deletion between gRNAs at skih-2 locus, introduces BglII site"]
["AF-JA-180","atacgcaaacaccacaaatgacg","AF-JA-180/181 make 1498bp amplicon over skih-2 locus (438bp is HR of AF-JA-179)"]
["AF-JA-181","TTATGTGCTTGACAAAGTGCAATTG","AF-JA-180/181 make 1498bp amplicon over skih-2 locus (438bp is HR of AF-JA-179)"]
["AF-JA-884","ACGCGATTTGATGAACATgtaag","AF-JA-884/885 make 1017bp amplicon overlapping C-terminal deletion of PD4148"]
["AF-JA-885","GCCCATTCATACACAACTTCCAT","AF-JA-884/885 make 1017bp amplicon overlapping C-terminal deletion of PD4148"]
["AF-JA-214","TCTTG ATGCATTCGGTTGCCTCTG","AF-JA-214/215 make gRNA #1 in y54e10a.11 exon 5"]
["AF-JA-215","AAAC CAGAGGCAACCGAATGCAT C","AF-JA-214/215 make gRNA #1 in y54e10a.11 exon 5"]
["AF-JA-216","TCTTG CGAAGATGGTCAAAGAGCT","AF-JA-214/215 make gRNA #2 in y54e10a.11 exon 6"]
["AF-JA-217","AAAC AGCTCTTTGACCATCTTCG C","AF-JA-214/215 make gRNA #2 in y54e10a.11 exon 6"]
["AF-JA-218","tttccagATTGTGGAAATGTCGAACCAATCGATGCACTTTAACACCGCAG AATAAA AGATCT GCTCGGAAGATTGGCTCTGTTCAGTGTGGCTTCGAATTgtcggttttagt","donor oligo for bridge deletion between gRNAs in y54e10a.11. Deletes 1092 nts and inserts BglII site, leaving N-tmerinal 436 AAs "]
["AF-JA-221","ctactctctttaaaggtgcatacc","AF-JA-221/222 make 1891bp amplicon that lacks both BglII and BamHI sites over y54e10a.11 locus"]
["AF-JA-222","aatctagtcagtaaatcgacctac","AF-JA-221/222 make 1891bp amplicon that lacks both BglII and BamHI sites over y54e10a.11 locus"]
["AF-JA-167","TCTTG AATTGGACTTAGAAGTCAG","AF-JA-167/168 make gRNA that cleaves over stop codon of unc-54"]
["AF-JA-168","AAAC CTGACTTCTAAGTCCAATT C","AF-JA-167/168 make gRNA that cleaves over stop codon of unc-54"]
["AF-JA-241","CTGCTGGGATTACACATGGCATGGATGAACTATACAAAGCCTCTGACTTCGACGTCCAATTACTCTTCAACATCCCTACATGCTCTTTCTCCCTGTGCTCCCA","donor oligo for HR over unc-54::gfp stop codon, to convert stop to AatII site and ablate stop codon"]
["AF-JA-796","CTGCTGGGATTACACATGGCATGGATGAACTATACAAAGCCTCTGACTTC GAA GGT CGa GGa TCT TTg TTA ACa TGT GGT GAT GTc GAA GAA AAT CCa GGa Cct GACGTCCAATTACTCTTCAACATCCCTACATGCTCTTTCTCCCTGTGCTCCCA","donor oligo removes unc-54 stop codon and introduces T2A peptide. Flanking homologies as with AF-JA-241 (for unc-54::GFP)"]
["AF-JA-987","agccttggccttgttgagttg","AF-JA-987/988 make 917bp amplicon over e1092, without AflII site."]
["AF-JA-988","CTCAACAAAAGGATGCCTCC","AF-JA-987/988 make 917bp amplicon over e1092, without AflII site."]
["AF-JA-260","TCTTG ATAGAAAGATCTTGAATGG","AF-JA-260/261 make gRNA in unc-54(nonstop) 3’UTR"]
["AF-JA-261","AAAC CCATTCAAGATCTTTCTAT C","AF-JA-260/261 make gRNA in unc-54(nonstop) 3’UTR"]
["AF-JA-262","AATAGAATTAATTCGTCAAGAAAAGTCGAAAAAAATTGTGCTCCCTCCCCgCAgCAAGATCTTTCTATCCCAAAATCTACACAATGTTCTGTGTACACTTCTTA","donor oligo with AF-JA-260/261 gRNA. Introduces BbvI site and a frameshift which should cause a stop codon to be encountered prior to poly(A) tail."]
["AF-JA-822","CATCGGCCGCTGTCATCAGATCGCCATCTCGCGCCCGTGCCTCTGACTTC GTCTCAAAGGGTGAAGAAGATAACATGGCAATTATTAAAG","AF-JA-822/823 are donor bridge ultramers to make unc-54::mCherry. Use with mCherry amplicon."]
["AF-JA-823","GAGCACAGGGAGAAAGAGCATGTAGGGATGTTGAAGAGTAATTGGACTTA CTTATACAATTCATCCATGCCACCTGTCGAGTGCCGCCCT","AF-JA-822/823 are donor bridge ultramers to make unc-54::mCherry. Use with mCherry amplicon."]
["AF-JA-989","AACGAATTATTTCACTGTGTGC","AF-JA-989/990 make 550bp amplicon over smg-1(e1228) mab-1 site. +MboII is 40,96,101,313bp if wt, 96,101,353 if e1228"]
["AF-JA-990","GAGAAATAGCTTGAAGTTTTAGCC","AF-JA-989/990 make 550bp amplicon over smg-1(e1228) mab-1 site. +MboII is 40,96,101,313bp if wt, 96,101,353 if e1228"]
["AF-JA-267","ACACGGAGUCGACAUGC","17nt RNA for loading standard. Similar but different from AF-MS-24 in sequence"]
["AF-MS-24","AUGUACACGGAGUCGACCCGCAACGCGA","28 nt RNA oligo for PAGE size selection of ribosome footprints"]

{"sheet":"Strains","index":1,"n_rows":41,"n_cols":3,"rows_kept":41,"cut":false,"hidden":false,"merged":[]}
["Strain Number (PD)","Description","Strain Construction"]
["PD 2859","unc-54(cc2859; unc-54::gfp::stop::nonstop)I","made by CRISPR using dpy-10 as a marker, donor was PCR product (GFP S65C from pPD95_69). LACKS two silent AAG->AAA mutations in unc-54 CDS, but contains a recoded 3'UTR (marked with BglII) without stop codons. JA from 60.27.1.3"]
["PD 2865","unc-54(cc2865; unc-54::gfp::AatII::nonstop)I","made by CRISPR using dpy-10 as coconversion marker in PD 2859 background. Inserts AatII site over stop codon, and has no in-frame stop codons with unc-54::gfp. JA from PD2859-63.19.4.1"]
["PD 2882","unc-54(cc2882; unc-54(e1301)::gfp::stop::nonstop)I","made by CRISPR using dpy-10 as marker in PD2859 background. Contains e1301 mutation conferring ts Unc behavior. JA from PD2859-74.13.2.2"]
["PD 4092","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I","made by CRISPR using dpy-10 as marker in PD2859 background. Has T2A site after GFP and no in-frame stop codons."]
["PD 2854","skih-2(cc2854)IV","made using CRISPR with dpy-10 as marker, deleted ~1060nt of skih-2, likely via NHEJ. JA from 57.19.16.2"]
["PD 2849","r74.6(cc2849)III","made using CRISPR with dpy-10 as a marker in the PD1074 background. Deletion of exons 3,4,5, partial deletion of exons 2,6. Deletion spanned by inserted BglII site. JA from PD1074-54.1.3.4"]
["PD 2862","y54e10a.11(cc2862)I","made by CRISPR using dpy-10 as coconversion marker, deletes 1092 bp and inserts stop codon. Leaves N-terminus of 436 AAs"]
["PD 2860","skih-2(cc2854)IV; r74.6(cc2849)III","made by crossing using qC1 balancer from PD5760, crossing with PD2854, then with PD2849. Both loci homozygous after following by PCR."]
["PD 2870","unc-54(cc2865; unc-54::gfp::AatII::nonstop)I; skih-2(cc2854)IV","made by mating PD3562 (source of nT1 GFP) with PD1074, then mating with PD2859 (unc-54::gfp), then with PD2854 (skih-2), then picking green Uncs, then non-green progeny"]
["PD 2884","unc-54(cc2865; unc-54::gfp::AatII::nonstop)I; r74.6(cc2849)III","made by crossing through PD2860 bg, and PD2865"]
["PD 2868","y54e10a.11(cc2862);unc-54(cc2865; unc-54::gfp::AatII::nonstop)I","made by mating PD2862 with ht2 GFP balancer (fromPD 8811), then mating with PD2865 and looking for y54e10a.11 homozygotes, then look for unc-54 homozygotes (by phenotype)"]
["PD 2873","y54e10a.11(cc2862) unc-54(cc2865;unc-54::gfp::AatII::nonstop)I; skih-2(cc2854)IV","made by some combination of matings between PD3562 (source of nT1 GFP), PD2854 (skih-2), PD2869 (y54e10a.11; unc-54(cc2865)) and screening by single worm PCR"]
["PD 2886","unc-54(cc2865; unc-54::gfp::AatII::nonstop)I; r74.6(cc2849)III; skih-2(cc2854)IV","made by crossing through PD2860 bg, and PD2865. Extremely slow growing"]
["PD 2888","unc-54(cc2888; unc-54::gfp::AatII::nonsto::Stop(BbvI)::p)I","made by CRISPR using dpy-10 as marker in PD2865/1074 heterozygote, then homozygoused from Rol F1. JA from PD2865-72.24.22.2"]
["PD 4141","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4141; G416D)IV","Obtained by mutagenesis of PD4092. JAA from 4092-5.1. GGT>GAT"]
["PD 4142","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4142; G627E)IV","Obtained by mutagenesis of PD4092. JAA from 4092-4.1. GGA>GAA"]
["PD 4143","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4143; 15SS5)IV","Obtained by mutagenesis of PD4092. JAA from 4092-6.1. Has a mutation of 5'SS of intron 15 (GT>AT)."]
["PD 4144","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4144; G367E)IV","Obtained by mutagenesis of PD4092. JAA from 4092-7.1. GGA>GAA"]
["PD 4145","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; ttc-37(cc4145; Q383*)V","Obtained by mutagenesis of PD4092. JAA from 4092-8.2. CAG>TAG"]
["PD 4146","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4146; Q162*)IV","Obtained by mutagenesis of PD4092. JAA from 4092-9.2. CAA>TAA"]
["PD 4147","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4147; 5SS5)IV","Obtained by mutagenesis of PD4092. JAA from 4092-10.2. Has a mutation of 5'SS of intron 5 (GT>GA)."]
["PD 4148","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4148; Del476)IV","Obtained by mutagenesis of PD4092. JAA from 4092-11.1. Has C-terminal 476bp deletion."]
["PD 4149","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4149; 13SS5)IV","Obtained by mutagenesis of PD4092. JAA from 4092-13.2. Has a mutation of 5'SS of intron 13 (GT>AT)."]
["PD 4150","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4150; S1132L)IV","Obtained by mutagenesis of PD4092. JAA from 4092-14.2. TCA>TTA"]
["PD 4151","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4151; L1002fs)IV","Obtained by mutagenesis of PD4092. JAA from 4092-15.1. Indel over Leu 1002 changing ELG...>ETWIILSES*"]
["PD 4152","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; ttc-37(cc4152; 3SS3)V","Obtained by mutagenesis of PD4092. JAA from 4092-16.1. Has a mutation of 3'SS of intron 3 (AG>AA)."]
["PD 4153","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4153; 10SS5)IV","Obtained by mutagenesis of PD4092. JAA from 4092-17.1. Has a mutation of 5'SS of intron 10 (GT>TT)."]
["PD 4154","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; ttc-37(cc4154; D564G)V","Obtained by mutagenesis of PD4092. JAA from 4092-18.1. GAT>GGT"]
["PD 4155","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4155; T381P)IV","Obtained by mutagenesis of PD4092. JAA from 4092-20.1. ACA>CCA"]
["PD 4156","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4156; R626*)IV","Obtained by mutagenesis of PD4092. JAA from 4092-21.1. AGA>TGA"]
["PD 4157","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc4157; R464*)IV","Obtained by mutagenesis of PD4092. JAA from 4092-22.1. CGA>TGA"]
["PD 4206","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; skih-2(cc2854)IV","Made by crossing PD1099 (VC2010/PD1074 males) with PD3562 (nT1). Resulting pharyngeal GFP males mated with PD4092. Non-Unc males with pharyngeal GFP mated to PD2854. Pharyngeal GFP hermaphrodites singled, isolated Unc, then pharyngeal GFP minus. Confirm skih-2 by PCR"]
["PD 4226","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; r74.6(cc2849)III","Made using qC1 balancer (from PD2840, PD5760) with PD2849, 4092."]
["PD 4233","unc-54(cc4092; unc-54::gfp::T2A::nonstop)I; r74.6(cc2849)III; skih-2(cc2854)IV","Mated PD2854 (skih-2) males (later frozen as PD4232) with PD4226. Singled animals until found homozygote for ski, pelo, and unc-54. JAA from 1.4.X.12.1"]
["PD 2071","unc-54(e1092) smg-1(e1228)I","Source of e1092, e1228 alleles."]
["PD 1748","unc-54(e1092)I; skih-2(cc2854)IV; r74.6(cc2849)III","Made by mating skih-2(cc2854) males with PD2860, mating those males with PD2071 (smg-1(e1228),unc-54(e1092)). Took cross progeny to 16C and homozygoused skih-2(cc2854) and r74.6(cc2849) mutations. Then homozygoused smg-1(+), then unc-54(e1092)"]
["PD 1749","unc-54(e1092)I smg-1(e1228)I; skih-2(cc2854)IV; r74.6(cc2849)III","Made by mating skih-2(cc2854) males with PD2860, mating those males with PD2071 (smg-1(e1228),unc-54(e1092)). Took cross progeny to 16C and homozygoused skih-2(cc2854) and r74.6(cc2849) mutations. Then homozygoused smg-1(e1228), then unc-54(e1092)"]
["PD 1767","smg-1(e1228)I; skih-2(cc2854)IV; r74.6(cc2849)III","Made by mating skih-2(cc2854) males with PD2860, mating those males with PD2071 (smg-1(e1228),unc-54(e1092)). Took cross progeny to 16C and homozygoused skih-2(cc2854) and r74.6(cc2849) mutations. Then homozygoused smg-1(e1228), then unc-54(+). JAA from B2.2.2.23.8.3"]
["PD 1768","skih-2(cc2854)IV; r74.6(cc2849)III","Made by mating skih-2(cc2854) males with PD2860, mating those males with PD2071 (smg-1(e1228),unc-54(e1092)). Took cross progeny to 16C and homozygoused skih-2(cc2854) and r74.6(cc2849) mutations. Then homozygoused smg-1(+), then unc-54(+). JAA from B2.2.2.12.10.2.1/2/3/4/5/7/8/11"]
["PD 4112","unc-54(cc4112;unc-54::mCherry)/unc-54(cc4106;unc-54::GFP)I","Source of unc-54::gfp, unc-54::mCherry males. Obtained by CRISPRing using dpy-10 as a marker in Hawaiian background (PD2182/CB4856). Crossed with PD4106 (Hw with unc-54::GFP) subsequently. Pick mCherry+/GFP+ male to maintain."]

{"sheet":"Plasmids","index":2,"n_rows":9,"n_cols":6,"rows_kept":9,"cut":false,"hidden":false,"merged":[]}
["pJA#","common name","antibiotic marker","backbone","r'aison d'etre","Cloning notes"]
[74.0,"unc-54 Cterminal gRNA","KAN","pRB1017","CRISPRing Cterminus of unc-54","AF-JA-167/168 cloned into pRB1017"]
[78.0,"skih-2 gRNA #1","KAN","pRB1017","CRISPRing skih-2","AF-JA-175/176 cloned into pRB1017"]
[79.0,"skih-2 gRNA #2","KAN","pRB1017","CRISPRing skih-2","AF-JA-177/178 cloned into pRB1017"]
[60.0,"r74.6 gRNA #1","KAN","pRB1017","CRISPR r74.6 locus in vicinity of Asp25","AF-JA-138/139 cloned into pRB1017"]
[61.0,"r74.6 gRNA #2","KAN","pRB1017","CRISPR r74.6 in vicinity of RGF motif","AF-JA-143/144 cloned into pRB1017"]
[84.0,"y54e10a.11 exon 5 gRNA","KAN","pRB1017","CRISPRing y54e10a.11","AF-JA-214/215 cloned into pRB1017"]
[85.0,"y54e10a.11 exon 6 gRNA","KAN","pRB1017","CRISPRing y54e10a.11","AF-JA-216/217 cloned into pRB1017"]
[130.0,"unc-54(nonstop) gRNA","KAN","pRB1017","cleavage in unc-54 recoded 3'UTR to insert back in stop codon. overlaps with BglII site, so only recoded cleaver","AF-JA-260/261 cloned into pRB1017"]