Worm Strain: RG3165
Allele Name: ve665
Gene: Y39E4B.13
Parent Strain: N2
Phenotype: Homozygous viable

Genotype: Y39E4B.13(ve665[LoxP + myo-2p::GFP::unc-54 3' UTR + rps-27p::neoR::unc-54 3' UTR + LoxP]) III.

Description: Homozygous viable. Deletion of 2573 bp with Calarco/Colaiacovo selection cassette conferring myo-2 GFP and G418 resistance inserted at break in parental strain N2. Left flanking Sequence: cttttttaaaaataaaatacgttattccca ; Right flanking sequence: gctacagtaacccgcgtggcgggacccaaa. sgRNA #1: atttattgtccgggaaatgt; sgRNA #2: accagtttcatctgtgtcga. Please reference Au et al., G3 9(1): 135-144 2019 in any work resulting from use of this mutation.

Y39E4B.13 sgRNA #1: atttattgtccgggaaatgt
Y39E4B.13 sgRNA #2: accagtttcatctgtgtcga

Left Flanking Sequence: cttttttaaaaataaaatacgttattccca
Right Flanking Sequence: gctacagtaacccgcgtggcgggacccaaa

Deletion: 2573 bp

Wildtype DNA File: Y39E4B.13_Wildtype
Insertion DNA File: Y39E4B.13_Insertion
Plasmid: Guides: pMLV-FY1 (Y39E4B.13 sgRNA #1), pMLV-FY2 (Y39E4B.13 sgRNA #2)
Plasmid: Template: pMLV-HA-FY (Y39E4B.13 repair)

QC: PPPP

Y39E4B.13 Primers:
Y39E4B.13-FGP: gcattcccgtagttgtccct
Y39E4B.13-RGP: ggcaaactgatgttcggcaa
Y39E4B.13-IFGP: gtgtgagacttcgccagtga
Y39E4B.13-IRGP: CTGACTTATCGGCCAACGGT
loxPmyo2NeoR-FCP: CTGCATGCGTCGACATAACT
loxPmyo2NeoR-RCP: CGTAGAGCTCGGTACCTCGT

Expected PCR results:
Primer Pair				WT		In/Del
Y39E4B.13-FGP	Y39E4B.13-IRGP		2756 bp		-
Y39E4B.13-RGP	Y39E4B.13-IFGP		2786 bp		-
Y39E4B.13-FGP	loxPMyo2NeoR-RCP	-		749 bp
Y39E4B.13-RGP	loxPMyo2NeoR-FCP	-		629 bp

PCR Parameters: (NEB Quick-load 2x Taq Master Mix)
(1) 95C/30 sec
(2) 95C/30 sec
(3) 58C/30 sec
(4) 68C/3:00 min
(5) To step (2) x 34 times
(6) 68C/5:00

