REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-KANSL3	Sigma	Cat# HPA035018, RRID: AB_10601763
Anti-HA	Cell Signaling	Cat# 3724 S, RRID: AB_1549585
Anti-GAPDH	Abcam	Cat# ab181602, RRID: AB_2630358
IRDye® 800CW Goat anti-Rabbit IgG	LI-COR Biosciences	Cat# 925-32211, RRID: AB_2651127
IRDye® 680RD Goat anti-Mouse IgG	LI-COR Biosciences	Cat# 926-68070, RRID: AB_10956588
Bacterial and virus strains
DH5alpha chemically competent cells	Lab prepred or Thermo Fisher	18265017
Chemicals, peptides, and recombinant proteins
dTAGV-1	Tocris	6914
dTAG-13-NEG	Tocris	6916
dTAG-13	Tocris	6605
Blasticidin S HCl, powder	Thermo Fisher	R21001
GlutaMAX	Thermo Fisher	35050061
2-Mercaptoethanol	Thermo Fisher	21985023
Sodium pyruvate (100 mM)	Thermo Fisher	11360070
MEM Non-Essential Amino Acids Solution (100×)	Thermo Fisher	11140050
10,000 I.U./mL Penicillin - 10,000 μg/mL Streptomycin (100×)	MSK	N/A
LIF	Lab prepared	N/A
Critical commercial assays
KAPA HiFi HotStart ReadyMix (2×)	Roche	KK2602
Eco53kI	NEB	R0116S
Calf Intestinal Alkaline Phosphatase (CIP)	NEB	M0290
QIAquick PCR & Gel Cleanup Kit (100)	QIAGEN	28506
In-Fusion® HD Cloning Plus	Takara	638911
QIAprep Spin Miniprep Kit (250)	QIAGEN	27106
X-Gal and IPTG Ready To Use Dropper	BioWorld	21530077-1
Taq DNA Polymerase Kit (1000 U)	QIAGEN	201205
BbsI-HF	NEB	R3539S
Quick Ligation Kit	NEB	M2200S
Alt-R® CRISPR-Cas9 crRNA	IDT	custom sequence
Alt-R® CRISPR-Cas9 tracrRNA	IDT	1072532
Alt-R® S.p. Cas9 Nuclease V3	IDT	1081058
SG Cell Line 4D-Nucleofector X Kit S	Lonza	V4XC-3032
P3 Primary Cell 4D-Nucleofector X Kit S	Lonza	V4XP-3032
FBS	MSK	N/A
Glasgow Minimum Essential Media	Sigma	G5154
Proteinase K	Roche	3115801001
NP-40 Substitute	Fisher Scientific	50-488-856
Tween 20	Fisher Scientific	BP337-500
Lipofectamine 2000	Thermo Fisher	11668030
Opti-MEM	Thermo Fisher	31985088
Experimental models: cell lines
E14 mouse embryonic stem cells	Joshua Brickman Lab	RRID: CVCL_C320
Oligonucleotides
M13 RP: CAGGAAACAGCTATGAC	This study	N/A
M13 FP: TGTAAAACGACGGCCAGT	This study	N/A
blast-dTAG-amp FP: TGCTTGACTACTGACA TGGCCAAGCCTTTGTCTCA	This study	N/A
blast-dTAG-amp RP: AGATCCGCCGCCACCCGACCC	This study	N/A
eGFP-dTAG-amp FP: TGCTTGACTACTGACAT GGTGTCTAAGGGCGAAGAG	This study	N/A
eSpCas9 FP: GCATATACGATACAAGGCTG	This study	N/A
K3-ki gDNA FP: CGTCCTGTTTAGGGGACGA	This study	N/A
K3-ki gDNA RP: CATGCACTGAAAGCTGGAAGAG	This study	N/A
K3-ki-sgRNA FP: CACCGGAAGTCCCTCTCCCCACCC	This study	N/A
K3-ki-sgRNA RP: AAACGGGTGGGGAGAGGGACTTCC	This study	N/A
K3 left homology arm: tccccgggtaccgagACCTTTAGC TTTTGATGAATTCATGTTTGTGATCCCTCTGG TTTGCAAAAACCTTTACCACCCAGGAAGAAAA TTGCCCAGCAAATTTAGGTTTTTGTTTTTGAGATGGGA TCTCAATATATTGCTCTGGCTGTCCTGGAACCCACTC TGTAGACTAGGCTGCCCTTGAACTCCCGGAGATCCG TCTGCTTCCGCCTCCCCAGTGCTGGGATTAAAGGCG TGCGTCACCACGCCTTGGCAATTGCAAAGGTCATG TTTTTGTGAACTGTGGAGTCTTGTCAGTTTACAGTTA TAAAATTGTGTATGGCACCGTTCTGTTTGGTGGCGG TGTGGGAGGACACATAGATGTGCATAATCAAAGA TCGAATGCTGAGCATAAAGACATGTTTTGATAG TGAAAGTTGGGAGAAATGGAAGCTATTGAATTCA CAAGCCCCCTTTCACTGCAGGTGACTAGCA TGCAGATATTCACACCCTGACTTGGCTGCTTGACTACTGAC	This study	N/A
K3 right homology arm: ggtggcggcggatctATGGCCCA TAGGGGTGGGGAGAGGGACTTCCAGACTTCAGC TCGGCGGATGGGTACCTCCCTGCTCTTCCAGC TTTCAGTGCATGAGCGGGAACTGGACCTCGTTTTCC TGGATCATAGCTATGCAAAACCGTGGAG TGCCCACCCAGATGCCAGTAGTGCCCGCCCC ACCCGCATGCTCTTTGTAACCCCCCG TCGGCAGCAGGAGAATACTATGTGAGTTTGAG AATACCATGTGAGTTTGGGTTTCCTTCTTAAGGAAGTGA TGCGTGGCGGGGCTTAGATGTTCTTAGGAGTGA TTGGGGCTGCAGGGTGAATGGAGAGCTTGGA TGAGCAGCATCAGAACGGCAGGGCAAAG TTGGACACCTACACAGACATTGCCAAACATGAG TTAGGAAAGGGAGATGCGCAAAGGAAAACAGCC TAGCTTGCTTAGTTCCCAGTAACTAGATAATTCCTTC TTCCCTTTACTGCAGCATCTAGTTTGTTTGGC AGTGGCTACAATctcgaattcactggc	This study	N/A
Recombinant DNA
pUC19	Addgene	50005
espCas9(1.1)-T2A-mCherry	A gift from Dr. Ian Chambers, Centre for Regenerative Medicine, University of Edinburgh	N/A
pCRIS-PITCH-BSD-P2A-dTAG	Addgene	91792
pLEX305-C-dTAG	Addgene	91798
pLEX305-N-dTAG	Addgene	91797
pUC19-eGFP-P2A-dTAG-Ing3	Addgene	170754
pET28a-Cas9-His	Addgene	98158
pUC19_dTAG-KANSL3-targeting	Addgene	170755
Software and algorithms
SnapGene	SnapGene	https://www.snapgene.com/
Image Studio Lite Software	LI-COR Biosciences	N/A
CHOPCHOP	http://chopchop.cbu.uib.no/	N/A
Other
KANSL3 dTAG knock-in locus.gb	Mendeley Data: https://doi.org/10.17632/mjd8zdmr6h.1	N/A