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URL: http://www.addgene.org/159743

Proper Citation: RRID:Addgene_159743

Insert Name: Nanog homeobox

Organism: Mus musculus

Bacterial Resistance: Kanamycin

Defining Citation: PMID:33091369

Vector Backbone Description: Backbone Size:2229; Vector Backbone:pENTR1a; Vector Types:Mouse Targeting; Bacterial Resistance:Kanamycin

Comments: Please note that this plasmid runs as a multimer (>14kb). While this may reduce DNA yield, the plasmid still functions as expected for generating ES cells. From original plasmid (Addgene 59995), 0.5kb of 5' and 3' homology arms were amplified with 2a-mCherry insert and cloned into pENTR backbone. A linker-venus sequence was inserted between the Nanog coding sequence and the 2a-mCherry cassette. To target Nanog, we used a "double cut donor" strategy (see DOI 10.1186/s13059-017-1164-8). In brief, we designed an sgRNA sequence to make a double strand break near the stop codon of the Nanog ORF. We then inserted flanking sgRNA recognition sequences at the 5' and 3' ends of the Nanog targeting sequence in the repair template. Cells were co-transfected with pMP108 and the Cas9/sgRNA expression plasmid.

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