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URL: http://www.addgene.org/199227

Proper Citation: RRID:Addgene_199227

Insert Name: Expression unit for PuroR.T2A.EGFP selectable and reporter markers

Organism: Synthetic

Bacterial Resistance: Ampicillin

Defining Citation: PMID:36928106

Vector Backbone Description: Backbone Size:2850; Vector Backbone:pBR322; Vector Types:Mammalian Expression, Gene targeting donor plasmid; Bacterial Resistance:Ampicillin

Comments: This target site-containing donor plasmid can be used to insert a transgene(s) at the human CLYBL safe harbor locus when delivered together with Sp-gRNA expression plasmid AY27_pU6.gRNA.CLYBL (Addgene plasmid #199238) or Sa-gRNA expression plasmid AM77_pU6.Sa-gRNA.CLYBL (Addgene plasmid #199237). Combining this donor with a S. pyogenes Cas9 or a S. aureus Cas9 nuclease allows for HMEJ-mediated gene targeting. Note: to reduce the chances for off-targeting donor DNA insertions when applying the Sp-gRNA for HMEJ-based genome editing it is preferable using AW01_pU.CAG.eSpCas9(1.1).rBGpA (Addgene plasmid #199255) or a similar construct, i.e., expressing a high specificity nuclease. Combining this donor with a S. pyogenes Cas9 nickase (e.g., Addgene plasmids #199256, #199253, #199254 or #199252) or a S. aureus Cas9 nickase (Addgene plasmid #199251) leads to seamless ITPN-mediated gene targeting.

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