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URL: http://www.addgene.org/114414

Proper Citation: RRID:Addgene_114414

Insert Name: MYL2 Homology Arms with linker-mEGFP and Cas9-excisable selection cassette

Organism: Homo sapiens

Bacterial Resistance: Ampicillin

Defining Citation: PMID:30956114

Vector Backbone Description: Vector Backbone:pUC57; Vector Types:Mammalian Expression, CRISPR, Donor Template; Bacterial Resistance:Ampicillin

Comments: This plasmid has been used with locus-specific CRISPR/Cas9 in a two-step editing process to add a mEGFP tag to the C-terminus of human MYL2 in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. After initial HDR, stable expression of CAGGS-driven mCherry will indicate cells with integrated donor sequence. This selection cassette is designed to be removed with subsequent CRISPR/Cas9-mediated excision as previously described (https://doi.org/10.1101/342881). A linker of 3-21 amino acids will be produced after excision. To enrich for edited cells, see our protocol for fluorsecence-assisted cell sorting and subcloning of hiPSCs (https://www.allencell.org/instructional-videos-and-tutorials-for-cell-methods.html). Further, we recommend PCR-based assays for identifying precisely edited clones as previously described (https://www.molbiolcell.org/doi/abs/10.1091/mbc.e17-03-0209). For more information on the entire plasmid collection, please see https://www.addgene.org/allen-institute-cell-science/. Please visit https://www.biorxiv.org/content/early/2018/06/09/342881 for the bioRxiv preprint.

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