|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||114414||Standard format: Plasmid sent in bacteria as agar stab||1||$85|
This material is available to academics and nonprofits only.
Vector typeMammalian Expression, CRISPR ; Donor Template
Growth in Bacteria
Bacterial Resistance(s)Ampicillin, 100 μg/mL
Copy numberHigh Copy
Gene/Insert nameMYL2 Homology Arms with linker-mEGFP and Cas9-excisable selection cassette
SpeciesH. sapiens (human)
Insert Size (bp)5708
Mutationhomology arms contain point mutations to disrupt crRNA binding sites used
Entrez GeneMYL2 (a.k.a. CMH10, MFM12, MLC-2, MLC-2s/v, MLC-2v, MLC2)
/ Fusion Protein
- linker-mEGFP (C terminal on insert)
- Cloning method Unknown
Terms and Licenses
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
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 BioRxiv preprint
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:AICSDP-49: MYL2-mEGFP was a gift from Allen Institute for Cell Science (Addgene plasmid # 114414 ; http://n2t.net/addgene:114414 ; RRID:Addgene_114414)
For your References section:Scarless gene tagging of transcriptionally silent genes in hiPSCs to visualize cardiomyocyte sarcomeres in live cells. Roberts B, Arakaki J, Gerbin K, Malik H, Nelson A, Hendershott M, Hookway C, Ludmann S, Mueller I, Yang R, Rafelski S, Gunawardane R. bioRxiv 342881 10.1101/342881