AICSDP-36: AAVS1-mEGFP (PGK)
(Plasmid #114404)

• Purpose: Homology arms and linker-mEGFP sequence for internal insertion of PGK-mEGFP at the AAVS1 safe harbor location in human cells as a reporter for cytoplasmic mEGFP
• Depositing Lab: Allen Institute for Cell Science
• Publication: Allen Institute for Cell Science Plasmids (unpublished)

Backbone

• Vector backbone: pUC57
• Vector type: Mammalian Expression, CRISPR ; Donor Template

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: AAVS1 Homology Arms with PGK driven mEGFP
• Species: H. sapiens (human)
• Insert Size (bp): 4717
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used
• Entrez Gene: AAVS1 (a.k.a. AAV)

Cloning Information

• Cloning method: Unknown

Resource Information

• Supplemental Documents:
  • annotated partial sequence from depositor
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Industry MTA

Depositor Comments

This plasmid has been used with locus-specific CRISPR/Cas9 to insert a PGK-mEGFP at the AAVS1 safe harbor locus in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. After insertion of the reporter using this donor template plasmid and CRISPR/Cas9 reagents, transfected cells may exhibit varying intensity levels of fluorescence, likely due to editing precision. To obtain cells of uniform intensity levels, see our protocol for fluorescence-assisted cell sorting and subcloning of transfected cells (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/ .