AICSDP-43: GJA1-mEGFP
(Plasmid #109120)

• Purpose: Homology arms and linker-mEGFP sequence for C-terminus tagging of human GJA1
• 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• Publication: Allen Institute for Cell Science Plasmids (unpublished)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: GJA1 Homology Arms with linker-mEGFP
• Alt name: CX43
• Species: H. sapiens (human)
• Insert Size (bp): 2732
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used
• Entrez Gene: GJA1 (a.k.a. AVSD3, CMDR, CX43, EKVP, EKVP3, GJAL, HLHS1, HSS, ODDD, PPKCA)
• Tag / Fusion Protein:
  • linker-mEGFP (C terminal on backbone)

Cloning Information

• Cloning method: Unknown

Resource Information

• Supplemental Documents:
  • annotated partial sequence from depositor

Terms and Licenses

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

Depositor Comments

This plasmid has been used with locus-specific CRISPR/Cas9 to add a mEGFP tag to the C-terminus of human GJA1 in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. Linker (AA) sequence: DPPVAT. After protein tagging 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/ .