AICSDP-8:TOMM20-mEGFP
(Plasmid #87423)

• Purpose: Homology arms and linker-mEGFP sequence for C-terminus tagging of human TOMM20
• Depositing Lab: Allen Institute for Cell Science
• Depositing Organization: Allen Institute
  Located in United States of America
• Publication: Roberts et al Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 10.1091/mbc.E17-03-0209. Epub 2017 Aug 16.
• Citations: 6 Articles

Backbone

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

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: TOMM20 Homology Arms with linker-mEGFP
• Alt name: TOMM20
• Alt name: TOM20
• Species: H. sapiens (human)
• Insert Size (bp): 2747
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used and point mutations specific to WTC genome
• Entrez Gene: TOMM20 (a.k.a. MAS20, MOM19, TOM20)
• Tag / Fusion Protein:
  • linker-mEGFP (C terminal on insert)

Cloning Information

• Cloning method: Unknown
• 5′ sequencing primer: M13-F20
• 3′ sequencing primer: M13-Rev

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 TOMM20 in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. Linker (AA) sequence: GGSGDPPVAT. 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/ .

Please visit https://www.biorxiv.org/content/early/2017/03/31/123042 for bioRxiv preprint.

DNA (Catalog # 87423-D50)

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

• Amount: 50 μg
• Concentration: 1 μg/μL
• Pricing: $495 USD
• Storage: 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

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

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.

DNA (Catalog # 87423-D100)

Purpose

Ready-to-use, high-purity DNA prep. DNA aliquots are suitable for use in mammalian cell transfections and other molecular biology applications.

Delivery

• Amount: 100 μg
• Concentration: 1 μg/μL
• Pricing: $585 USD
• Storage: 4 ℃ (short-term) or -20 ℃ (long-term)

Terms and Licenses

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

Quality Control

Every DNA prep is verified by next-generation sequencing, confirming plasmid identity, sequence integrity, and absence of DNA contaminants. Concentration is confirmed to be within ±2.5% of the stated concentration.

Our production process has been validated to consistently yield endotoxin levels below 1.0 EU/μg. Individual lots are not tested for endotoxin.