AICSDP-28:TUBA1B-mTagRFP-T
(Plasmid #101785)

• Purpose: Homology arms and mTagRFPT-linker sequence for N-terminus (second exon) tagging of human TUBA1B
• Depositing Lab: Allen Institute for Cell Science
• Publication: Allen Institute for Cell Science Plasmids (unpublished)

Backbone

• Vector backbone: pUC19
• Backbone manufacturer: New England BioLabs
• 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: TUBA1B Homology Arms with mTagRFPT-linker
• Alt name: TUBA1B
• Species: H. sapiens (human)
• Insert Size (bp): 2746
• Mutation: homology arms contain point mutations to disrupt crRNA binding sites used
• Entrez Gene: TUBA1B (a.k.a. K-ALPHA-1)
• Tag / Fusion Protein:
  • mTagRFPT-linker (N terminal on backbone)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: unknown

Resource Information

• Supplemental Documents:
  • Annotated genbank file
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Industry MTA
  • Evrogen Ancillary Agreement for FP Distribution to Industry

Depositor Comments

This plasmid has been used with locus-specific CRISPR/Cas9 to add a mTagRFP-T tag to the N-terminus of human TUBA1B in WTC human induced pluripotent stem cells by the Allen Institute for Cell Science. Linker (AA) sequence: GGSGGS. 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/ .