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AICSDP-55:DMD-mEGFP
(Plasmid #159744)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 159744 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

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
    DMD Homology Arms with linker-mEGFP and Cas9-excisable selection cassette
  • Alt name
    DMD
  • Species
    H. sapiens (human)
  • Insert Size (bp)
    6410
  • Mutation
    homology arms contain point mutations to disrupt crRNA binding sites used, and WTC-11 specific homozygous SNP(s)
  • Entrez Gene
    DMD (a.k.a. BMD, CMD3B, DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270, DXS272, MRX85)
  • Tag / Fusion Protein
    • linker-mEGFP (C terminal on insert)

Cloning Information

  • Cloning method Unknown

Terms and Licenses

Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

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 DMD 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 fluorescence-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

How to cite this plasmid ( Back to top)

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-55:DMD-mEGFP was a gift from Allen Institute for Cell Science (Addgene plasmid # 159744 ; http://n2t.net/addgene:159744 ; RRID:Addgene_159744)