P3-dCas9-Tet1-GFP-Puro
(Plasmid #190728)

• Purpose: Expression of human spdCas9-Tet1 fusion for targeted DNA methylation in mammalian cell. EGFP-puromycin double marker under an independent CMV promoter. Cloning backbone for spgRNA (BbsI)
• Depositing Lab: Maja Jagodic
• Publication: Implication of DNA methylation changes at chromosome 1q21.1 in the brain pathology of Primary Progressive Multiple Sclerosis (unpublished)

Backbone

• Vector backbone: pdCas9-DNMT3A-EGFP
• Total vector size (bp): 12965
• Vector type: Mammalian Expression, CRISPR
• Selectable markers: Puromycin ; EGFP

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: dCas9-tet1
• Species: H. sapiens (human)
• Mutation: Changed Pro434 codon from CCG to CCC and Gly468 from GGA to GGT to remove the BbsI sites
• Promoter: pCAG
• Tags / Fusion Proteins:
  • 3XFLAG (N terminal on insert)
  • SV40 NLS (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: BamHI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: gcctgtacgagacacggat

Resource Information

• Supplemental Documents:
  • P3-dCas9-Tet1-GFP-Puro.gb
  • P3-dCas9-Tet1-GFP-Puro.gb
• A portion of this plasmid was derived from a plasmid made by: A portion of this plasmid was derived from a plasmid made by the plasmid derived from Addgene plasmids #71666 and #84475

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Undesired BbsI restriction sites were removed from the Tet1 sequence, without affecting the amino acid sequence.