CRISPRoff-v2.1
(Plasmid #167981)

• Purpose: Expresses CRISPRoff-v2.1 (DNMT3A-DNMT3L-XTEN80-dCas9-HA-2xNLS-BFP-KRAB) downstream of the CAG promoter
• Depositing Lab: Luke Gilbert
• Publication: Nuñez et al Cell. 2021 Apr 7. pii: S0092-8674(21)00353-6. doi: 10.1016/j.cell.2021.03.025.

Backbone

• Vector backbone: CAG expression plasmid
• Backbone size w/o insert (bp): 4836
• Total vector size (bp): 11874
• Vector type: Mammalian Expression

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: CRISPRoff-v2.1 (DNMT3A-DNMT3L-dCas9-BFP-KRAB)
• Species: H. sapiens (human)
• Insert Size (bp): 7038
• Promoter: CAG
• Tags / Fusion Proteins:
  • HA (C terminal on insert)
  • tagBFP (C terminal on insert)
  • 2xNLS (C terminal on insert)
  • DNMT3A-DNMT3L (N terminal on insert)
  • KRAB (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: ggcaaagaattctgcagtcg
• 3′ sequencing primer: ccaccaccttctgataggc

Resource Information

• Supplemental Documents:
  • jknp112-cag-dnmt3a-3l-dcas9-bfp-krab.gb
• A portion of this plasmid was derived from a plasmid made by: The dCas9 and KRAB sequences were obtained from a previous CRISPRi construct (Gilbert et al., 2013). The DNMT3A and DNMT3L sequences, including the D3A-D3L fusion, originated from Stepper et al. 2017. XTEN linker sequences were previously published (Schellenberger et al., 2009).
• Articles Citing this Plasmid:
  • 7 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Evrogen Limited Use Label License for FPs
• Industry Terms:
  • Not Available to Industry