pPB-Ins-TRE3Gp-KRAB-dCas9-ecDHFR-IRES-GFP-EF1Ap-Puro
(Plasmid #183410)

• Purpose: piggyBAC-based doxycycline- and trimethoprim-inducible KRAB-dCas9 CRISPR interference plasmid for enhancer and promoter repression in mammalian cells
• Depositing Lab: Azim Surani
• Publication: Tang et al Nat Cell Biol. 2022 Apr;24(4):448-460. doi: 10.1038/s41556-022-00878-z. Epub 2022 Apr 11.

Backbone

• Vector backbone: PB510B-1
• Backbone manufacturer: System Biosciences
• Backbone size w/o insert (bp): 4422
• Total vector size (bp): 13019
• Modifications to backbone: The CMV promoter (cut by SpeI and NotI) was replaced by a TRE3G promoter fragment using In-fusion cloning. Subsequently, KRAB-dCas9-ecDHFR and IRES-EGFP fragments were inserted downstream of the TRE3G promoter (NotI).
• Vector type: Mammalian Expression, CRISPR, Synthetic Biology ; piggyBAC
• Selectable markers: Puromycin

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: KRAB-dCas9-ecDHFR
• Species: Synthetic
• Insert Size (bp): 4977
• Promoter: TRE3G

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: CTTTAGGCGTGTACGGTGGG
• 3′ sequencing primer: cctaggaatgctcgtcaaga

Resource Information

• Supplemental Documents:
  • 346 pPB-Ins-TRE3Gp-Krab-dCas9(1.1)-ecDHFR-IRES-EGFP-EF1a-Puro.gbk
• A portion of this plasmid was derived from a plasmid made by: Tetracycline controllable expression system by TET Systems
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • piggyBac Limited Use Label License
  • Tet Systems Limited Use Label License
• Industry Terms:
  • Not Available to Industry