pSLPB2B-ecDHFR-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin
(Plasmid #187950)

• Purpose: ecDHFR degron-tagged dCas9 fused with tagRFPt, P2A site and tagBFP, expressed under EF1-alpha promoter in a piggyBac plasmid. Expresses Blasticidin resistance under PGK promoter.
• Depositing Lab: Edda Schulz
• Publication: Noviello et al Nat Commun. 2023 Jun 3;14(1):3225. doi: 10.1038/s41467-023-38909-4.

Backbone

• Vector backbone: pSLPB2-SpdCas9-tagRFPt-P2A-tagBFP-PGK-Blasticidin
• Backbone size w/o insert (bp): 11406
• Total vector size (bp): 12190
• Vector type: Mammalian Expression, CRISPR, Synthetic Biology
• Selectable markers: Blasticidin

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: ecDHFR-SpdCas9-tagRFPt-P2A-tagBFP
• Alt name: S. pyogenes Cas9 (D10A; N863A)
• Alt name: dihydrofolate reductase (DHFR) destabilization domain from E.coli
• Species: H. sapiens (human), Synthetic; E.coli
• Insert Size (bp): 2357
• Tag / Fusion Protein:
  • GGS linker (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: ATCAGTCTGATTGCGGCGT
• 3′ sequencing primer: TCGCCGCTCCAGAATCTC

Resource Information

• Supplemental Documents:
  • pSLPB2B-NLS-ecDHFR-linker-dCas9-tagRFPt-P2A-tagBFP-pGK-Blasticidin Bv2.gb
• A portion of this plasmid was derived from a plasmid made by: The ecDHFR degron domain was amplified from CAG-DDdCas9VP192-T2A-EGFP-ires-puro (Addgene plasmid # 69534 ; http://n2t.net/addgene:69534 ; RRID:Addgene_69534, a gift from Timo Otonkoski).

Terms and Licenses

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

Depositor Comments

Please visit https://www.biorxiv.org/content/10.1101/2022.10.05.511019v1 for bioRxiv preprint.