pGG369
(Plasmid #165606)

• Purpose: Vector for expression of ω-dCas9 and sgRNA in E. coli: lacUV2-ω-1xFLAG-dCas9(Wt PID)-1xNLS-3xHA-1xNLS and UV5-BsaI-sgRNA (BsaI sites in place of the spacer)
• Depositing Lab: Marcus Noyes
• Publication: Goldberg et al Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.

Backbone

• Vector backbone: pB1H2_UV2-ω-dCas9::K2_UV5-sgRNA
• Backbone manufacturer: Marcus Noyes Lab
• Vector type: Bacterial Expression, CRISPR, Synthetic Biology

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): XL1 Blue
• Growth instructions: Low or intermediate copy number is expected (high copy is suppressed by the intact rop gene on this plasmid).
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: ω-1xFLAG-dCas9(PAM-interacting domain and overall coding scheme from Wt SpCas9)-1xNLS-3xHA-1xNLS and BsaI-sgRNA
• Mutation: Recoding of BsaI site in the AmpR cassette, two BsaI sites in place of the sgRNA's spacer, and KanR disruption in PID replaced with intact PID from Wt SpCas9
• Promoter: lacUV2 driving ω-dCas9 and UV5 driving sgRNA
• Tags / Fusion Proteins:
  • Omega (ω) subunit of RNA polymerase (N terminal on insert)
  • 1x FLAG (N terminal on insert)
  • SV40 NLS (C terminal on insert)
  • 3x HA (C terminal on insert)
  • c-Myc-like NLS (C terminal on insert)

Cloning Information

• Cloning method: Gibson Cloning
• 5′ sequencing primer: n/a (multiple primers used)
• 3′ sequencing primer: n/a (multiple primers used)

Resource Information

• Supplemental Documents:
  • pGG369.gbk
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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