pPMQAK1-Ptrc-[E*](104)-Cas9-B0015-lacZ-PnrsB-mazF-LVA
(Plasmid #190790)

• Purpose: [E*](104) construct. Theophylline inducible Cas9 (S. pyogenes) expression. Two BsaI-sites allow for simultaneous insertion of sgRNA and donor DNA. Includes a nickel-inducible curing system.
• Depositing Lab: Paul Hudson
• Publication: Cengic et al ACS Synth Biol. 2022 Aug 15. doi: 10.1021/acssynbio.2c00375.

Backbone

• Vector backbone: pPMQAK1
• Backbone size w/o insert (bp): 7662
• Total vector size (bp): 13148
• Modifications to backbone: A silent mutation of a BsaI-site found in the AmpR gene.
• Vector type: Bacterial Expression, CRISPR, Synthetic Biology

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin and Kanamycin, 100 & 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: Cas9
• Species: S. pyogenes
• Insert Size (bp): 4107
• Mutation: silent mutations of BpiI-sites in original Cas9 gene
• Promoter: Ptrc-[E*](104)

Cloning Information for Gene/Insert 1

• Cloning method: Restriction Enzyme
• 5′ cloning site: BpiI (destroyed during cloning)
• 3′ cloning site: BpiI (destroyed during cloning)
• 5′ sequencing primer: -
• 3′ sequencing primer: -

Gene/Insert 2

• Gene/Insert name: mazF-LVA
• Species: E. coli
• Insert Size (bp): 369
• Promoter: PnrsB
• Tag / Fusion Protein:
  • LVA (C terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BpiI (destroyed during cloning)
• 3′ cloning site: BpiI (destroyed during cloning)
• 5′ sequencing primer: -
• 3′ sequencing primer: -

Resource Information

• Supplemental Documents:
  • pPMQAK1-Ptrc-_E__(104)-Cas9-B0015-lacZ-PnrsB-mazF-LVA.gb

Terms and Licenses

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