pYDE009
(Plasmid #194151)

• Purpose: Delivery of dcas9 into A. baumannii via Tn7 for CRISPRi. Tn7 element has tetR-tetP-dcas9, GmR; plasmid backbone is CarbR.
• Depositing Lab: Edward Geisinger
• Publication: Bai et al J Bacteriol. 2021 May 20;203(12):e0056520. doi: 10.1128/JB.00565-20. Epub 2021 Mar 29.

Backbone

• Vector backbone: pUC18T-mini-Tn7T-Gm
• Backbone size w/o insert (bp): 4569
• Total vector size (bp): 9673
• Modifications to backbone: The tetR-tetP-dcas9-rrnB T1-T7 Te fragment from pdCas9 bacteria was amplified and cloned using SpeI and PstI sites in pUC18T-mini-Tn7T-Gm.
• Vector type: Bacterial Expression
• Selectable markers: Gentamicin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Can propagate using carbenicillin (or ampicillin) 100. Note, also has gentamicin resistance provided by the Tn7 insert. Gentamicin need not be used for propagation; rather, GmR should be verified by screening.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: tetR-tetP-dcas9-rrnB T1-T7Te
• Insert Size (bp): 5126
• Promoter: tetp

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: SpeI (not destroyed)
• 3′ cloning site: PstI (not destroyed)
• 5′ sequencing primer: na
• 3′ sequencing primer: na

Resource Information

• Supplemental Documents:
  • pYDE009(pUC18TminiTn7TGm-dCas9).gbk

Terms and Licenses

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

Depositor Comments

Note that delivery by mating requires transposase (eg pTNS3) and helper (eg RK600) plasmids.