pUCmini-iCAP-AAV.CAP-Mac
(Plasmid #200658)

• Purpose: Synthetic construct isolate AAV.CAP-Mac VP1 gene
• Depositing Lab: Viviana Gradinaru
• Publication: Chuapoco et al Nat Nanotechnol. 2023 Jul 10. doi: 10.1038/s41565-023-01419-x.

Backbone

• Vector backbone: pUC57-mini
• Vector type: Mammalian Expression, AAV

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: Synthetic construct isolate AAV.CAP-Mac VP1 gene
• Alt name: AAV.CAP-Mac
• Alt name: CAP-Mac
• Species: Synthetic
• Mutation: 7 amino acid insertion after Q588 of the AAV9 CAP gene
• Promoter: p41

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AgeI (not destroyed)
• 3′ cloning site: BsiWI (not destroyed)
• 5′ sequencing primer: unknown

Resource Information

• Supplemental Documents:
  • pUCmini-iCAP-CAP-Mac.gbk

Terms and Licenses

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

Depositor Comments

Please note that this is a non-standard rep-cap construct. It uses a tTA-TRE amplification loop to increase Capsid expression and AAV production. We find that this system increases AAV titers 1.5-5 fold (Ben Deverman, Bryan Simpson and Paul Patterson, unpublished data). This is a tet-off system, so no dox or tet is needed to turn it on. It can be used like any other rep-cap plasmid. This system should only present a problem if the rAAV genome to be packaged has a tet responsive element that directs expression of a protein that affected the health of the production cells. The introduction of an XbaI restriction site for ease of cloning introduces a K449R mutation, which does not have an overt effect on vector production or transduction.

This work was supported by funding from:
NIH BRAIN Armamentarium UF1MH128336
Michael J Fox Foundation for Parkinson's Research ASAP-020495