pUCmini-iCAP-PHP.N
(Plasmid #127851)

• Purpose: non-standard AAV2 rep-AAV-PHP.N cap plasmid with AAV cap expression controlled by a tTA-TRE amplification system
• Depositing Lab: Viviana Gradinaru
• Publication: Ravindra Kumar et al Nat Methods. 2020 May;17(5):541-550. doi: 10.1038/s41592-020-0799-7. Epub 2020 Apr 20.

Backbone

• Vector backbone: pUC57-mini
• Backbone size w/o insert (bp): 6314
• 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-PHP.N VP1 gene
• Alt name: AAV-PHP.N Cap
• Species: Synthetic
• Insert Size (bp): 2229
• Promoter: p41

Cloning Information

• Cloning method: Gibson Cloning

Resource Information

• Supplemental Documents:
  • pUCmini-iCAP-PHP.N.gb

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 We find that this system increases AAV titers 1.5-5 fold (Ben Deverman, Bryan Simpson and Paul Patterson, unpublished data).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.