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Purposenon-standard AAV2 rep-AAV.CAP-B22 cap plasmid with AAV cap expression controlled by a tTA-TRE amplifcation system
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Depositing Lab
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Sequence Information
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | |
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Plasmid | 175005 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $85 |
Backbone
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Vector backbonepUC57-mini
- Backbone size w/o insert (bp) 6311
- Total vector size (bp) 8543
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Vector typeMammalian Expression, AAV
Growth in Bacteria
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Bacterial Resistance(s)Ampicillin, 100 μg/mL
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Growth Temperature37°C
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Growth Strain(s)NEB Stable
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Copy numberUnknown
Gene/Insert
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Gene/Insert nameSynthetic construct isolate AAV.CAP-B22 VP1 gene
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Alt nameAAV.CAP-B22 Cap
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SpeciesSynthetic
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Insert Size (bp)2232
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Mutation7 amino acid substitution between VP1 452 and VP1 460 from NGSGQNQ to DGQSSKS
- Promoter p41
Cloning Information
- Cloning method Gibson Cloning
- 5′ sequencing primer unknown (Common Sequencing Primers)
Resource Information
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Supplemental Documents
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Article Citing this Plasmid
Terms and Licenses
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Academic/Nonprofit Terms
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Industry Terms
- Not Available to Industry
Trademarks:
- Zeocin® is an InvivoGen trademark.
Depositor Comments
This plasmid is based on the pUCmini-iCAP-PHP.eB plasmid. 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 We find that this system increases AAV titers 1.5-5 fold (Ben Deverman, Bryan Simpson and Paul Patterson, unpublished data).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. This work supported by NIH BRAIN Initiative grant UF1MH128336 . 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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
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For your Materials & Methods section:
pUCmini-iCAP-AAV.CAP-B22 was a gift from Viviana Gradinaru (Addgene plasmid # 175005 ; http://n2t.net/addgene:175005 ; RRID:Addgene_175005) -
For your References section:
AAV capsid variants with brain-wide transgene expression and decreased liver targeting after intravenous delivery in mouse and marmoset. Goertsen D, Flytzanis NC, Goeden N, Chuapoco MR, Cummins A, Chen Y, Fan Y, Zhang Q, Sharma J, Duan Y, Wang L, Feng G, Chen Y, Ip NY, Pickel J, Gradinaru V. Nat Neurosci. 2022 Jan;25(1):106-115. doi: 10.1038/s41593-021-00969-4. Epub 2021 Dec 9. 10.1038/s41593-021-00969-4 PubMed 34887588