pAAV-flex-taCasp3-TEVp
(Plasmid #45580)

• Depositing Labs: Nirao Shah, Jim Wells
• Publication: Yang et al Cell. 2013 May 9;153(4):896-909. doi: 10.1016/j.cell.2013.04.017.

Backbone

• Vector backbone: pAAV-MCS
• Backbone manufacturer: Stratagene
• Backbone size w/o insert (bp): 5347
• Total vector size (bp): 7225
• Vector type: AAV ; Adeno Associated Viral Vector

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): Stbl2
• Growth instructions: For liquid cultures grow in LB:2XYT (1:1) overnight at 30 C.
• Copy number: Low Copy

Gene/Insert 1

• Gene/Insert name: Caspase 3
• Alt name: Casp3
• Species: H. sapiens (human), Synthetic
• Insert Size (bp): 847
• Mutation: Linker replaced with a TEV protease cleavage site
• Entrez Gene: CASP3 (a.k.a. CPP32, CPP32B, SCA-1)
• Promoter: EF1a

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: AAA GCA GCG TAT CCA C
• 3′ sequencing primer: AAT CAT GTC CCT GCC GTC GAT C

Gene/Insert 2

• Gene/Insert name: Tobacco Etch Virus protease
• Alt name: TEV protease
• Insert Size (bp): 705
• Promoter: EF1a

Cloning Information for Gene/Insert 2

• Cloning method: Unknown
• 5′ sequencing primer: TTC TCA AGC CTC AGA CAG TGG
• 3′ sequencing primer: AGA GGG GAT CGT TGT AGA AGT C

Resource Information

• Articles Citing this Plasmid:
  • 31 References

Terms and Licenses

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

Depositor Comments

The taCasp3-T2A-TEVp transgene was generated by overlapping PCR of plasmids harboring taCasp3 and TEVp (Gray et al., 2010; Cell 142, 637-646 PMID: 20723762). This transgene was inserted in reverse orientation into the plasmid pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-pA such that it replaced hChR2(H134R)-EYFP

Information for AAV5 (Catalog # 45580-AAV5)

Purpose

Ready-to-use AAV5 particles produced from pAAV-flex-taCasp3-TEVp (#45580). In addition to the viral particles, you will also receive purified pAAV-flex-taCasp3-TEVp plasmid DNA.

EF1a-driven, Cre-dependent, bicicstronic expression of designer pro-taCasp3 and TEVp. These AAV preparations are suitable purity for injection into animals.

Delivery

• Volume: 100 µL
• Titer: ≥ 7×10¹² vg/mL
• Pricing: $375 USD for preparation of 100 µL virus + $30 USD for plasmid.
• Storage: Store at -80℃. Thaw just before use and keep on ice.
• Shipment: Viral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.

Viral Production & Use

• Packaging Plasmids: encode adenoviral helper sequences and AAV rep gene, AAV5 cap gene
• Buffer: PBS + 0.001% Pluronic F-68 + 200 mM NaCl
• Serotype: AAV5
• Purification: Iodixanol gradient ultracentrifugation

Biosafety

Requestor is responsible for compliance with their institution's biosafety regulations. Lentivirus is generally considered BSL-2. AAV is generally considered BSL-1, but may require BSL-2 handling depending on the insert. Biosafety Guide

Terms and Licenses

• Academic/Nonprofit Terms:
  • Terms of Use for Viral Vectors
• Industry Terms:
  • Not Available to Industry

Viral Quality Control

Quality Control:

• Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). The specific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
• Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.

Visit our viral production page for more information.

Addgene Comments

Using FLEX vectors in vivo: LoxP sites in FLEX plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Cre-activated (i.e., "flipped") viral vectors. Addgene has measured this occurs in 0.1-0.8% of viral particles in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, it is necessary to optimize the injection volume and viral titer to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral particle dosage in order to reduce the likelihood of Cre-independent expression.