pAAV-FLEX-ArchT-tdTomato
(Plasmid #28305)

• Purpose: Cre-dependent viral expression of ArchT-tdTomato for neuronal inhibition
• Depositing Lab: Edward Boyden
• Publication: Han et al Front Syst Neurosci. 2011;5:18. Epub 2011 Apr 13.

Backbone

• Vector backbone: AAV-FLEX
• Backbone manufacturer: Scott Sternson
• Backbone size w/o insert (bp): 6482
• Vector type: Mammalian Expression, AAV, Cre/Lox ; Adeno-associated virus

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 30°C
• Growth Strain(s): Stbl3
• Growth instructions: Use recombinase-free E. coli (Stbl3, XL-10, Sure2 et.al), grow at 30C
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: ArchT-tdTomato
• Alt name: archaerhodopsin
• Alt name: archaerhodopsin TP009
• Alt name: ArchT
• Species: H. strain TP009
• Insert Size (bp): 744
• Mutation: N/A
• Promoter: CAG
• Tag / Fusion Protein:
  • tdTomato (C terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: KpnI (not destroyed)
• 3′ cloning site: BsrGI (not destroyed)
• 5′ sequencing primer: cttctggcgtgtgaccgg

Resource Information

• Addgene Notes:
  • Addgene diagnostic digest
• Articles Citing this Plasmid:
  • 8 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

PLEASE CONTACT ED BOYDEN () FOR DETAILS ON THIS REAGENT AND FURTHER REAGENTS IN THIS LINE.

Information for AAV5 (Catalog # 28305-AAV5)

Purpose

Ready-to-use AAV5 particles produced from pAAV-FLEX-ArchT-tdTomato (#28305). In addition to the viral particles, you will also receive purified pAAV-FLEX-ArchT-tdTomato plasmid DNA.

CAG-driven Cre-dependent ArchT-tdTomato expression for optogenetic neural inhibition. 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% Poloxamer 188
• Serotype: AAV5
• Purification: Iodixanol gradient ultracentrifugation
• Reporter Gene: tdTomato (Cre-dependent)

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.