PurposeFlp-dependent Chr2-mCherry encoded in an AAV vector for rAAV production and expression in mammalian cells
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||75470||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
|AAV1||75470-AAV1||Virus (100 µL at titer ≥ 7×10¹² vg/mL) and Plasmid.|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 5667
- Total vector size (bp) 7413
Vector typeMammalian Expression, AAV, Synthetic Biology
Growth in Bacteria
Growth Strain(s)NEB Stable
Gene/Insert nameReversed ChR2(H134R)-mCherry
Insert Size (bp)1647
- Promoter CAG
- Cloning method Unknown
- 5′ sequencing primer CTCTGCTAACCATGTTCATGC
- 3′ sequencing primer GCCATACGGGAAGCAATAGC (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
Tang JC, Drokhlyansky E, Etemad B, Rudolph S, Guo B, Wang S, Ellis EG, Li JZ, Cepko CL (2016) Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. Elife 5. doi:10.7554/eLife.15312
Information for AAV1 (Catalog # 75470-AAV1) ( Back to top )
Ready-to-use AAV1 particles produced from pAAV-CAG-FLEXFRT-ChR2(H134R)-mCherry (#75470). In addition to the viral particles, you will also receive purified pAAV-CAG-FLEXFRT-ChR2(H134R)-mCherry plasmid DNA.CAG-driven, Flp recombinase-dependent expression of channelrhodopsin H134R mutant fused to mCherry. These AAV preparations are suitable purity for injection into animals.
- Volume 100 µL
- Titer ≥ 7×10¹² vg/mL
- Pricing $350 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, AAV1 cap gene
- Buffer PBS + 0.001% Pluronic F-68 + 200 mM NaCl
- Serotype AAV1
- Purification Iodixanol gradient ultracentrifugation
- Reporter Gene mCherry
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
Viral 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.
Using recombinase-dependent vectors in vivo: FRT sites in fDIO plasmids are known to recombine during DNA amplification and viral vector production, which may result in a minority of Flp-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 Flp-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 Flp-dependent transgene expression and function in vivo. This may include reducing the viral particle dosage in order to reduce the likelihood of Flp-independent expression.
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.
For your Materials & Methods section:pAAV-CAG-FLEXFRT-ChR2(H134R)-mCherry was a gift from Connie Cepko (Addgene plasmid # 75470 ; http://n2t.net/addgene:75470 ; RRID:Addgene_75470)
For viral preps, please replace (Addgene plasmid # 75470) in the above sentence with: (Addgene viral prep # 75470-AAV1)
For your References section:Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. Tang JC, Drokhlyansky E, Etemad B, Rudolph S, Guo B, Wang S, Ellis EG, Li JZ, Cepko CL. Elife. 2016 May 20;5. pii: e15312. doi: 10.7554/eLife.15312. 10.7554/eLife.15312 PubMed 27205882