pAAV-Ef1a-fDIO-GCaMP6s
(Plasmid #105714)

• Purpose: Can be used to express GCaMP6s in the presence of Flp recombinase. Can be used to create adeno-associated virus to express Flp-dependent GCaMP6s.
• Depositing Lab: Rylan Larsen
• Publication: AAV GCaMP6 Plasmids (unpublished)

Backbone

• Vector backbone: pAAV-Ef1a-fDIO hChR2(H134R)-EYFP
• Backbone manufacturer: Karl Deisseroth
• Backbone size w/o insert (bp): 5609
• Total vector size (bp): 6857
• Vector type: Mammalian Expression, AAV ; Flp/Frt

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: GCaMP6s
• Alt name: GCaMP3-K78H T302L R303P D380Y T381R S383T R392G
• Alt name: GCaMP3 variant 641
• Species: R. norvegicus (rat); A. victoria (jellyfish)
• Insert Size (bp): 1274
• Promoter: Ef1a, Human elongation factor-1 alpha

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: AscI (not destroyed)
• 5′ sequencing primer: cCTATTCTTCaAAAGGTATAGGAACTTC
• 3′ sequencing primer: tTCTAGAGAATAGGAACT

Resource Information

• Supplemental Documents:
  • pAAV-Ef1a-fDIO-GCaMP6s.gb
  • Restriction Digest - SmaI
• A portion of this plasmid was derived from a plasmid made by: GCaMP6s was synthesized de novo based on sequences published by Douglas Kim et al, Janelia Research Campus in Chen et al, 2013 PMID: 23868258.
• Articles Citing this Plasmid:
  • 4 References

Terms and Licenses

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

Information for AAV8 (Catalog # 105714-AAV8)

Purpose

Ready-to-use AAV8 particles produced from pAAV-Ef1a-fDIO-GCaMP6s (#105714). In addition to the viral particles, you will also receive purified pAAV-Ef1a-fDIO-GCaMP6s plasmid DNA.

EF1a-driven, Flp recombinase-dependent expression of GCaMP6s calcium sensor. These AAV preparations are suitable purity for injection into animals

Delivery

• Volume: 100 µL
• Titer: ≥ 1×10¹³ 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, AAV8 cap gene
• Buffer: PBS + 0.001% Poloxamer 188
• Serotype: AAV8
• 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 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 0.1-0.6% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Flp-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Flp-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Flp-independent expression.