pAAV-Ef1a-DIO-Synaptophysin-GCaMP6s
(Plasmid #105715)

• Purpose: In the presence of Cre, the plasmid can be used to express GCaMP6s fused to the presynaptic protein synaptophysin. Drives enriched expression of GCaMP6s in neuronal axon boutons.
• Depositing Lab: Rylan Larsen
• Publication: AAV GCaMP6 Plasmids (unpublished)

Backbone

• Vector backbone: pAAV-EF1a-Flpo
• Backbone manufacturer: Karl Deisseroth
• Backbone size w/o insert (bp): 6109
• Total vector size (bp): 7465
• Modifications to backbone: The post-transcriptional regulatory WPRE was replaced by WPRE3. The hGH polyA sequence was replaced by SV40pA.
• Vector type: Mammalian Expression, AAV, Cre/Lox

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: synaptophysin-GCaMP6s
• Alt name: synaptophysin-GCaMP3-K78H T302L R303P D380Y T381R S383T R392G
• Species: R. norvegicus (rat); A. victoria (jellyfish)
• Insert Size (bp): 1274
• GenBank ID: 24804
• Promoter: Ef1a, Human elongation factor-1 alpha

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: AscI (not destroyed)
• 3′ cloning site: RsrII (destroyed during cloning)
• 5′ sequencing primer: acttctctccaaggtttgtc
• 3′ sequencing primer: ttgatatcgaattcataact

Resource Information

• Supplemental Documents:
  • Restriction Digest - SmaI
  • AAV_Ef1a_DIO_SynGCaMP6s_WPRE3_SV40_.gb
• 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. The Synaptophysin-GCaMP6s fusion sequence was previously published by Ofer Yizhar's lab in Mahn et al, 2016 PMID: 26950004

Terms and Licenses

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

Information for AAV5 (Catalog # 105715-AAV5)

Purpose

Ready-to-use AAV5 particles produced from pAAV-Ef1a-DIO-Synaptophysin-GCaMP6s (#105715). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO-Synaptophysin-GCaMP6s plasmid DNA.

EF1a-driven, Cre-dependent GCaMP6s fused to the presynaptic protein synaptophysin. 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.