AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE
(Plasmid #51509)

• Purpose: Can be used to generate AAV virus that will express cytoplasmic tdTomato and presynaptic (synaptophysin-fused) EGFP in the presence of Cre in neurons from the synapsin promoter
• Depositing Lab: Hongkui Zeng
• Publication: Oh et al Nature. 2014 Apr 10;508(7495):207-14. doi: 10.1038/nature13186. Epub 2014 Apr 2.

Backbone

• Vector backbone: pAAV-MCS
  (Search Vector Database)
• Backbone manufacturer: Stratagene
• Total vector size (bp): 7634
• Modifications to backbone: A fragment containing the phSyn promoter-FLEX-tdTomato-T2A-synaptophysin fused EGFP-WPRE-bGHpA was swapped in to replace the CMV promoter-MCS-hGHpA sequence. Total insert size including the two ITRs is 5037 bp.
• Vector type: AAV

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: tdTomato-T2A-SypEGFP
• Alt name: tdTomato
• Alt name: EGFP
• Alt name: Synaptophysin
• Insert Size (bp): 1428
• Promoter: phSyn1

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: Not1 (not destroyed)
• 3′ cloning site: Not1 (not destroyed)
• 5′ sequencing primer: none
• 3′ sequencing primer: none

Resource Information

• Terms and Licenses:
  • UBMTA
  • Ancillary Agreement for Plasmids Containing FP Materials
  • genOway Notice of RIghts
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry
• Articles Citing this Plasmid:
  • 8 References

Information for AAV1 (Catalog # 51509-AAV1)

Purpose

Ready-to-use AAV1 particles produced from AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE (#51509). In addition to the viral particles, you will also receive purified AAV phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE plasmid DNA.

hSyn-driven, Cre-dependent expression of cytoplasmic tdTomato and presynaptic EGFP (synaptophysin-fused). These AAV preparations are suitable purity for injection into animals.

Delivery

• Volume: 100 µL
• Titer: ≥ 5×10¹² 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: tdTomato (cytoplasmic, Cre-dependent), EGFP (presynaptic, 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

Resource Information

• Terms and Licenses:
  • Ancillary Agreement for Penn Vectors
  • 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 0.01-0.03% of viral vectors in our typical production protocol. This can lead to a small number of cells exhibiting Cre-independent transgene expression in vivo. To address this, we recommend titrating to find the optimal AAV dosage required for Cre-dependent transgene expression and function in vivo. This may include reducing the viral vector dosage in order to reduce the likelihood of Cre-independent expression.