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.

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• Serotype: Select serotype for details See details about
• Pricing: Select serotype and quantity $ USD for preparation of µL virus + $30 USD for plasmid.
• How this works:
  • Place a request for a quantity of 10 (1 mL), 25 (2.5 mL), or 50 (5 mL). Our all-inclusive pricing includes DNA production and QC.
  • Addgene will quickly confirm that we can produce a high-quality prep for you.
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  • Receive your prep in 6–9 weeks after the MTA is approved by your organization.
  • Learn more about our Packaged on Request Service.

Backbone

• Vector backbone: pAAV-MCS
• 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, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): NEB Stable
• Growth instructions: Test 2-3 colonies to ensure the full sized plasmid is selected.
• 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

• Articles Citing this Plasmid:
  • 26 References

Terms and Licenses

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

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: ≥ 4×10¹² 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, AAV1 cap gene
• Buffer: PBS + 0.001% Poloxamer 188 + 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

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.