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pAAV-Ef1a-DIO-PPO-Venus
(Plasmid #139505)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 139505 Standard format: Plasmid sent in bacteria as agar stab 1 $75
AAV9 139505-AAV9 Virus (100 µL at titer ≥ 1×10¹³ vg/mL) and Plasmid.
$380

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pAAV-Ef1a-DIO-eYFP
  • Backbone manufacturer
    Deisseroth lab
  • Backbone size w/o insert (bp) 5681
  • Total vector size (bp) 7439
  • Vector type
    Mammalian Expression, AAV

Growth in Bacteria

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

Gene/Insert

  • Gene/Insert name
    parapinopsin
  • Alt name
    PPO
  • Species
    Lethenteron camtschaticum
  • Insert Size (bp)
    1029
  • GenBank ID
    AB116380.1
  • Promoter Ef1a
  • Tag / Fusion Protein
    • Venus tag following 3x Ala linker (C terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site AscI (not destroyed)
  • 3′ cloning site NheI (not destroyed)
  • 5′ sequencing primer T7 forward primer
  • 3′ sequencing primer 5’-GGCACAGTCGAGGCGCGCCAGCGGGTTTAAACG-3’
  • (Common Sequencing Primers)

Resource Information

  • Supplemental Documents
  • A portion of this plasmid was derived from a plasmid made by
    Akihisa Terakita, Osaka University, Japan

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

See Koyanagi et al., PNAS 2004 (PMID: 15096614) for original cloning of lamprey parapinopsin. Please visit https://www.biorxiv.org/content/10.1101/2021.02.19.432008v1 for bioRxiv preprint.

Information for AAV9 (Catalog # 139505-AAV9) ( Back to top )

Purpose

Ready-to-use AAV9 particles produced from pAAV-Ef1a-DIO-PPO-Venus (#139505). In addition to the viral particles, you will also receive purified pAAV-Ef1a-DIO-PPO-Venus plasmid DNA.

EF1a-driven, Cre-dependent expression of Venus-tagged parapinopsin for spatiotemporal control of inhibitory GPCR signaling cascades. These AAV preparations are suitable purity for injection into animals.

Delivery

  • Volume 100 µL
  • Titer ≥ 1×10¹³ 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, AAV9 cap gene
  • Buffer PBS + 0.001% Pluronic F-68
  • Serotype AAV9
  • Purification Iodixanol gradient ultracentrifugation
  • Reporter Gene Venus (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

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.

How to cite this plasmid ( Back to top)

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-Ef1a-DIO-PPO-Venus was a gift from Michael Bruchas (Addgene plasmid # 139505 ; http://n2t.net/addgene:139505 ; RRID:Addgene_139505)

    For viral preps, please replace (Addgene plasmid # 139505) in the above sentence with: (Addgene viral prep # 139505-AAV9)

  • For your References section:

    A photoswitchable GPCR-based opsin for presynaptic inhibition. Copits BA, Gowrishankar R, O'Neill PR, Li JN, Girven KS, Yoo JJ, Meshik X, Parker KE, Spangler SM, Elerding AJ, Brown BJ, Shirley SE, Ma KKL, Vasquez AM, Stander MC, Kalyanaraman V, Vogt SK, Samineni VK, Patriarchi T, Tian L, Gautam N, Sunahara RK, Gereau RW 4th, Bruchas MR. Neuron. 2021 Jun 2;109(11):1791-1809.e11. doi: 10.1016/j.neuron.2021.04.026. Epub 2021 May 11. 10.1016/j.neuron.2021.04.026 PubMed 33979635