AAVS1-iSAM
(Plasmid #211495)

• Purpose: AAVS1 targeting vector containing an all-in-one DOX-inducible system for CRISPRa
• Depositing Labs: Antonella Fidanza, Lesley Forrester
• Publication: Petazzi et al eLife 2024, 13:RP94884 [Reviewed Preprint]

Backbone

• Vector backbone: pz Donor
• Total vector size (bp): 15822
• Vector type: Mammalian Expression, CRISPR, Synthetic Biology
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): Stbl3
• Copy number: Unknown

Gene/Insert 1

• Gene/Insert name: UniSAM insert
• Alt name: SV40NLS- dCas9-VP64-MS2-SV40NLS-p65-HSF1-T2A-mCherry
• Species: Synthetic
• Insert Size (bp): 6669
• Promoter: TREGV promoter
• Tag / Fusion Protein:
  • T2A-mCherry Tag

Cloning Information for Gene/Insert 1

• Cloning method: Gibson Cloning
• 5′ sequencing primer: NA

Gene/Insert 2

• Gene/Insert name: TET-ON
• Alt name: EF1a-TET-ON
• Species: Synthetic
• Insert Size (bp): 747
• Promoter: EF1a

Cloning Information for Gene/Insert 2

• Cloning method: Gibson Cloning
• 5′ sequencing primer: NA

Gene/Insert 3

• Gene/Insert name: Neo Resistance
• Alt name: SplicingAcceptor-T2A-NeoR
• Species: Synthetic
• Insert Size (bp): 858
• Promoter: Splicing acceptor

Cloning Information for Gene/Insert 3

• Cloning method: Gibson Cloning
• 5′ sequencing primer: NA

Gene/Insert 4

• Gene/Insert name: Insulator
• Alt name: chicken b-globin insulator
• Species: Synthetic
• Insert Size (bp): 574
• Promoter: NA

Cloning Information for Gene/Insert 4

• Cloning method: Gibson Cloning
• 5′ sequencing primer: NA

Resource Information

• Supplemental Documents:
  • AAVS1 iSAM.gbk

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Please note that the Addgene verified sequence identified differences in the TRE3GV promoter region; however, the plasmid still functions as expected and inducible expression has been tested variety of cell lines (HepG2, iPSCs, hESC, HeLa, HEK).