pIF1079
(Plasmid #237445)

• Purpose: Expression SpCas9 and Efe1-RT from bidirectional CAG promoters. SpCas9 sgRNA and Efe1 non-coding expression is driven by U6 and H1 respecitvely.
• Depositing Lab: IIya Finkelstein
• Publication: Buffington et al bioRxiv 2024.07.21.604473; doi: 10.1101/2024.07.21.604473

Backbone

• Vector backbone: RT14
• Backbone size w/o insert (bp): 5700
• Total vector size (bp): 11400
• Modifications to backbone: SpCas9 flanked by BbsI cut sites. Efe1-RT flanked by SapI cut sites. sgRNA and template region flanked by BsaI cut sites.
• Vector type: Mammalian Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: SpCas9
• Insert Size (bp): 4269
• Promoter: CAG
• Tag / Fusion Protein:
  • 3xFLAG (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Golden Gate
• 5′ sequencing primer: TTCCGTCTTCTGCCACCA

Gene/Insert 2

• Gene/Insert name: Efe1-RT
• Insert Size (bp): 1029
• Promoter: CAG

Cloning Information for Gene/Insert 2

• Cloning method: Golden Gate
• 5′ sequencing primer: TTGGAAGAGCGCCACCAT

Gene/Insert 3

• Gene/Insert name: EMX1 sgRNA
• Insert Size (bp): 96
• Promoter: U6

Cloning Information for Gene/Insert 3

• Cloning method: Golden Gate
• 5′ sequencing primer: CTTGGGTAGTTTGCAGTT

Gene/Insert 4

• Gene/Insert name: Efe1-ncRNA targeting EMX1
• Insert Size (bp): 211
• Promoter: H1

Cloning Information for Gene/Insert 4

• Cloning method: Golden Gate
• 5′ sequencing primer: GTGGCGCCCTGCAATATTT

Resource Information

• Supplemental Documents:
  • pIF_Retron Editor User-Friendly.gbk

Terms and Licenses

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

Depositor Comments

Please visit https://doi.org/10.1101/2024.07.21.604473 for bioRxiv preprint.