pCAGGs-NLS-TIR1_P2A_NES-TIR1
(Plasmid #117699)

• Purpose: Expression of NLS-(Os)TIR1-HA and Myc-NES-(Os)TIR1from one mRNA
• Depositing Lab: Joerg Mansfeld
• Publication: Daniel et al Nat Commun. 2018 Aug 17;9(1):3297. doi: 10.1038/s41467-018-05855-5.

Backbone

• Vector backbone: custom
• Backbone manufacturer: Francis Stewart
• Backbone size w/o insert (bp): 6399
• Total vector size (bp): 10062
• Vector type: Mammalian Expression
• Selectable markers: Puromycin

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: TRANSPORT INHIBITOR RESPONSE 1
• Alt name: TIR1
• Species: Oryza sativa
• Insert Size (bp): 1794
• Mutation: codon optimized for murine expression
• Promoter: chicken beta-actin promoter
• Tags / Fusion Proteins:
  • HA tag (C terminal on insert)
  • NLS (N terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Unknown
• 5′ sequencing primer: unknown
• 3′ sequencing primer: unknown

Gene/Insert 2

• Gene/Insert name: TRANSPORT INHIBITOR RESPONSE 1
• Alt name: TIR1
• Species: Oryza sativa
• Insert Size (bp): 1794
• Mutation: codon optimized for murine expression
• Promoter: None, 2A peptide from porcine teschovirus-1 polyprotein
• Tag / Fusion Protein:
  • Myc-NES (N terminal on insert)

Cloning Information for Gene/Insert 2

• Cloning method: Restriction Enzyme
• 5′ cloning site: BsrG1 (not destroyed)
• 3′ cloning site: BsrG1 (not destroyed)
• 5′ sequencing primer: GGCTACCCATACGATGTTCC
• 3′ sequencing primer: ATGGTGGAAAATAACATATAGAC

Resource Information

• Supplemental Documents:
  • pCAGGS-NLS-TIR-HA-P2A-Myc-NES-Tir1_IRES-puro.gb
• Addgene Notes:
  • Addgene Diagnostic Digest
• A portion of this plasmid was derived from a plasmid made by: We received the backbone containing NLS-Tir1 from Francis Stewart (TU Dresden, BIOTEC Institute, Tatzberg 47-49, 01307 Dresden, Germany
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

The codon optimized NLS-TIR1 is described in:

Baker, O. et al. RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses. Sci Rep 6, 25529 (2016).

Some ambiguous bases and differences are present between the depositor sequence and Addgene's NGS sequence, but they are not expected to impact plasmid functionality.