pJF15
(Plasmid #253746)

• Purpose: Encodes GFP_AID* and pRPL18B-OsTIR1(F74G) expression cassettes. The OsTIR1(F74G) coding sequence is codon optimized for S. cerevisiae.
• Depositing Lab: Matthias Heinemann
• Publication: Heinemann Lab GFP_AID Plasmids 2026 (unpublished)

Backbone

• Vector backbone: pYTK-DN4
• Backbone manufacturer: Andreas Milias-Argeitis, Addgene Plasmid #180285 as described by Novarina et al. (2022).
• Vector type: Yeast Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert 1

• Gene/Insert name: GFP_AID*
• Alt name: pTEF1-GFP_AID*-tCYC1
• Insert Size (bp): 4569
• Promoter: TEF1
• Tag / Fusion Protein:
  • mAID*-tag (C terminal on insert)

Cloning Information for Gene/Insert 1

• Cloning method: Golden Gate
• 5′ sequencing primer: n/a

Gene/Insert 2

• Gene/Insert name: OsTIR1
• Alt name: pRPL18B-OsTIR1(F74G)-tPGK1
• Mutation: F74G
• Promoter: RPL18B

Cloning Information for Gene/Insert 2

• Cloning method: Golden Gate
• 5′ sequencing primer: n/a

Resource Information

• A portion of this plasmid was derived from a plasmid made by: GFP-AID* sequence was obtained from plasmid pG2J (Addgene plasmid #102882) described in Papagiannakis et al. (2017) Molecular Cell, 10.1016/j.molcel.2016.11.018.

Terms and Licenses

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

Depositor Comments

The golden gate (Multi-gene) plasmid assembly was performed according to the MoClo-Cloning standards described in: Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology, 4(9), 975-986.

Parts from the MoClo Yeast Toolkit (Addgene kit #1000000061) were used for MoClo assembly.

The OsTIR1(F74G) variant was originally described in Yesbolatova et al., 2020 (https://doi.org/10.1038/s41467-020-19532-z)