KG#908
(Plasmid #110914)

• Purpose: Expresses the plant proteins TIR1 (necessary for Auxin-Inducible Degradation) pan-neuronally in C. elegans
• Depositing Lab: Kenneth Miller
• Publication: An Intron-Compatible Marker for Long Distance CRISPR-Mediated Gene Editing in Caenorhabditis elegans (unpublished)

Backbone

• Vector backbone: pUC
• Vector type: Bacterial 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: rab-3 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 1208

Cloning Information for Gene/Insert 1

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

Gene/Insert 2

• Gene/Insert name: TIR1 cDNA
• Species: A. thaliana (mustard weed)
• Insert Size (bp): 1887

Cloning Information for Gene/Insert 2

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

Gene/Insert 3

• Gene/Insert name: unc-54 3' control region with 1 artificial intron just upstream and 1 artificial intron in control region
• Species: C. elegans (nematode)
• Insert Size (bp): 996

Cloning Information for Gene/Insert 3

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

Resource Information

• Supplemental Documents:
  • Annotated ApE and Strider formatted file--download free ApE plasmid editor to view

Terms and Licenses

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

Depositor Comments

We used Q5 polymerase and primers engineered with restriction sites to amplify the 1885 bp TIR1 sequence from pLZ31 and cloned it into Nhe I/ Kpn I cut KG#59 (4.9 Kb). Transformed into XL1-Blue electrocompetent cells. Did Qiagen preps on 2 clones to find one with correct size insert for sequence verification.

Features of the construct: The rab-3:: promoter drives expression pan-neuronally. AAAA provides a consensus ribosome binding site. TAA AT provides a consensus stop translation site. This is the Arabidopsis thaliana (mustard weed) TIR1 sequence published in the Zhang et al., 2015 Auxin Inducible Degradation paper from the Dernburg lab. It has been codon optimized for C. elegans and contains 2 synthetic introns to boost expression. It also contains 2 point mutations (D170E and M473L) shown to increase the affinity of the Arabidopsis TIR1 protein for its substrates and to increase auxin sensitivity without causing auxin-independent activity. There are also introns in the 5' and 3' UTR in this vector.