KG#797
(Plasmid #110916)

• Purpose: Expresses the active zone - enriched protein Sentryn-GFP in a subset of cholinergic ventral nerve cord motor neurons
• Depositing Lab: Kenneth Miller
• Publication: Edwards et al Genetics. 2018 Nov;210(3):947-968. doi: 10.1534/genetics.118.301466.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2617
• Total vector size (bp): 8562
• 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: unc-129 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 2642

Cloning Information for Gene/Insert 1

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

Gene/Insert 2

• Gene/Insert name: STRN-1 (Sentryn)-GFP
• Species: C. elegans (nematode)
• Insert Size (bp): 2277
• Tag / Fusion Protein:
  • GFP (contains 3 artificial introns) (C terminal on insert)

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 intron just upstream and 1 intron in control region
• Species: C. elegans (nematode)
• Insert Size (bp): 944

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 Herculase II DNA polymerase and primers engineered with restriction sites to amplify and clone the 1.4 Kb strn-1 (C16E9.2) cDNA (minus its stop codon) from KG#720 rab-3::strn-1-FLAG into Nhe I/ Kpn I cut KG#367 (unc-129::___-GFP expression vector; 7.2 Kb) such that it is in-frame with the GFP. Transformed into XL1-Blue electrocompetent cells. Did Qiagen preps on 2 clones to find one with correct size insert for sequence verification. Verified sequence.

Features of the construct: The unc-129:: promoter drives expression in a subset of 9 DA/ DB cholinergic motor neurons. AAAA precedes the start codon to provide a consensus ribosome binding site. A non-structured linker (SSGSSG) replaces the strn-1 stop codon and is immediately followed by the Fire vector GFP with 3 artificial introns (there are also introns in the 5' and 3' UTR in this vector). 2 bases are added to maintain the reading frame with the downstream GFP when cloning into the Kpn I site (this encodes an Ala).