KG#371
(Plasmid #110882)

• Purpose: Expresses the lysosome marker CTNS-1-mCherry cDNA in a subset of ventral cord cholinergic motor neurons
• Depositing Lab: Kenneth Miller
• Publication: Edwards et al Genetics. 2013 May;194(1):143-61. doi: 10.1534/genetics.112.147348.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2610
• Total vector size (bp): 8210
• 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: CTNS-1a-mCherry cDNA
• Species: C. elegans (nematode)
• Insert Size (bp): 1976
• Tag / Fusion Protein:
  • intron-mCherry (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): 887

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
  • Takara Bio Limited Use Label License (formerly Clontech)
• Industry Terms:
  • Not Available to Industry

Depositor Comments

We used Herculase II polymerase and primers engineered with restriction sites to amplify the 1.2 Kb ctns-1 cDNA "a" isoform (minus its stop codon and in frame with downstream RFP) from KG#354 and cloned it into Nhe I/ Age I cut KG#240 (7.2 Kb; unc-129::____-mCherry). Transformed into XL1-Blue electrocompetent cells. Qiagen miniprepped 2 clones to find those with correct insert, then confirmed insert by sequencing.

Reference: Edwards SL, Yu S, Hoover, CM, Phillips BC, Richmond JE, and Miller KG. (2013). An Organelle Gatekeeper Function for Caenorhabditis elegans UNC-16 (JIP3) at the Axon Initial Segment. Genetics. 194(1): 143-161.PMCID: PMC3632462.

Features of the construct: ctns-1a is a marker for lysosomes. An AAAA precedes the start codon for a good consensus ribosome binding site. This expression construct has a promoter (unc-129::) that will drive strong and specific expression (of whatever gene is inserted in the MCS) in 9 DA/ DB cholinergic neurons of the ventral nerve cord. The unc-129 promoter sequence includes the region from the native Sma I site (2.6 Kb upstream of the start codon) down to the 7th nucleotide before the A of the ATG. This includes a 19 bp 5' UTR (out of 25 bp 5' UTR total). The stop codon is left off of the ctns-1 cDNA for in-frame fusion to the downstream RFP (mCherry). The Age I site encodes 2 amino acids that are in frame with the downstream RFP. There is an CAG following the restriction site that is not part of the following intron. The restriction site + these 3 nucleotides will code for the amino acids Thr, Gly, and Gln and those last 3 nucleotides (CAG) provide a good consensus for preceding an intron. After this is the 44 bp intron 3 from T07H6.4 followed by the mCherry cDNA sequence (711 bp; no further introns; this is the mCherry cDNA obtained directly from the Tsien lab at UCSD) and stop codon, then the unc-54 3' control region with the 2 introns (1 just upstream of the unc-54 sequence and 1 in the unc-54 sequence) in it because the Apa I site is the 3' boundary of all of this. This vector also includes the 5' UTR intron found in most Fire lab vectors. Another feature of the expression vector include a "decoy" 5' to the promoter to reduce non-specific expression in the gut (see Fire Lab 1995 Kit documentation for description).