KG#121
(Plasmid #110879)

• Purpose: Expresses the C. elegans unc-31 cDNA pan-neuronally
• Depositing Lab: Kenneth Miller
• Publication: Charlie et al Genetics. 2006 Feb;172(2):943-61. doi: 10.1534/genetics.105.049577. Epub 2005 Nov 4.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2617
• Total vector size (bp): 8694
• 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): 1223

Cloning Information for Gene/Insert 1

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

Gene/Insert 2

• Gene/Insert name: unc-31 cDNA
• Species: C. elegans (nematode)
• Insert Size (bp): 3783
• Mutation: Missing first 408bp

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): 993

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

Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 3780 bp unc-31 cDNA coding region from KG#104 and cloned into Nhe I/ Age I cut KG#59 (rab-3:: expression vector). Transformed into XL1-Blue electrocompetent cells. Miniprepped 6 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and made glycerol stock.

Features of the expression construct: This expression construct has a promoter (rab-3) that will drive strong and specific expression of unc-31 cDNA in the entire nervous system of juvenile and adult animals. Other features 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), the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns (as we are) as opposed to a gene with introns. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG to provide a consensus translation start site. The sequence AT follows the stop codon and immediately precedes the 3' restriction site to provide a consensus stop translation signal. The vector provides the unc-54 3' UTR and control region.

Note: this unc-31 cDNA from lacks the first 408 bp of the full-length unc-31 cDNA, but confers full rescue activity for locomotion.