KG#65
(Plasmid #110933)

• Purpose: Expresses proteins in cholinergic neurons in the ventral nerve cord of C. elegans
• 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): 2632
• Total vector size (bp): 4198
• 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-17 beta promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 491

Cloning Information for Gene/Insert 1

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

Gene/Insert 2

• Gene/Insert name: unc-54 3' control region
• Species: C. elegans (nematode)
• Insert Size (bp): 940

Cloning Information for Gene/Insert 2

• 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

Unique multi-cloning sites: Nhe I, Sal I, Acc I, Kpn I/ Age I/ Eco RV, Xho I, Sac I, Bgl II

Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 0.5 Kb unc-17 beta promoter from RM#605p DNA (gift of Jim Rand) and cloned into Pst I/ Bam HI cut pPD96.52 (C. elegans body wall muscle expression vector; this digestion will remove the myo-3 promoter in 2 fragments and leave the 3.7 kb 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 a glycerol stock.

Features of the expression construct: This expression construct has a promoter (unc-17 beta) that will drive strong and specific expression (of whatever gene is inserted in the MCS) in the A and B class motor neurons and the AS cells of the ventral nerve cord of juvenile and adult animals (does not drive expression in VC's, but still gives full behavioral rescue of unc-17 beta mutants (by thrashing assay). The promoter is an artificial fusion of the unc-17 promoter beta region (115 bp Hind III - Xba I fragment with the unc-17 basal promoter region (378 bp region from Nar I to middle of the non-coding exon 1 of cha-1/ unc-17. 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 opposed to a gene with introns.

Note: this vector can be converted to a vector that fuses GFP, YFP, or CFP onto the C-terminus of the protein as follows:

Remove the ~1000 bp (Kpn I or Age I) + (Spe I or BsiW I [expensive, 55 C cutter with 50% activity at 37 C; heat inactivate 80 C] or Apa I) fragment containing the unc-54 3’ control region from this plasmid and replace it with the ~1800 bp like-digested fragment containing 3-intron S65C GFP (see pPD94.81 in Fire Lab plasmids), YFP (see pPD136.64 in Fire Lab plasmids) or CFP (see pPD136.61 in Fire Lab plasmids) + the unc-54 3’ UTR and control region. Note if Spe I is used to cut these Fire Lab vectors, you get 3 bands: 1100, 1800, and 4000 (total size 6.9 Kb). The 1800 bp band is the XFP-containing band.

For C-terminal fusions, remember to leave the stop codon off of the upstream protein. For the above-mentioned Fire Lab vectors, the reading frame for the downstream XFP relative to the Kpn I and Age I sites is as follows (triplets are the reading frame codons):

Kpn I
G GTA CCG GT
Age I

Alternatively, use Gibson Assembly/ NEBuilder to insert any genetically encoded fluorescent protein at the N- or C-terminus of your protein.