KG#493
(Plasmid #110932)

• Purpose: Expresses proteins in C. elegans cholinergic neurons in head, ventral nerve cord, and tail
• Depositing Lab: Kenneth Miller
• Publication: Williams et al Genes Dev. 2007 Nov 1;21(21):2731-46. doi: 10.1101/gad.1592007. Epub 2007 Oct 17.

Backbone

• Vector backbone: pUC
• Backbone size w/o insert (bp): 2632
• Total vector size (bp): 6940
• 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 promoter
• Species: C. elegans (nematode)
• Insert Size (bp): 3244

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, Sbf I, Kpn I/ Age I/ Eco RV, Bgl II

Note: this vector is the same as KG#94, except a Sbf I site replaces the Sal I site.

Synthesized 2 complementary oligos containing Nhe I and Age I sticky ends and Sbf I and Kpn I sites in between. Hybridization of the 2 oligos to each other should produce Nhe I and Age I sticky ends. After hybridization, this synthetic insert (which is non-phosphorylated on its 5' termini) was cloned into Nhe I/ Age I cut KG#94. Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones and tested for the Sbf I site by cutting with Sbf I. Chose one clone that has the Sbf I site with correct band size (and only a single band) and made a glycerol stock.

Features of the expression construct: This is a C. elegans expression vector with the unc-17 3.2 Kb promoter + a large MCS for cloning in cDNAs. Includes cha-1/unc-17 upstream region from Sna-1 (=Bst1107 I = GTA/TAC) down to first half of the 66 bp first exon (which is common to unc-17 and cha-1 transcripts but is not translated). Includes beta site so that expression occurs in most cholinergic cells. Expression still missing from VC's and some cholinergic head and tail neurons. In this construct the GFP/ 3' control region of RM#349p is replaced with the MCS II + 3' control region of pPD96.52. This includes 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.