Skip to main content
This website uses cookies to ensure you get the best experience. By continuing to use this site, you agree to the use of cookies.

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more

Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at [email protected]. Learn more

KG#244
(Plasmid #110927)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 110927 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pUC
  • Backbone size w/o insert (bp) 2632
  • Total vector size (bp) 6360
  • 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

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

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

Unique multi-cloning sites: Nhe I/ Sal I/ Kpn I/ Age I/ Not I/ Bgl II

We used Pst I/ Bam H I to cut out the ~1200 bp rab-3 promoter from KG#208, leaving the 3700 bp vector fragment. To this vector fragment, we ligated the 2600 bp Pst I/ Bam H I unc-129 promoter fragment cut from KG#230. Picked 4 colonies for minipreps. Chose 1 clone with correct size insert and made glycerol stock.

Features of the expression construct: This is the same as KG#230 except it has a Not I site between the Xho I and Bgl II sites in the MC.

Features of the expression construct: 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 of the DA/ DB cholinergic neurons of the ventral nerve cord. 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). This promoter is a shortened version of the unc-129 promoter. This shortened version drives expression in 9 ventral cord motor neurons that Sieburth et al identified as the DA's. The promoter itself is described in Colavita et al., 1998, although they claim expression in both DA's and DB's. The DA motor neurons form NMJ's in the dorsal nerve cord and receive synaptic inputs in the ventral cord (therefore proteins localized to the ventral cord would be dendritically localized/ postsynaptic). 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.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    KG#244 was a gift from Kenneth Miller (Addgene plasmid # 110927 ; http://n2t.net/addgene:110927 ; RRID:Addgene_110927)
  • For your References section:

    Impaired dense core vesicle maturation in Caenorhabditis elegans mutants lacking Rab2. Edwards SL, Charlie NK, Richmond JE, Hegermann J, Eimer S, Miller KG. J Cell Biol. 2009 Sep 21;186(6):881-95. doi: 10.1083/jcb.200902095. 10.1083/jcb.200902095 PubMed 19797080