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KG#83
(Plasmid #110877)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 110877 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) 2617
  • Total vector size (bp) 8673
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Growth instructions
    Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).
  • Copy number
    High Copy

Gene/Insert 1

  • Gene/Insert name
    rab-3 promoter
  • Species
    C. elegans (nematode)
  • Insert Size (bp)
    1208

Cloning Information for Gene/Insert 1

Gene/Insert 2

  • Gene/Insert name
    acy-1 P260S gain-of-function cDNA
  • Species
    C. elegans (nematode)
  • Insert Size (bp)
    3762
  • Mutation
    Changed Proline to Serine at amino acid 260

Cloning Information for Gene/Insert 2

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)
    978

Cloning Information for Gene/Insert 3

Resource Information

Terms and Licenses

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

Depositor Comments

Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).

Used Age I/ Xho I to cut out the 3800 bp acy-1(P260S) cDNA from KG#81 and cloned into the like-digested rab-3 expression vector KG#59 (4900 bp). Transformed into XL1-Blue electrocompetent cells. Miniprepped 4 clones to find one with correct size insert and vector and made glycerol stock.

Features of the construct: This expression construct has a promoter (rab-3) that will drive strong expression of the acy-1(P260S) cDNA in the nervous system. An unc-54 3' end containing a poly A addition signal is downstream of the MCS to provide a 3' UTR for the transcript. Three introns in the UTR's are included to help expression (1 in the 5' UTR, 1 just upstream of the unc-54 3' end, and 1 in the unc-54 3' end sequence). This construct includes only the acy-1 coding region. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG. An AT immediately follows the TAA stop codon. Note: the acy-1 cDNA from which this clone was produced corresponds to the single acy-1 cDNA predicted by wormbase, which is F17C8.1 except for the gf mutation in this clone.

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#83 was a gift from Kenneth Miller (Addgene plasmid # 110877 ; http://n2t.net/addgene:110877 ; RRID:Addgene_110877)
  • For your References section:

    Mutations that rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the G alpha(s) pathway and define a third major branch of the synaptic signaling network. Schade MA, Reynolds NK, Dollins CM, Miller KG. Genetics. 2005 Feb;169(2):631-49. doi: 10.1534/genetics.104.032334. Epub 2004 Oct 16. 10.1534/genetics.104.032334 PubMed 15489510