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Addgene

pMM287
(Plasmid #46035)

Ordering

This material is available to academics and nonprofits only.
Item Catalog # Description Quantity Price (USD)
Plasmid 46035 Standard format: Plasmid sent in bacteria as agar stab 1 $85

Backbone

  • Vector backbone
    pBI
  • Backbone manufacturer
    Clontech
  • Total vector size (bp) 11707
  • Modifications to backbone
    insertion of polylinker containing Bsp1D1 and XhoI sites
  • Vector type
    Mammalian Expression ; TetOn, bidirectional promoter
  • Selectable markers
    Puromycin

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Copy number
    Low Copy

Gene/Insert

  • Gene/Insert name
    WRN K577M
  • Alt name
    Werner syndrome, RecQ helicase-like
  • Alt name
    RECQ3
  • Alt name
    RECQL2
  • Species
    H. sapiens (human)
  • Insert Size (bp)
    4514
  • Mutation
    K577M helicase-dead mutant; S2G introduced during cloning
  • GenBank ID
    NM_000553.4
  • Entrez Gene
    WRN (a.k.a. RECQ3, RECQL2, RECQL3)
  • Promoter CMV2
  • Tag / Fusion Protein
    • 5x myc (N terminal on insert)

Cloning Information

  • Cloning method Restriction Enzyme
  • 5′ cloning site BspD1 (not destroyed)
  • 3′ cloning site Xho1 (not destroyed)
  • 5′ sequencing primer Unknown
  • (Common Sequencing Primers)

Terms and Licenses

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

Depositor Comments

Bidirectional vector expresses WRN and GFP at similar expression levels. In the associated article, cells were transiently transfected with this plasmid together with a tTA transactivator plasmid (Gossen, 1992).

This is a mammalian expression vector for helicase-dead human WRN protein. It was constructed from pBI, a Clontech plasmid with a bi-directional promoter+ TRE backbone, modified by insertion of a polylinker containing BspD1 and Xhol sites followed by insertion of a myc-WRN K577M ORF- created by making K577M (A>T) mutation in pMM290 sequence.

The S2G mutation was a result of the cloning strategy to add the myc tag. The WRN protein containing S2G was biochemically verified and is wild-type in terms of activities, as described in the associated publication.

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:

    pMM287 was a gift from Raymond Monnat (Addgene plasmid # 46035 ; http://n2t.net/addgene:46035 ; RRID:Addgene_46035)
  • For your References section:

    The Werner syndrome protein has separable recombination and survival functions. Swanson C, Saintigny Y, Emond MJ, Monnat RJ Jr. DNA Repair (Amst). 2004 May 4;3(5):475-82. 10.1016/j.dnarep.2004.01.002 PubMed 15084309