pMM289
(Plasmid #46037)

• Depositing Lab: Raymond Monnat
• Publication: Swanson et al DNA Repair (Amst). 2004 May 4;3(5):475-82.

Backbone

• Vector backbone: pBI
• Backbone manufacturer: Clontech
• Backbone size w/o insert (bp): 4400
• Total vector size (bp): 11707
• Modifications to backbone: insertion of polylinker containing Bsp1D1 and XhoI sites
• Vector type: Mammalian Expression ; TRE, 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 E84A/K577M
• Alt name: Werner syndrome, RecQ helicase-like
• Alt name: RECQ3
• Alt name: RECQL2
• Species: H. sapiens (human)
• Insert Size (bp): 4514
• Mutation: E84A/K577M exonuclease and 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

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

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 human WRN protein that lacks exonuclease or helicase activity by virtue of EB4A (exo) and K577M (helicase) inactivating residue substitutions. 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 myc-WRN E84A K577M- created by making E84A (A>C) mutation in pMM287 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.