pMM290
(Plasmid #46038)

• 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
• 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
• Alt name: Werner syndrome, RecQ helicase-like
• Alt name: RECQ3
• Alt name: RECQL2
• Species: H. sapiens (human)
• Insert Size (bp): 4514
• Mutation: S2G introduced during cloning
• GenBank ID: NM_000553.4
• Entrez Gene: WRN (a.k.a. RECQ3, RECQL2, RECQL3)
• Promoter: CMV
• 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

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

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 catalytically active human WRN protein. lt 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 WT. Vector backbone is pMM286, myc-WRN WT came from DspD1-Xhol fragment from pMM229.

The WRN sequence in this plasmid contains a number of variations from the WTWRN sequence (NM_000553.4). These polymorphisms are either silent or do not to alter the function of the WRN protein. *The A>G nucleotide change at position 235 was introduced into the WRN sequence during the cloning to generate myc epitope-tagged WRN. The subsequent amino acid change (S2G) does not appear to have any functional consequence on the WRN problem.