pcDNA3.1 His-GFP-EWSR1-myc-His
(Plasmid #46385)

• Purpose: expresses EGFP tagged EWSR1 in mammalian cells
• Depositing Lab: Heinz Gehring
• Publication: Pahlich et al Proteins. 2008 Sep;72(4):1125-37. doi: 10.1002/prot.22004.

Backbone

• Vector backbone: pcDNA3.1(-)B/myc-His
• Backbone manufacturer: Invitrogen
• Backbone size w/o insert (bp): 5500
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

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

Gene/Insert

• Gene/Insert name: EWSR1
• Alt name: EWS
• Alt name: EWS RNA-binding protein 1 isoform CRA_e
• Species: H. sapiens (human)
• Insert Size (bp): 2600
• Entrez Gene: EWSR1 (a.k.a. EWS, EWS-FLI1, bK984G1.4)
• Promoter: CMV
• Tags / Fusion Proteins:
  • His (N terminal on insert)
  • EGFP (N terminal on insert)
  • myc (C terminal on backbone)
  • His (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NheI (not destroyed)
• 3′ cloning site: EcoRI (not destroyed)
• 5′ sequencing primer: CMV-F
• 3′ sequencing primer: BGHrev

Resource Information

• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

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

Depositor Comments

The EWS-myc-His sequence was amplified by PCR using a XhoI-NotI-forward primer, a SacII His-reverse primer, and pcDNA3.1(-)B-EWS-myc-His (Addgene plasmid 46386) expression vector as a template. The resulting fragment was digested with XhoI and SacII restriction enzymes, and incorporated into the pEGFP-N2 plasmid (Clontech). The His-GFP fragment from pEGFP-N2-EWS-myc-His was amplified using a NheI-His forward and NotI-GFP-reverse primer and cloned into the pcDNA3.1(-)B-EWS-myc-His plasmid to create the final His-EGFP-EWS-myc-HIS plasmid.

This plasmid contains the Y583F point mutation which was specially introduced to disrupt NLS function. This construct can serve as a negative control for nuclear localization, as well as for tyrosine phosphorylation.