Purposeexpresses EGFP tagged EWSR1 in mammalian cells
Full plasmid sequence is not available for this item.
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||46385||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 5500
Vector typeMammalian Expression
Selectable markersNeomycin (select with G418)
Growth in Bacteria
Alt nameEWS RNA-binding protein 1 isoform CRA_e
SpeciesH. sapiens (human)
Insert Size (bp)2600
Entrez GeneEWSR1 (a.k.a. EWS, EWS-FLI1, bK984G1.4)
- Promoter CMV
/ Fusion Proteins
- His (N terminal on insert)
- EGFP (N terminal on insert)
- myc (C terminal on backbone)
- His (C terminal on backbone)
- 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 (Common Sequencing Primers)
Terms and Licenses
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.
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:pcDNA3.1 His-GFP-EWSR1-myc-His was a gift from Heinz Gehring (Addgene plasmid # 46385 ; http://n2t.net/addgene:46385 ; RRID:Addgene_46385)
For your References section:Identification of proteins interacting with protein arginine methyltransferase 8: the Ewing sarcoma (EWS) protein binds independent of its methylation state. Pahlich S, Zakaryan RP, Gehring H. Proteins. 2008 Sep;72(4):1125-37. doi: 10.1002/prot.22004. 10.1002/prot.22004 PubMed 18320585