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URL: http://www.addgene.org/34703

Proper Citation: RRID:Addgene_34703

Insert Name: eGFP

Bacterial Resistance: Kanamycin

Defining Citation: PMID:22113462

Vector Backbone Description: Backbone Marker:Created by Köster and Fraser; Vector Backbone:act-GVP-UG; Vector Types:Xenopus expression; Bacterial Resistance:Kanamycin

Comments: The Sox2/Oct3-4 enhancer elements and a minimal promoter from the murine FGF4 were subcloned from the plasmid described in Figure 1 of Ambrosetti et al. (2000). The fragment contains six tandem repeats of the 35-bp fragment containing the Sox2/Oct3-4 heterodimer transcription factor binding domain (bd) followed by a 165-bp minimal FGF4 promoter (mFGF4). This plasmid was a gift of Claudio Basilico (New York University). To increase the FP signal, the Sox2/Oct 3-4-mFGF regulatory fragment was moved to the “act-GVP-UG” concatenated plasmid created by Köster and Fraser (2001), which was adapted from the Drosophila Gal4-UAS system (Brand and Perrimon,1993) and shown to function in Xenopus (Chae et al.,2002; Hartley et al.,2002). We replaced the minimal promoters of the act-GVP-UG plasmid with our Sox2/Oct3-mFGF regulatory fragment to control the expression of the transcriptional activator, Gal4-VP16, which in turn drives the expression of genes controlled by the upstream activating sequence (UAS). The final expression vector (abbreviated as pSox2-bd::FP) contains: 6 repeats of the Sox2/Oct3-4 transcription factor binding domain, mFGF4, Gal4-VP16, a polyadenylation site, 14 repeats of UAS, mFGF4, and eGFP (ClonTech, Palo Alto, CA). Sequencing with EGFP-N identified 1nt deletion at position 1733, 1 nt insertion at position 1214, 2 nt insertion at 1557 compared to the depositor provided sequence. These mutations are all in non-coding regions and have been shown by the depositing lab to not effect the function of the plasmid.

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