Part:BBa_K5107004

ERE5

ERE5 is the five tandem repeats of the ERE minimal:(BBa_K5107001) to increase the interaction surface of the hormone receptor with the DNA.

Usage and Biology

Previously, in-cell systems have successfully used five tandem repeats of the REs for different human hormonal receptors[1]. Inserted in plasmids such as those we used for our in cell-assay, it is shown efficient to express Lac-Z when the associated hormone-receptor complex is present in the cell. In this manner, hormone work as trancription activator.

Figure 1:Schematic overview of the in cell system which uses the Lac-Z as reporter gene when hormone/edc i spresent in the cell.

Assemply

• These elements are part of the plasmid fist presented by (Miller et all,2010)[1]. For the list of the plasmid check BBa_K5107000.
• We used specific primers to amplify the REs from two of these plasmids(pRR-ERalpha-5Z,pRR-MR-5Z). The plamsids were a gift from Charles Miller (Addgene plasmid # 23061, # 23059 ; http://n2t.net/addgene:23061,http://n2t.net/addgene:23059  ; RRID:Addgene_23061,RRID:Addgene_23059).

Primers for HRE5 and ERE5

	Forward Primer	Reverse Primer
HRE5	ataggtagaNCCGTCTGGTACAGGGTGTTCTT	atgtgggNCCCCGGGAAAAAGAACACCCTGTA
ERE5	ataggtagaNCCCAGGTCAGAGTGACCTG	atgtgggNCCGACCCCGGGAAAAAGC

• After that, USER Cloning was performed so the final constucts are derived. BBa_K5107006 and BBa_K5107012

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 28
  Illegal PstI site found at 45
  Illegal PstI site found at 85
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 28
  Illegal PstI site found at 45
  Illegal PstI site found at 85
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 28
  Illegal PstI site found at 45
  Illegal PstI site found at 85
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 28
  Illegal PstI site found at 45
  Illegal PstI site found at 85
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Miller, C. A., Tan, X., Wilson, M., Bhattacharyya, S., & Ludwig, S. (2010). Single plasmids expressing human steroid hormone receptors and a reporter gene for use in yeast signaling assays. Plasmid, 63(2), 73–78. https://doi.org/10.1016/j.plasmid.2009.11.003