Part:BBa_K2100032

pEXPR pHybrid:eYFP

This construct is an expression vector with our hybrid promoter upstream of a yellow fluorescent protein (eYFP). The hybrid promoter entry vector contains 6 estrogen responsive elements (EREs) of the sequence 5′-GGTCAnnnTGACC-3′ and 6 progesterone responsive elements (PREs) of the sequence 5′-G/A G G/T AC A/G TGGTGTTCT-3′. The EREs and the PREs are rotated one by one upstream of a minimal CMV promoter. There is no spacing between the response elements. The entry vector backbone contains an L4 site on the 5' end and an R1 site on the 3' end so that an LR reaction can be carried out to combine the hybrid promoter with another construct.

The hybrid promoter functions as both a progesterone and estrogen responsive promoter. When estrogen diffuses through the cell membrane, the ligand binds the estrogen receptor alpha (ERa) to expose the DNA binding domain. The DBD binds to the EREs on the hybrid promoter. The PRE sites act as random nucleotide spacing between the EREs, and the promoter recognizes the bound ERE sites and transcribes the eYFP gene downstream of the mimCMV. Thus, the promoter functions the same way as a pERE in the precense of estrogen.

When progesterone diffuses through the cell membrane, the ligand binds the Progesterone Receptor beta (PgR-B) to expose the DNA binding domain. The DBD binds to the PREs on the hybrid promoter. The ERE sites act as random nucleotide spacing between the PREs, and the promoter recognizes the bound PRE sites and transcribes the gene downstream of the mimCMV. Thus, the promoter functions the same way as a pPRE in the precense of progesterone.

In the presence of estrogen, the Estrogen Receptor alpha conforms to expose a DNA binding domain that can bind to the EREs, activating transcription. In the presence of progesterone, the progesterone receptor beta conforms to expose a DNA binding domain that can bind to the PREs, activating transcription of eYFP as well. Thus, no matter which ligand (estrogen or progesterone) is present in the cell, the hybrid promoter will be activated.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 301
  Illegal PstI site found at 559
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 301
  Illegal PstI site found at 559
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 301
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 301
  Illegal PstI site found at 559
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 301
  Illegal PstI site found at 559
  Illegal AgeI site found at 342
  
• 1000
  COMPATIBLE WITH RFC[1000]