Difference between revisions of "Part:BBa K2100030"
 
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<partinfo>BBa_K2100030 short</partinfo>
 
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The entry vector contains the PREx3 promoter, consisting of 3 progesterone responsive elements of the sequence 5'-G/A G G/T AC A/G TGGTGTTCT-3' upstream of a minimal CMV promoter. The PREs are interspaced according to the spacing on the TRE promoter.
 
  
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When progesterone diffuses through the cell membrane of an endometrial cell, it binds to the ligand binding domain on PgR-beta (Progesterone Receptor beta) and exposes its DNA binding domain. The DBD can then bind to the PREs on the promoter, recruiting transcriptional machinery and activating transcription.  
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This construct is an expression vector containing the pPREx3 promoter upstream of a yellow fluorescent protein (eYFP). pPREx3 is a synthetic mammalian promoter that responds to the progesterone receptor activated by medoxyprogesterone (MPA). pPREx3 consists of three repeats of the progesterone response element (PRE) consensus sequence upstream of a minimal promoter (minCMV).
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In endometrial cells, progesterone receptors facilitate the cell's response to progesterone, initiating the secretory phase of the menstrual cycle. Mechanistically, progesterone diffuses through the cell membrane and binds to the ligand binding domain of the PgR-beta isoform. The DNA-binding domain of the progesterone receptor becomes exposed. The receptor then relocalizes to the nucleus from the cytoplasm, recruits co-activators, and acts as a transcription factor. The eYFP gene is then transcribed. There are two isoforms of the progesterone receptor, PgR alpha and PgR beta. In the context of our project, we are working with PgR beta.
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Our team tested different possible variations of an estrogen-responsive promoter by varying the amount of progesterone response elements (PREs) present for the activated PgR complex to bind to. This particular construct contains three progesterone responsive elements with the sequence 5′-G/A G G/T AC A/G TGGTGTTCT-3′. We interspaced these elements with 22 randomly selected bases in between, the same spacing between TREs in the TRE promoter.  Since we wanted to decrease the probability of helical turns inhibiting the transcriptional activity of promoters, we modeled the spacing after the TRE promoter.
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Latest revision as of 05:32, 19 October 2016


pEXPR pPRE3:eYFP

This construct is an expression vector containing the pPREx3 promoter upstream of a yellow fluorescent protein (eYFP). pPREx3 is a synthetic mammalian promoter that responds to the progesterone receptor activated by medoxyprogesterone (MPA). pPREx3 consists of three repeats of the progesterone response element (PRE) consensus sequence upstream of a minimal promoter (minCMV).

In endometrial cells, progesterone receptors facilitate the cell's response to progesterone, initiating the secretory phase of the menstrual cycle. Mechanistically, progesterone diffuses through the cell membrane and binds to the ligand binding domain of the PgR-beta isoform. The DNA-binding domain of the progesterone receptor becomes exposed. The receptor then relocalizes to the nucleus from the cytoplasm, recruits co-activators, and acts as a transcription factor. The eYFP gene is then transcribed. There are two isoforms of the progesterone receptor, PgR alpha and PgR beta. In the context of our project, we are working with PgR beta.

Our team tested different possible variations of an estrogen-responsive promoter by varying the amount of progesterone response elements (PREs) present for the activated PgR complex to bind to. This particular construct contains three progesterone responsive elements with the sequence 5′-G/A G G/T AC A/G TGGTGTTCT-3′. We interspaced these elements with 22 randomly selected bases in between, the same spacing between TREs in the TRE promoter. Since we wanted to decrease the probability of helical turns inhibiting the transcriptional activity of promoters, we modeled the spacing after the TRE promoter.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 211
    Illegal PstI site found at 466
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 211
    Illegal PstI site found at 466
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 211
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 211
    Illegal PstI site found at 466
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 211
    Illegal PstI site found at 466
    Illegal AgeI site found at 249
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 7
    Illegal BsaI.rc site found at 225