Part:BBa_K2100031
pEXPR pPRE4:eYFP
This construct is an expression vector containing the pPREx4 promoter upstream of a yellow fluorescent protein (eYFP). pPREx4 is a synthetic mammalian promoter that responds to the progesterone receptor activated by medoxyprogesterone (MPA). pPREx4 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 four 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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal EcoRI site found at 251
Illegal EcoRI site found at 278
Illegal PstI site found at 516 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal EcoRI site found at 251
Illegal EcoRI site found at 278
Illegal PstI site found at 516 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal EcoRI site found at 251
Illegal EcoRI site found at 278 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal EcoRI site found at 251
Illegal EcoRI site found at 278
Illegal PstI site found at 516 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal EcoRI site found at 251
Illegal EcoRI site found at 278
Illegal PstI site found at 516
Illegal AgeI site found at 299 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 19
Illegal BsaI.rc site found at 265
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