Difference between revisions of "Part:BBa K1400000"

Revision as of 21:57, 17 October 2014

PTRE(4)GX Dual input promoter. Activation at tetO binding sites, repression at gal4 sites.

This is an engineered variant of the pGAL1 promoter native to S. cerevisiae. This dual input promoter has four upstream activating sequences (UAS) and two repressing sequences. The four UAS sites are tetO binding sites that can bind to the tetracycline responsive activator protein, rtTA (reverse tetracycline-controlled transactivator), to induce transcription. The third and fourth GAL4 binding sites of the native pGAL1 promoter are replaced with tetO sites in this version and the first two GAL4 sites are replaced with random sequences with identical C-G content . The Mig1 sequences that are native to the pGAL1 promoter are replaced with two tetO sites. The two repressing sequences are binding sites for the GAL4 DNA binding domain proximal to the TATA box, causing transcriptional repression by steric hindrance and prevention of transcription machinery assembly at the promoter. In cells expressing rtTA and GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline). The level of transcription can be modulated or repressed with the addition of β-estradiol.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 35
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 166

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 <partinfo>BBa_K1400000 short</partinfo>
-This is a dual input promoter that can be activated and repressed by two different activating proteins (Tet and Gal4). Is based off the Gal promoter in Saccharomyces cerevisiae, which is a relatively strong promoter. It has four Tetr sites upstream of the TATA box. These can be bound by the tet repressor, or by  activating variants using the Tetr binding domain like rtTA (tet binding domain, vp16 activating domain). 10bp downstream of the TATA box two Gal4 binding sites are added, which can be bound by Gal4 binding proteins or phusion activators like GEV (Gal4 binding domain, human estrogen receptor, vp16). The close proximity of these sites to the TATA box cause any binding protein (activating or repressing) to repress expression. The close proximity of these sites all seem to affect transcription rates of this promoter, as it has significantly less expression than the native Gal promoter. Thus this promoter can be used for complex control of expression.
+This is an engineered variant of the pGAL1 promoter native to S. cerevisiae. This dual input promoter has four upstream activating sequences (UAS) and two repressing sequences. The four UAS sites are tetO binding sites that can bind to the tetracycline responsive activator protein, rtTA (reverse tetracycline-controlled transactivator), to induce transcription. The third and fourth GAL4 binding sites of the native pGAL1 promoter are replaced with tetO sites in this version and the first two GAL4 sites are replaced with random sequences with identical C-G content . The Mig1 sequences that are native to the pGAL1 promoter are replaced with two tetO sites. The two repressing sequences are binding sites for the GAL4 DNA binding domain proximal to the TATA box, causing transcriptional repression by steric hindrance and prevention of transcription machinery assembly at the promoter.
+In cells expressing rtTA and GEV (GAL4 binding domain-human estrogen receptor-VP16 activator domain), this promoter can be used to drive transcription of a downstream gene by the addition of aTc (anhydrotetracycline). The level of transcription can be modulated or repressed with the addition of β-estradiol.
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