Part:BBa_K203110:Design
Constitutive promoter; 0.4 REU
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We performed [http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters RA-PCR] with oligos containing binding sites for some well known generally activating transcription factors (Sp1, Ap1, CREB, NF-Y) which we identified from literature search [1],[2],[3]. We also added NF-κB responsive oligos as NF-κB has non-specific activity and is therefore used by a variety of viral constitutive promoters, e.g. the HIV promoter [4]. We then cloned the construct into Part:BBa_K203112 by SpeI and HindIII to obtain a core promoter. We picked 24 colonies, two of which we dismissed after a test digest. This promoter corresponds to clone S5.
| Oligo name | Oligo Sequence | µL of Oligo used |
|---|---|---|
| NFkB responsive forward 1 | GCGATCGGCAGATCAGGGGACTTTGCCGGGTGACGGGTTCA | 0,5 |
| NFkB responsive forward 2 | GCGATCGGCAGATCAGGGGRWYYCCCGGGTGACGGGTTCA | 0,5 |
| NFkB responsive reverse | TGATCTGCCGATCGCCCGTTTCAGGGGTGAACCCGTCACCC | 1 |
| Spacer reverse | TGATCTGCCGATCGCNNNNNNNNNNTGAACCCGTCACCC | 3 |
| Sp1 responsive | GCGATCGGCAGATCAGGGGCGGGGCGGGTGACGGGTTCA | 2 |
| Ap1 responsive | GCGATCGGCAGATCATGACTCAGGGTGACGGGTTCA | 1 |
| CREB responsive | GCGATCGGCAGATCABNBVNTGACGTCAGGGTGACGGGTTCA | 1 |
| NF-Y responsive | GCGATCGGCAGATCANNCCAATNNGGGTGACGGGTTCA | 1 |
| Stop 5' | CAGTACTAGTGGGTGACGGGTTCA | 1 |
| Stop 3' | TGACAAGCTTAGTGAACCCGTCACCC | 1 |
Source
Generated in our laboratory.[5]
References
[1] Edelmann, G.M. et al. Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. PNAS 97, 3038-43 (2000).
[2] Ogawa, R. Construction of strong mammalian promoters by random cis-acting element elongation. Biotechniques 42, 628-632 (2007).
[3] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002).
[4] Rattner, A. NF-kappa B activates the HIV promoter in neurons. EMBO 12, 4261–4267 (1993).
[5] RA-PCR, a method for the generation of randomized promoter libraries. igem 2009 Heidelberg team wiki. Available online at http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters#Results