Part:BBa_K1119010:Design

Human Elongation Factor-1alpha Promoter

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 83
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

At the time of regional jamboree, no GFP signal could be observed in cells transfected with GFP reporter driven by EF-1alpha promoter. Originally, we thought that the sequence of EF-1alpha promoter cloned from iDUET101a contained the full functional promoter region annotated in pBudCE4.1 (Invitrogen). We believed that EF-1alpha did trigger transcription but failed to translate the GFP coding sequence due to insufficient 5’ untranslated region (UTR). After the regional jamboree, the promoter was re-cloned with additional 3' sequences after the identified TATA box to allow a longer 5’ untranslated region before the GFP coding DNA sequence. From the the results above, we believed that translation of GFP is successful this time.

Source

The EF-1alpha promoter sequence was cloned out from plasmid iDUET101a (http://www.addgene.org/17629/) using PCR with primers that includes prefix and suffix in RFC10 standard.

References

Qin, Jane Yuxia, Li Zhang, et al. "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter." PLoS ONE. 5.5 (2010) <http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010611>.

Zhou, B. Y., Ye, Z., Chen, G., Gao, Z. P., Zhang, Y. A., & Cheng, L. (2007). Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer. Stem Cells, 25(3), 779-789. doi:10.1634/stemcells.2006-0128 <http://onlinelibrary.wiley.com/doi/10.1634/stemcells.2006-0128/abstract>