Difference between revisions of "Part:BBa K1119010:Design"

(→‎References)
(→‎References)
 
Line 14: Line 14:
 
===References===
 
===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>.
 
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>
 
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>

Latest revision as of 03:29, 29 October 2013


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>