Part:BBa_K2217013

CAG Promoter

We tried to improve CAG promoter at BBa_K1179069 by making it RFC 10 compatible. CMV enhancer + chicken beta-actin promoter, the part was improved by sequence adjustment to gene block synthesis requirements The sequence GGC was repeated 6 consecutive times starting at base 538 and a homopolymer run of 16 G bases is present starting at base 413 was reduced less than 9.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

In 2017, AFCM-Egypt submitted the CAG promoter ( Part:BBa_K2217013), which is made up of CMV enhancer + chicken beta-actin promoter, and CAG promoter is recognized as a strong hybrid mammalian promoter. However, we found that the CAG promoter ( Part:BBa_K2217013) of AFCM-Egypt had no quantitative data until now. Therefore, we planned to measure the strength of CAG promoter (pCAG) and compare it with other three promoters: CMV promoter (pCMV), EF1a promoter (pEF1a) and Ubiquitin promoter (pUbiquitin) to make contributions to the quantitative characterization data of pCAG. Our team built four kinds of plasmids: which are built based on pGuide, with the pCAG, pCMV, pEF1a, pUbiquitin activating the expression of GFP, and then the expression of GFP strength can reflect the promoter strength after building complete four plasmids. Transfect the plasmids respectively into 293 cells by liposome transfection, then we collected samples after 24/48 hours. Finally, comparing GFP expression of CAG promoter, CMV, EF1a, Ubiquitin in 293 cells by flow cytometry, we obtained the result of the diagram below:

Fig.1 the absolute fluorescence for four different promoters. The measurement units are arbitrary units of fluorescence.

The experimental procedures used in this assay involved measuring fluorescence using Median Fluorescence Intensity . Thus, the absolute values are arbitrary units, and cannot be directly compared to other systems. Our experiment, however, does reveal the relative strength of the CAG promoter device as compared to three promoters.

Fig.2 relative promoter strength of CAG, comparing with CMV, Ubiquitin, EF1a. (The fluorescence in 24h were weaker than that in 48h among all of these promoters.In 48h,PCMV had about 2-fold greater fluorescence than the pCAG. pUbiqutin had about 1.1-fold to 1.2-fold greater fluorescence than the pCAG. pEF1a had about 1.5-fold greater fluorescence than the pCAG. The measurement units are arbitrary units of fluorescence.)