Part:BBa_K2805002:Design

(C120op)x5-minCyc1-mRuby2-tCyc1

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal suffix found in sequence at 1086
  Illegal BglII site found at 369
  Illegal BamHI site found at 126
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1406
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The primary concern was Dueber MoClo and GoldenGate compatibility. These sequences were designed to be robustly assembled and integrated into the S. cerevisiae genome. Additionally, the number of C120op repeats is variable throughout E. litoralis; 5 was selected for its high activity, as described by Motta-Mena et al.

Source

The C120op EL222-binding sequence was first identified in E. litoralis by Giomar Rivera-Cancel, Laura B. Motta-Mena, and Kevin H. Gardner (Biochemistry 2012, doi: 10.1021/bi301306t). mRuby2 is derived from Entacmaea quadricolor, best described by Aj et al. (Nature Methods 2012, doi: 10.1038/nmeth.2171).

References

1. Benzinger D, Khammash M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nature Communications. 2018;9(1):3521. https://doi.org/10.1038/s41467-018-05882-2. doi: 10.1038/s41467-018-05882-2.

2. Zhao EM, Zhang Y, Mehl J, et al. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature. 2018;555:683. http://dx.doi.org/10.1038/nature26141.

3. Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol. 2015;4(9):975-986. https://doi.org/10.1021/sb500366v. doi: 10.1021/sb500366v.