Part:BBa_K3859014

Ura3 upstream homologous region-Promoter-sfGFP-terminator-Ura3 downstream homologous region

The sfGFP is used in our construction of yeast barcode plasmid(fig.15A). sfGFP is a robustly folded green fluorescent protein which folds when even when fused to poorly folded polypeptides[1].

Characterization

We designed a plasmid in Ecoil which contains sfGFP cassette first, then replaced the sfGFP with our barcode using golden gate assembly. Next we cultured the E.coli with the edited plasmid on a LB agar plate and grew overnight at 37°C. Colonies that show no fluorescence contains the plasmids that have been successfully edited(fig.15B). The sfGFP cassette is used for us to estimate whether the barcodes were successfully transfered into plasmids.

【fig.15A】LB plate grows with E.coil which contain sfGFP(before golden gate assembly)

【fig.15B】LB plate grows with E.coil which does not contain sfGFP(after golden gate assembly)

Reference

1. Pedelacq, J. D., & Cabantous, S. (2019). Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology. International journal of molecular sciences, 20(14), 3479. https://doi.org/10.3390/ijms20143479

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 3
  Illegal NotI site found at 3523
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1842
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1731
  Illegal BsaI.rc site found at 713
  Illegal SapI.rc site found at 897
  Illegal SapI.rc site found at 2628