Part:BBa_K3402051

yeGFP expression cassette with NLS

This device is composed of promoter Ptef1 (BBa_K3402007), reporter gene yeGFP (BBa_K3402000), NLS (BBa_K3402008) and terminator Tsyn7 (BBa_K3402001).

Usage and Biology

We hope our CRISPR/Cas9 system can work in Starmerella bombicola and give play to its high efficiency. Firstly, we constructed a yeGFP expression device, we add nuclear localization sequence (NLS) to the 3’ ending of yeGFP gene and use Ptef1 as the promoter to express yeGFP. Besides, the hygromycin resistance gene was also built into the plasmid as the marker gene to determine if the transformation is successful.
We set two groups, control group and experimental group, to observe if there is green fluorescence in the nucleus to know if it will work as we suspected. For the control group, the green fluorescence is diffused in the cytoplasm. For the experimental group, we observed the green fluorescence in the nucleus, then this sequence could be used for the subsequent construction of the CRISPR/Cas9 system.

Fig.1 Without the NLS (nuclear localization signal peptide)

Fig.2 With the NLS (nuclear localization signal peptide)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 2279
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1051
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 2279
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 2279
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1492