Part:BBa_K3402057

Double-gene editing cassette

This device is composed of 50bp-upPXA1(BBa_K3402037), hph(BBa_K3402012), 50bp-doPXA1(BBa_K3402038), Ptef1(BBa_K3402007), sgPXA1(BBa_K3402039), sgGFP(BBa_K3402024), Tsyn7(BBa_K3402001), upGFP(BBa_K3402025), doGFP(BBa_K3402026).

Usage and Biology

We knocked out the PXA1 and GFP gene by this device. In the meantime, we inserted hygromycin resistance gene at the origin PXA1 site by homology directed repair, which was to used as the marker gene to know if it is successful to knock PXA1.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were extracted genomes for PCR, gel electrophoresis analysis and conducted green fluorescence observation. As a result, all of the target fragments were verified to be right in gel electrophoresis analysis and only two positive colonies showed fluorescence.

Fig. 1 Green fluorescence observation in fluorescence

Fig. 2 Gel electrophoresis analysis of positive transformants

The double gene-editing efficiency was 99%.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 2171
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2807