Part:BBa_K3402058

Triple-gene editing cassette

This device is composed of up-doLeu (BBa_K3402048), sgLeu (BBa_K3402049), 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

Based on Double-gene editing expression cassette(BBa_K3402057), we designed the sgRNA that targeted at Leu site. We constructed the Pxa1, GFP, Leu editing expression cassette, and transformed it into the recombinant strain.
The transformants were cultured on solid YPD medium with hygromycin. Then the positive colonies were conducted green fluorescence observation. After that, the positive transformants without fluorescence were dotted on the Complete Medium (CM) and Minimal Medium (MM). Then the colonies grown on the CM but did not grow on the MM were the correct transformants.

Fig. 1 Green fluorescence observation in fluorescence

Fig. 2 Colony observation a)Complete Medium b)Minimal Medium

The triple gene-editing efficiency was 30%.

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 776
  Illegal XhoI site found at 3223
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 174
  Illegal NgoMIV site found at 203
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 3859