Part:BBa_K3402053

Self-excising hygromycin marker cassette

This device is composed of upSBLE(BBa_K3402030), Six site(BBa_K3402032), Pgalk(BBa_K3402033), Rec(BBa_K3402034), Tgki(BBa_K3402019), hph(BBa_K3402012), Ptef1(BBa_K3402007), Cas9(BBa_K3402023), NLS(BBa_K3402008), Tsyn7(BBa_K3402001), doSBLE(BBa_K3402031).

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Usage and Biology

We designed the self-excising hygromycin marker cassette which can be used to excise hygromycin resistance gene after screening. upSBLE and doSBLE are homologous arms which are used to construct device by homology directed repair. Pgalk is a galactose-induced promoter, which can control the expression of serine recombinant enzyme (Rec). According to the β-Rec/six self-excising system, serine recombinant enzyme will recognize two adjacent six sites in the same DNA strand and specifically cut off the fragments between two adjacent six sequences.
As a result, the resistance gene can be recycled and the engineered yeast will never show hygromycin resistance.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 858
  Illegal XbaI site found at 2596
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1380
  Illegal BamHI site found at 445
  Illegal BamHI site found at 3783
  Illegal XhoI site found at 2214
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 858
  Illegal XbaI site found at 2596
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 858
  Illegal XbaI site found at 2596
  Illegal NgoMIV site found at 523
  
• 1000
  COMPATIBLE WITH RFC[1000]