Part:BBa_K2429180

U6:LacZ Direct Repeat

The U6 promoter is a constitutively active promoter. The direct repeat (DR) portion of the LacZ backbone contains the code for the RNA loop where the CRISPR protein binds. The DR portion is specific to the bacterial species, which is why we have LacDR for L. Shahii. We used golden gates to insert the desired ASO sequence into the LacZ gene. This allowed us to select for the colonies that contained the ASO gene sequence because when the LacZ gene was disrupted, the colonies no longer appeared blue.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 800
  Illegal EcoRI site found at 1496
  Illegal XbaI site found at 1523
  Illegal PstI site found at 1535
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 800
  Illegal EcoRI site found at 1496
  Illegal PstI site found at 1535
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 800
  Illegal EcoRI site found at 1496
  Illegal BamHI site found at 1517
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 800
  Illegal EcoRI site found at 1496
  Illegal XbaI site found at 1523
  Illegal PstI site found at 1535
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 800
  Illegal EcoRI site found at 1496
  Illegal XbaI site found at 1523
  Illegal PstI site found at 1535
  Illegal NgoMIV site found at 2002
  Illegal AgeI site found at 794
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 1762
  Illegal BsaI.rc site found at 1142