Part:BBa_K1750000

loxP-flanked RFP upstream from NPTII

Red fluorescent protein (encoded by EforRED), which produces red light, is flanked by loxP sites that are recombined by Cre-recombinase to excise the intervening gene. The kanamycin-resistance gene NPTII is positioned downstream. This part should be used in conjuction with Cre-recombinase as a reporter for its activity.

In the absence of Cre-recombinase expression, RFP is expressed, thus cells appear red. Upon induction of Cre-recombinase expression, loxP recombination excises the EforRED gene, allowing expression of the kanamycin-resistance gene, therefore cells can grow on kanamycin-supplemented media.

Separate ribosomal binding sites and a terminator following RFP allow independent expression of both genes. This construct is under the control of the T7 promoter.

Despite addition of a terminator sequence downstream of RFP, this construct shows leaky read-through of NPTII. Furthermore, expression in the high copy number vector pSB1C3 indicated mild toxicity to E. coli; however expression in the low copy number pSB3C5 showed no toxicity.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 103
  Illegal BamHI site found at 851
  Illegal XhoI site found at 1667
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 517
  
• 1000
  COMPATIBLE WITH RFC[1000]