Part:BBa_K152009

Zea mays Actin1 Intron RNAi Production Tool

This short construct was designed to allow ANY biobrick (likely size dependant) to be used in the production of an RNAi construct for expression in bacteria. It replaces the standard biobrick restriction site conformation with an altered one for sense (EcoRI and XbaI) and antisense (SpeI and NotI) fragment placement. The small 100 bp intron of the Actin1 gene (Genebank Index 168403) of Zea mays was picked as the heart of this construct since plant RNAi constructs with hairpin loops made by introns produce much higher rates of transcriptional silencing [Nature 407, 319-320 (21 September 2000)]. On either side of the intron are cloning sites for sense and antisense fragments. The sense fragment has to be inserted first, by cutting the plasmid with EcoRI and XbaI, while cutting the sense fragment with EcoRI and SpeI and ligating them together. Next, the antisense fragment is meant to be inserted by cutting the RNAi construction tool (now containing the same fragment in the sense orientation) with NotI and SpeI, while cutting the sense fragment with NotI and SpeI and ligating the two together. There will be no SpeI sites present anymore, and two XbaI sites on either end but internal to EcoRI and PstI. As there is a transcriptional termination signal programmed into the 3' end, it would be convenient to cut out a cassette using NdeI or EcoRI and PstI for insertion into another plasmid downstream of a promoter. Alternatively, there are two XbaI sites present just outside of the sense and antisense fragments, and these can used to remove a promoterless, transcription terminatorless construct for placement downstream of a strong promoter in another plasmid.

Note: the intron was reprogrammed to contain an efficient rbs and NcoI and AvrII restriction sites for the possible inclusion of a reporter gene in the loop region of the construct.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 14
  Illegal XbaI site found at 23
  Illegal SpeI site found at 154
  Illegal PstI site found at 218
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 14
  Illegal SpeI site found at 154
  Illegal PstI site found at 218
  Illegal NotI site found at 162
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 14
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 14
  Illegal XbaI site found at 23
  Illegal SpeI site found at 154
  Illegal PstI site found at 218
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 14
  Illegal XbaI site found at 23
  Illegal SpeI site found at 154
  Illegal PstI site found at 218
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 112