Part:BBa_K3893010

Up part of cassette for dsRNA constitutive production 2

dsRNA cassette production to improve stability Upper part of the cassette for constitutive dsRNA production, which is composed of a terminator (BBa_K3893007), promoter T7 (BBa_I712074), RBS (BBa_B0034) andGFP transcription unit that functions as a dropout (BBa_K3893017).

Assembly system

Before constructing the modified plasmids and using them, we took into account the following: ○ Have a Biobrick-compatible backbone, in case you don't have it you could modify it with PCR overhang and add RFC10 prefix and suffix. ○ The following restriction enzymes are used: EcoRI, PstI, SpeI, XbaI, BglII and KpnI. Therefore the dsRNA sequence should not contain these restriction sites. ○ Previously carry out PCR overhang to the dsRNA sequence to add the restriction sites: BglII and KpnI.

Once the above considerations have been met, the following assemblies are performed: 1) We synthesized the up (UP) and down (DP) parts and cloned them into Biobrick-compatible backbones with EcoRI and PstI. 2) Assembly 3A adapted: We digested the UP plasmid with EcoRI - SpeI, the DP plasmid with XbaI - PstI and a Biobrick compatible backbone with EcoRI - PstI. Then we ligated and obtained as product the modified plasmid (dsRNA receptor). 3) We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 164
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 918
  Illegal SpeI site found at 164
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 158
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 164
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 164
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 834