Part:BBa_K3893008

Up part of cassette for dsRNA constitutive production 1

Part of Modular Platform for dsRNA production

Upper part of the cassette for constitutive dsRNA production, which is composed of a terminator BBa_K3893007, promoter T7 BBa_I712074, RBS BBa_B0034 and GFP 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 165
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 921
  Illegal SpeI site found at 165
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 159
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 165
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 165
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 837