Part:BBa_K3893012

Up part of cassette for dsRNA constitutive production 3

Part of Modular Platform for dsRNA production

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.

Step 1

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 a product the modified plasmid (dsRNA receptor).

Step 2

3. We inserted the dsRNA into the plasmid by digesting and ligating both sequences with BglII and KpnI.

Step 3

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 2525
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 749
  Illegal NheI site found at 1665
  Illegal NheI site found at 2470
  Illegal NheI site found at 3279
  Illegal SpeI site found at 2525
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2519
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 2525
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 2525
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 665
  Illegal BsaI.rc site found at 877
  Illegal BsaI.rc site found at 1581
  Illegal BsaI.rc site found at 2386
  Illegal BsaI.rc site found at 3195