Part:BBa_K3893009
Down part of cassette for dsRNA constitutive production 1
Part of Modular Platform for dsRNA production
Lower part of the cassette for constitutive dsRNA production, which is composed of a GFP transcription unit that functions as a dropout, RBS (BBa_K3893019), promoter T7 (BBa_K3893020) and terminator (BBa_K3893006).
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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 798
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 798
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 798
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 698
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