Part:BBa_K3893012:Design
Up part of cassette for dsRNA constitutive production 3
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 2525
- 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2519
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 2525
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 2525
- 1000INCOMPATIBLE 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
Design Notes
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.
Source
Assembly system