Part:BBa_K4630102:Design
stgRNA-barcode-cassette (1+2)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 55
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 94
Design Notes
We designed the restriction site for the ligation of the two basic cassettes. We used SpeI and EcoRI to cleave the stgRNA cassette 1 plasmid. Then, XbaI and EcoRI were used to cleave the recording fragments in the stgRNA cassette 2 plasmid. Due to the ability of SpeI and XbaI to form the same viscous end, we are able to connect the carrier and fragment to each other through T4 ligase.
The principle of its recording is that when the system responds to the first signal and the first level knockout occurs, the terminator before the second level stgRNA2 disappears. Therefore, when the system receives the second signal stimulation, it can activate the expression of secondary stgRNA2, targeting self-knockout. Thus achieving multi-level recording. The entire system presents an unlocking model. The terminator in the first level sequence is the lock of the second level sequence. Only when the first level performs self-knocking can the lock of the second level be opened, expressing the second level normally.
Design Graph
Figure. 1 Design Graph of stgRNA-barcode-cassette (1+2)
Source
The subcloning of BBa_K4630100 and BBa_K4630101.