Part:BBa_K4630103:Design
stgRNA-barcode-cassette (0+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]
- 1000COMPATIBLE WITH RFC[1000]
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.
Unlike stgRNA barcode cassette (1+2), the first level sequence in stgRNA barcode cassette (0+2) has undergone self-targeting knockout of the first level, resulting in a loss of 215bp.In addition, the pCas plasmid involved in the knockout contained a low-copy temperature-sensitive replicon oriR101. It can replicate normally when cultured at 30 ℃, but will automatically lose when cultured above 37 ℃. This facilitates the elimination of plasmids.
Design Graph
Figure. 1 Design of stgRNA barcode cassette (0+2)
Source
The subcloning of the induction product of stgRNA-barcode-cassette 1 and stgRNA-barcode-cassette 2.