BBa_ K4335021 consists of a terminator, a cloning site, SpScaffold and a terminator
PCrU6.3 is a previously characterized RNA from chromosome 8 of Chlamydomonas reinhardtii [1]
Template of sgRNA. Use Golden Gate (BsaⅠ) to replace the template sequence with a ~20bp guide sequence. Add 'ACTT' on the 5'-terminal of your guide sequence and 'AAAC' on the reverse 5'-terminal.
sgRNA
Usage
pCrU6.3+insert site+SpScaffold+polyT Term is one of the core functional components of plasmid pTX2038 and pTX2040 Its main function is to express specific sgRNA, and combine with
HSP70A Promoter+3×Flag+NLS+SpCas9+NLS+Rbcs2 Term [BBa_K4335020]works together to cut specific genes in the nuclear genome of Chlamydomonas reinhardtii, so as to knock out negative lipid related regulatory genes.
Result
Verification of the effect of Golden Gate inserting specific sgRNA
The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.
Gel run of samples from colony PCR.Primer length ~660 bp. M: 2000 bp DNA Marker; 38: pTX2038 vector; 40: pTX2040 vector; sgRNA1-3: different sgRNAs is designed.
Figure 1 shows that the colony PCR has successfully amplified about 660 bp bands, which correspond to the vector corresponding to the sgRNA of each target gene (pTX2038/pTX2040). It preliminarily indicates that we have successfully inserted the sgRNA of the target gene into the vector (pTX2038/pTX2040), and then Sanger sequencing of each insert site has been carried out, which proves that we have successfully constructed.
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 48 Illegal PstI site found at 121
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 48 Illegal PstI site found at 121 Illegal NotI site found at 453
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 48 Illegal PstI site found at 121
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 48 Illegal PstI site found at 121
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 551 Illegal BsaI.rc site found at 533
