Difference between revisions of "Part:BBa K4335012"
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<h2>Usage</h2> | <h2>Usage</h2> | ||
− | pCrU6.3 was constructed into plasmid pTX2038 and pTX2040 as the promoter of gRNA, and was successfully introduced into Chlamydomonas reinhardtii to obtain the positive transformant. | + | pCrU6.3 was constructed into plasmid <a href="https://parts.igem.org/Part:BBa_K4335038" width=">[pTX2038]</a> and<a href="https://parts.igem.org/Part:BBa_K4335040" width=">[pTX2040]</a> as the promoter of gRNA, and was successfully introduced into Chlamydomonas reinhardtii to obtain the positive transformant. |
<h2>Result</h2> | <h2>Result</h2> |
Revision as of 07:37, 11 October 2022
pCrU6.3
pCrU6.3 is a previously characterized RNA polymerase III promoter from the chromosome 8 of Chlamydomonas reinhardtii.
The first prerequisite for CRISPR/Cas9 experiments is sgRNA transcription under the control of the RNA polymerase III promoter (RNAPII) . In general, the RNAPII transcription factor binding site is intrinsic to the transcriptional sequence and therefore not amenable to sgRNA transcription. U6snRNA is one of the few examples of sgRNAs with proximal RNAPII promoter elements. Therefore, we used previously characterized U6snRNA sequences from chromosome 8.
Usage
pCrU6.3 was constructed into plasmidPSY1 was selected as the target gene.Detection of chlamydia U6 promoter (CrU6.3) drive sgRNA transcription.Rhine chlamydomonas wild type and guide
In addition, we found that the promoter had never appeared in the iGEM database before, so we will use the results in the literature to append to it for future reference. (the following results are from literature [2])
Reference
[1Jakab, G., Mougin, A., Kis, M., Pollák, T., Antal, M., Branlant, C., and Solymosy, F. (1997). Chlamydomonas U2, U4 and U6 snRNAs. An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac-U6atac snRNA duplex. Biochimie 79: 387–395]
[2] Greiner, A. et al. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. Plant Cell 29, 2498–2518 (2017).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 48
Illegal PstI site found at 121 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 48
Illegal PstI site found at 121
Illegal NotI site found at 453 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 48
Illegal PstI site found at 121 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 48
Illegal PstI site found at 121 - 1000COMPATIBLE WITH RFC[1000]