Difference between revisions of "Part:BBa K2340000"
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The part was validated with two screening primers 200bp up- and downstream of insert and then seperated on a 1% agarose gel using electrophoresis. | The part was validated with two screening primers 200bp up- and downstream of insert and then seperated on a 1% agarose gel using electrophoresis. | ||
| − | [[File: | + | [[File:DCAS13a-GFP-AGE.jpeg|600px|thumb|left|Figure 1: Agarose gel of a screening PCR of BBa_K2340000 in pSB1C3. We used two primer approximately 200bp up- and downstream of our insert]] |
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Revision as of 23:45, 31 October 2017
dCAS13a linked to GFP with NLS
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1112
Illegal BglII site found at 2048
Illegal BglII site found at 2312
Illegal BglII site found at 2816
Illegal BglII site found at 3302
Illegal BglII site found at 3410
Illegal BglII site found at 3740 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 662
Illegal SapI.rc site found at 3421
Usage and Biology
'dead' CAS13a with a double HEPN nuclease 1 and 2 inactive mutation, linked to a green fluorescent protein (wtGFP) via a linker sequence (ggctcctccggc). On the N-terminal and the C-terminal of the construct there are nuclear localization sequences (NLS; DNA sequence: cccaagaaaaaacgcaaggtg. Amino acid sequence: PKKKRKV) that will reduce background noise when expressed and used in mammalian cell lines.
When supplied with a guide RNA (gRNA), this enzyme will process it into a CRISPR RNA (crRNA). The crRNA contains a sequence that is complementary to the RNA it is targetting.
Validation
The part was validated with two screening primers 200bp up- and downstream of insert and then seperated on a 1% agarose gel using electrophoresis.
