Difference between revisions of "Part:BBa K4041006"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K4041006 short</partinfo> | <partinfo>BBa_K4041006 short</partinfo> | ||
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https://static.igem.org/mediawiki/parts/0/08/HKIS--crRNA_dem.png | https://static.igem.org/mediawiki/parts/0/08/HKIS--crRNA_dem.png | ||
| − | + | <br>Figure 1: Demonstration for a 2 oligo anneal system that is similar to a plasmid double stranded transcription system. The upper strand is annealed to the “T7 promoter” primer for reverse transcription. Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation. | |
| − | Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation. | + | |
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https://static.igem.org/mediawiki/parts/c/c9/HKIS--ureapage.png | https://static.igem.org/mediawiki/parts/c/c9/HKIS--ureapage.png | ||
| + | <br>15% UREA Page (Run in room temperature with 80V), indicating that the crRNA is successfully transcribed | ||
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https://static.igem.org/mediawiki/parts/a/a9/HKIS--crRNAbuff.png | https://static.igem.org/mediawiki/parts/a/a9/HKIS--crRNAbuff.png | ||
| − | + | <br>crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly. | |
| − | crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly. | + | |
<table style="border-color:383737;" bgcolor="white"> | <table style="border-color:383737;" bgcolor="white"> | ||
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| − | + | <tr> | |
| − | + | <td>The following crRNA were proved to work in this block in in Vibrio</td> | |
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| − | + | </tr> | |
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<tr> | <tr> | ||
| − | + | <td>TDH forward (crRNA1)</td> | |
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<td>ACTGTGAACATTAATGATAA</td> | <td>ACTGTGAACATTAATGATAA</td> | ||
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| − | + | </tr> | |
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<tr> | <tr> | ||
| − | + | <td>vcgC forward (crRNA3)</td> | |
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<td>ATAGCACTAATGTGTCATCT</td> | <td>ATAGCACTAATGTGTCATCT</td> | ||
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| − | + | </tr> | |
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<tr> | <tr> | ||
| − | + | <td>vcgC reverse (crRNA4, with PAM)</td> | |
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<td>GCGGAGACGAGATCGCTATC</td> | <td>GCGGAGACGAGATCGCTATC</td> | ||
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| − | + | </tr> | |
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<tr> | <tr> | ||
| − | + | <td>ctxA Forward (crRNA5)</td> | |
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<td>TGATCATGCAAGAGGAACTC</td> | <td>TGATCATGCAAGAGGAACTC</td> | ||
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| − | + | </tr> | |
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<tr> | <tr> | ||
| + | <td>crRNA 2 (high background) and crRNA 6 (no cleavage signal) is not functional from design, and is discarded</td> | ||
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| − | + | </tr> | |
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| − | + | </table> | |
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Latest revision as of 20:15, 21 October 2021
vcgC cas12a crRNA transcription device (crRNA3)
Figure 1: Demonstration for a 2 oligo anneal system that is similar to a plasmid double stranded transcription system. The upper strand is annealed to the “T7 promoter” primer for reverse transcription. Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation.
15% UREA Page (Run in room temperature with 80V), indicating that the crRNA is successfully transcribed
crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly.
| The following crRNA were proved to work in this block in in Vibrio | |
| TDH forward (crRNA1) | ACTGTGAACATTAATGATAA |
| vcgC forward (crRNA3) | ATAGCACTAATGTGTCATCT |
| vcgC reverse (crRNA4, with PAM) | GCGGAGACGAGATCGCTATC |
| ctxA Forward (crRNA5) | TGATCATGCAAGAGGAACTC |
| crRNA 2 (high background) and crRNA 6 (no cleavage signal) is not functional from design, and is discarded |
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]