Difference between revisions of "Part:BBa K1150047"
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<partinfo>BBa_K1150047 short</partinfo> | <partinfo>BBa_K1150047 short</partinfo> | ||
| − | Behind a strong CMV promoter a HA-tag, a NLS and dCas9 followed by another NLS and a BGH terminator were assembled via the iGEM cloning method. | + | Behind a strong CMV promoter a [https://parts.igem.org/Part:BBa_K1150016 HA-tag], a [https://parts.igem.org/Part:BBa_K1150010 NLS] and [https://parts.igem.org/Part:BBa_K1150000 dCas9] followed by another NLS and a [https://parts.igem.org/Part:BBa_K1150012 BGH terminator] were assembled via the iGEM cloning method. |
The truncation of the huge dCas9 gene was performed by PCR over the backbone (=dCas9 in pSB1C3). The nucleotides between the primer pair is not amplified. One of the primers has an overlap compatible to the first bases of the other one, so that after this PCR a one fragment Gibson assembly could be performed. | The truncation of the huge dCas9 gene was performed by PCR over the backbone (=dCas9 in pSB1C3). The nucleotides between the primer pair is not amplified. One of the primers has an overlap compatible to the first bases of the other one, so that after this PCR a one fragment Gibson assembly could be performed. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This part can be used in the uniCAS Toolkit of team Freiburg as a shorter version of dCas9. | + | This part can be used in the uniCAS Toolkit of team Freiburg as a shorter version of dCas9. |
| − | Truncation 1: here | + | |
| + | Truncation 1: here 336 bp at the 3'-end of dCas9 are missing. | ||
Latest revision as of 00:54, 29 October 2013
Truncated CMV dCas9 Device #1
Behind a strong CMV promoter a HA-tag, a NLS and dCas9 followed by another NLS and a BGH terminator were assembled via the iGEM cloning method. The truncation of the huge dCas9 gene was performed by PCR over the backbone (=dCas9 in pSB1C3). The nucleotides between the primer pair is not amplified. One of the primers has an overlap compatible to the first bases of the other one, so that after this PCR a one fragment Gibson assembly could be performed.
Usage and Biology
This part can be used in the uniCAS Toolkit of team Freiburg as a shorter version of dCas9.
Truncation 1: here 336 bp at the 3'-end of dCas9 are missing.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 576
Illegal BglII site found at 900 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
To verify the expression of the truncated dCas9 version we performed westernblot with anti-HA antibody.
