Difference between revisions of "Part:BBa K3971010"
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<partinfo>BBa_K3971010 short</partinfo> | <partinfo>BBa_K3971010 short</partinfo> | ||
| − | This composite part has been designed to characterize the transport | + | This composite part has been designed to characterize the sucrose transport efficiency through the cscB (sucrose permease) transporter in <i>S. elongatus</i> UTEX 2973. The cassette contains two homology arms to the Neutral Site 1 of the <i>S. elongatus</i> UTEX 2973 genome which allows for the homology-repair-based insertion of the cassette into the genome. This cassette is to be cloned into the pSL2680 plasmid [1], which is a Cpf1 based CRISPR plasmid and can be used to insert the cassette into the Neutral Site 1 using a gRNA target within that region. |
The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another. Thus one can study the effect of different promoters on the cscB transport efficiency. | The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another. Thus one can study the effect of different promoters on the cscB transport efficiency. | ||
Latest revision as of 18:46, 20 October 2021
cscB (sucrose permease) transport efficiency characterization with different promoters.
This composite part has been designed to characterize the sucrose transport efficiency through the cscB (sucrose permease) transporter in S. elongatus UTEX 2973. The cassette contains two homology arms to the Neutral Site 1 of the S. elongatus UTEX 2973 genome which allows for the homology-repair-based insertion of the cassette into the genome. This cassette is to be cloned into the pSL2680 plasmid [1], which is a Cpf1 based CRISPR plasmid and can be used to insert the cassette into the Neutral Site 1 using a gRNA target within that region.
The promoter in this part is PcpcB-m6 (BBa_K3971000). In order to allow for modularity in the promoter region of the cassette, two restriction sites Nde1 and BamH1 flank it, so that the promoter can be easily excised out and replaced with another. Thus one can study the effect of different promoters on the cscB transport efficiency.
Both ends of the composite part contain Kpn1 restriction sites since this cassette is designed to be cloned into the pSL2680 plasmid which has a Kpn1 site and a Sal1 site to allow for cloning of cassettes.
References
1: Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Ungerer J, Pakrasi HB. Sci Rep. 2016 Dec 21;6:39681. doi: 10.1038/srep39681. 10.1038/srep39681 PubMed 28000776
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 2764
Illegal PstI site found at 362 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2577
Illegal PstI site found at 362 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1103
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 2764
Illegal PstI site found at 362 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 2764
Illegal PstI site found at 362
Illegal NgoMIV site found at 1589 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1633
Illegal BsaI.rc site found at 152