Difference between revisions of "Part:BBa K3629013:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | There is an EcoRI site within | + | There is an EcoRI site within the XRP2 terminator making this part RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) could be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly. |
The promoter [https://parts.igem.org/Part:BBa_K3629001 (BBa_K3629001)] is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional [https://parts.igem.org/Part:BBa_K2983050 BBa_K2983050] promoter were made to remove theses sites. | The promoter [https://parts.igem.org/Part:BBa_K3629001 (BBa_K3629001)] is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional [https://parts.igem.org/Part:BBa_K2983050 BBa_K2983050] promoter were made to remove theses sites. | ||
| + | |||
| + | The native signal peptide from <i>P. funiculosum</I> was removed so it would not interfere with the fused Lip2 secretion tag native to <i>Y. lipolytica.</i> | ||
===Source=== | ===Source=== | ||
Latest revision as of 04:43, 19 October 2020
Modified PfCBHI expression construct with gibson homology sequences and 6X His tag
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2725
Illegal EcoRI site found at 2546 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2546
Illegal NheI site found at 75
Illegal SpeI site found at 2726
Illegal PstI site found at 2740
Illegal NotI site found at 7
Illegal NotI site found at 2733 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2546
Illegal BglII site found at 1322
Illegal BamHI site found at 2631
Illegal XhoI site found at 87 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2726
Illegal EcoRI site found at 2546 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal EcoRI site found at 2546
Illegal XbaI site found at 16
Illegal SpeI site found at 2726
Illegal PstI site found at 2740
Illegal NgoMIV site found at 804 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
There is an EcoRI site within the XRP2 terminator making this part RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) could be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly.
The promoter (BBa_K3629001) is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional BBa_K2983050 promoter were made to remove theses sites.
The native signal peptide from P. funiculosum was removed so it would not interfere with the fused Lip2 secretion tag native to Y. lipolytica.
Source
The coding sequence of the CBHI is from Penicillium funiculosum, however it has been codon-optimized and mutated to have optimal activity in Yarrowia lipolytica based on our modelling. The promoter and terminator sequences are from wild-type Y. lipolytica.