Difference between revisions of "Part:BBa K3629013:Design"
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===Design Notes=== | ===Design Notes=== | ||
EcoRI site within coding sequence and XRP2 terminator making it RFC10 incompatible. However we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) can be used to clone this part into an iGEM plasmid or another plasmid. | EcoRI site within coding sequence and XRP2 terminator making it RFC10 incompatible. However we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) can be used to clone this part into an iGEM plasmid or another plasmid. | ||
| + | |||
| + | The promoter (BBa_K3629001) is an intronic promoter, however the wild-type sequence contains a BsaI, PstI, and SpeI restriction site 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 the BsaI, PstI and SpeI sites. | ||
===Source=== | ===Source=== | ||
Revision as of 01:37, 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
EcoRI site within coding sequence and XRP2 terminator making it RFC10 incompatible. However we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) can be used to clone this part into an iGEM plasmid or another plasmid.
The promoter (BBa_K3629001) is an intronic promoter, however the wild-type sequence contains a BsaI, PstI, and SpeI restriction site 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 the BsaI, PstI and SpeI sites.
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.