Difference between revisions of "Part:BBa K3629016:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | There is an EcoRI site within coding sequence and 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) can be used to clone this part into an iGEM plasmid or another plasmid. | + | There is an EcoRI site within coding sequence and 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) can 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 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 [https://parts.igem.org/Part:BBa_K2983050 BBa_K2983050] promoter were made to remove the BsaI, PstI and SpeI sites. | The promoter [https://parts.igem.org/Part:BBa_K3629001 (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 [https://parts.igem.org/Part:BBa_K2983050 BBa_K2983050] promoter were made to remove the BsaI, PstI and SpeI sites. | ||
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===Source=== | ===Source=== | ||
Revision as of 02:55, 19 October 2020
Modified T. reesei EGI 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 2385
Illegal EcoRI site found at 2204 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2204
Illegal NheI site found at 81
Illegal SpeI site found at 2386
Illegal PstI site found at 2400
Illegal NotI site found at 7
Illegal NotI site found at 2393 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2204
Illegal BamHI site found at 2334
Illegal XhoI site found at 132 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2386
Illegal EcoRI site found at 2204 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal EcoRI site found at 2204
Illegal XbaI site found at 16
Illegal SpeI site found at 2386
Illegal PstI site found at 2400
Illegal NgoMIV site found at 1326 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
There is an EcoRI site within coding sequence and 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) can 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 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 EGI is from Trichoderma reesei, 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.