Difference between revisions of "Part:BBa K3629014:Design"
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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. | ||
| − | 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. | + | 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. |
===Source=== | ===Source=== | ||
Revision as of 02:32, 19 October 2020
N. crassa CBHI expression construct with gibson homology sequences and Myc tag
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2751
Illegal EcoRI site found at 1054
Illegal EcoRI site found at 2570 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal EcoRI site found at 1054
Illegal EcoRI site found at 2570
Illegal NheI site found at 75
Illegal SpeI site found at 2752
Illegal PstI site found at 2766
Illegal NotI site found at 7
Illegal NotI site found at 2759 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal EcoRI site found at 1054
Illegal EcoRI site found at 2570
Illegal BglII site found at 1343
Illegal BglII site found at 1703
Illegal BamHI site found at 2700
Illegal XhoI site found at 2745 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2752
Illegal EcoRI site found at 1054
Illegal EcoRI site found at 2570 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal EcoRI site found at 1054
Illegal EcoRI site found at 2570
Illegal XbaI site found at 16
Illegal SpeI site found at 2752
Illegal PstI site found at 2766
Illegal AgeI site found at 2211 - 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.
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 Neurospora crassa, however it has been codon-optimized for optimal activity in Yarrowia lipolytica. The promoter and terminator sequences are from wild-type Y. lipolytica.