Part:BBa_K3629012:Design
T. reesei CBHII expression construct with gibson homology sequences and FLAG tag
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2737
Illegal EcoRI site found at 2561 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2561
Illegal NheI site found at 75
Illegal SpeI site found at 2738
Illegal PstI site found at 2752
Illegal NotI site found at 7
Illegal NotI site found at 2745 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal EcoRI site found at 2561
Illegal BamHI site found at 2686
Illegal XhoI site found at 124
Illegal XhoI site found at 1198 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2738
Illegal EcoRI site found at 2561 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal EcoRI site found at 2561
Illegal XbaI site found at 16
Illegal SpeI site found at 2738
Illegal PstI site found at 2752
Illegal NgoMIV site found at 1458 - 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) 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.
Source
The coding sequence of the CBHI is from Trichoderma reesei, however it has been codon-optimized for optimal activity in Yarrowia lipolytica. The promoter and terminator sequences are from wild-type Y. lipolytica.