Difference between revisions of "Part:BBa K3629014:Design"

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===Design Notes===
 
===Design Notes===
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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 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.
  
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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 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.
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The native signal peptide from <I>N. crassa</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 04:44, 19 October 2020


N. crassa CBHI expression construct with gibson homology sequences and Myc tag


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE 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 N. crassa 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 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.

References