Difference between revisions of "Part:BBa K1362424"
 
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<b>Summary</b>
 
<b>Summary</b>
  
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*We aimed to use this part for circularization of the cyclotide protein. To build the biobrick we preferred restriction-free cloning but we realised that the biobrick was not compatible with restriction-free cloning.
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We aimed to use this part for circularization of the cyclotide protein. For using this we found that it had 7 nucleotide sequences that made it incompatible for Gibson's assembly/Restriction free cloning. The 7 length sequence makes it out of frame with other components of the composite structure. So without disturbing the restriction site we designed a similar part that overcomes this problem: BBa_K3582021.
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In the translation product, we observe that the amino acid sequence does not fully match the claimed sequence. The first few amino acids are missing. We also note that the first few nucleotide sequences are not inframe; meaning that to create a scarless assembly of different components using Gibson' assembly or RF cloning, this part would not be compatible.  
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So we modified the part and built the modified version of N-Intein which is compatible with restriction-free cloning technique: BBa_K3582022.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:48, 27 October 2020


BsaI restriction site for RFC[105] cloning

This is the reverse complement of a BsaI restriction site headed by an Adenine as a spacer-base to separate the recognition sequence from the outward-lying cutting sequence. It was used by us for scarless golden-gate cloning to fuse inteins to other proteins and thereby implement a variety of possible post-translational modifications.

Information added by Team IISER-Pune-India 2020

Team: IISER-Pune-India 2020

Author: Avadhoot Jadhav

Summary

We aimed to use this part for circularization of the cyclotide protein. For using this we found that it had 7 nucleotide sequences that made it incompatible for Gibson's assembly/Restriction free cloning. The 7 length sequence makes it out of frame with other components of the composite structure. So without disturbing the restriction site we designed a similar part that overcomes this problem: BBa_K3582021.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1