Part:BBa_K5401010

RNAPAnc302 Blast250

Ancestral sequence reconstruction has been a long-standing technique used in evolutionary biology to infer sequences of ancient proteins based on existing sequences. By using ancestral reconstruction, we are able to generate functional distant homologs through in silico methods. A programme called Molecular Evolutionary Genetics Analysis version X (MEGAX) was utilised for this purpose.

The ancestral sequence (RNAPAnc302 Blast250) was inferred after a series of workflow using a larger dataset of ~250 sequences. The ancestral sequence was subsequently cloned into stable T7-expressing plasmid (plasmid 1c) for downstream testing.

Usage and Biology

The ancestral sequence (RNAPAnc302 Blast250) was derived based of using T7 RNA polymerase as the selection marker.

Characterization

The ancestral sequence was initially cloned into the plasmid 1c for testing and evaluation. However, Sanger sequencing of our cloned constructs showed deletion/truncation of the RNA polymerase sequence.

Fig 1: Sanger sequencing results from cloned constructs showed deletion of the entire ancestral RNA polymerase sequence

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 647
  Illegal PstI site found at 2027
  
• 12
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  Illegal EcoRI site found at 647
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• 21
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  Illegal EcoRI site found at 647
  Illegal BamHI site found at 1491
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• 23
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  Illegal EcoRI site found at 647
  Illegal PstI site found at 2027
  
• 25
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  Illegal EcoRI site found at 647
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• 1000
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  Illegal SapI.rc site found at 508