Difference between revisions of "Part:BBa K3075001:Design"

 
 
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<partinfo>BBa_K3075001 short</partinfo>
 
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<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
  
  
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===Design Notes===
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===Design===
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Design
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The following gene construct was designed to enable the cloning and expression of the recombinant proteins DBAT<sup>G38R/F301V</sup>-SnoopT-His within a T7 expression system.
  
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Additions to the gene are as follows:
  
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===Source===
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:*'''Gibson forward and reverse overhangs''' – complementary overhangs outside the protein coding region enables cloning into pET19b via Gibson assembly
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:*'''Hexahistidine peptide[MT1] tag''' – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.
  
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Source
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Additionally, GSG linkers are included between the peptide sequences for additional fluidity to allow individual, unhindered protein folding of each component (enzyme and tag).
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[[File:DBAT_anottated.png]]
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'''Figure 1:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the DBAT<sup>G38R/F301V</sup>-SnoopT-His gene construct. Image by Linda Chen.
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===Source===
  
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===References===
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Originated from ''Taxus cuspidata'' (Japanese yew)

Latest revision as of 00:41, 22 October 2019


DBATG38R/F301V-SnoopT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 28
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

The following gene construct was designed to enable the cloning and expression of the recombinant proteins DBATG38R/F301V-SnoopT-His within a T7 expression system.

Additions to the gene are as follows:

  • Gibson forward and reverse overhangs – complementary overhangs outside the protein coding region enables cloning into pET19b via Gibson assembly
  • Hexahistidine peptide[MT1] tag – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.

Additionally, GSG linkers are included between the peptide sequences for additional fluidity to allow individual, unhindered protein folding of each component (enzyme and tag).

DBAT anottated.png

Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the DBATG38R/F301V-SnoopT-His gene construct. Image by Linda Chen.


Source

Originated from Taxus cuspidata (Japanese yew)