Part:BBa_K2790005

Three gene construct of smmoB, smmoX and smmoZ linked with viral 2A peptide.

This part contains the coding sequence, codon optimised for P. pastoris, for the three subunits of soluble methane monooxygenase (sMMO) namely, sMMO B (regulatory subunit), sMMO X (Hydroxylase component) and sMMO Z (Hydroxylase Component). The coding sequences are linked via viral 2A peptides and are tagged with a hexa-His tag at their C-terminus. These parts will be expressed as a polycistronic mRNA but during translation, ribosome will skip over a peptide bond in the 2A sequence leading to the formation of separate individual protein subunits. This part can be expressed under any promoter (constitutive or inducible) and equimolar quantities of each subunit is expected. This part was synthesized so as to reduce the number of antibiotic selection markers required to clone all 6 subunits of sMMO enzyme complex in a eukaryotic system as this eliminates the need of individual promoter and terminator sequences flanking the genes. The other 3 sMMO subunits (sMMO C, sMMO Y and sMMO D), codon optimised for P. pastoris and linked via viral 2A peptides, can be found at<a href="https://parts.igem.org/Part:BBa_K2790004"> BBa_K2790004. </a>

<img src="">

Figure 1<b> Cloning of P2A2 in pSB1C3 vector and clone confirmation by restriction digestion with EcoRI and PstI- g block P2A2 (2721bp) cloned in pSB1C3 Plasmid (2Kb) and the resultant clone P2A2_pSB1C3 (~5Kb) was run on 1% Agarose gel for validation. </li>

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 176
  Illegal BglII site found at 1513
  Illegal BglII site found at 1990
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 496
  Illegal NgoMIV site found at 2164
  
• 1000
  COMPATIBLE WITH RFC[1000]