Part:BBa_K2271107:Design

PEX5 variant R19 with Promotor/Terminator

Assembly Compatibility:

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

  Illegal NheI site found at 1341
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1128
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1633
  
• 1000
  COMPATIBLE WITH RFC[1000]

Part design

This part was designed in the course of a targeted mutagenesis approach. For this Pex5 variant we considered recently published literature (Towards designer organelles by subverting the peroxisomal import pathway - Baker et al., 2017) and designed a receptor that should interact with a PTS1 variant. We performed molecular dynamics simulations to test how benefitial the mutations were and synthesized the Pex5 gene by IDT.
After gene synthesis we performed golden gate cloning and made it compatible to the biobrick standard via overhang PCR with the appropriate primers. This product then was ligated into the biobrick backone pSB1C3.
This part contains the promotor PR and the terminator TR.

Source

The original sequence was taken from NCBI (GenBank: KZV12481.1). After mutagenesis we performed the IDT codon optimization for Saccharomyces cerevisiae, removed any restrictions sites that would interfere with the biobrick or golden gate standard and synthesized the gene with appropiate Golden Gate overhangs.