Part:BBa_K3765020

SpyCatcher002+dCBD under T7 promoter

SpyCatcher002-dCBD fusion protein generator.

Usage and Biology

This protein generator can be used for expressing the SpyCatcher002-dCBD fusion protein under a strong bacteriophage T7 promoter by the T7 RNA Polymerase in suitable bacterial strains like E. coli BL21(DE3). Transcriptional regulation is by the lac operator sequence, and expression can be switched on via IPTG induction. Also contains a strong T7 RBS upstream and wild type T7 terminator downstream of the CDS for initiation and termination of translation.

Fig 1. Feature Map

To construct this protein generator, we cloned the SpyCatcher002-dCBD CDS into the high copy number pETMCN vector. The vector contains SpeI and XbaI restriction sites just downstream of the RBS and upstream of the terminator, respectively. We used Gibson Assembly for cloning, so there were no hindrances with regard to the presence of these restriction sites. In case someone wants to use this part, it is suggested to get it synthesized and use Gibson Assembly (or other such assembly techniques) for cloning insread of restriction digestion-ligation.

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Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 981
  Illegal SpeI site found at 45
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 987
  Illegal SpeI site found at 45
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 723
  Illegal BamHI site found at 969
  Illegal XhoI site found at 365
  Illegal XhoI site found at 951
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 981
  Illegal SpeI site found at 45
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 981
  Illegal SpeI site found at 45
  Illegal AgeI site found at 195
  Illegal AgeI site found at 561
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 450