Part:BBa_K777006:Design

Tar receptor under the control of constitutive promoter J23112

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1323
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 152
  

Design Notes

• Genomic sequence was amplified using the following primers.
  • Primers PCR: Base pairs in caps represent the respective restriction sites followed by either prefix or suffix. Primers were provided by SIGMA.
    • Fwd: Tar for + prefix: 5´ actGAATTCgcggccgctTCTAGAtgattaaccgtatccgcgtagtc 3´
    • Rev: Tar rev + suffix: 5´ tcCTGCAGcggccgctACTAGTcaaaatgtttcccagtttggatc 3´

• One XbaI site was found at position 420. The restriction site was changed from TCTAGt to CCTAGt via QuikChange PCR.
  • Primers QuikChange (QC): Primers were provided by SIGMA.
    • Fwd: TarQC for: 5´ ACTGATTGATTAcCTAGATTATGGCAATACTGGAG 3´
    • Rev: TarQC rev: 5´ TGCCATAATCTAGgTAATCAATCAGTTCAGTTAAC 3´
      (the uncapitalized letter induces the mutation for removal of the XbaI site)

Source

• The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B, complete genome (CP000948.1).
• J23112 information was taken from the parts.igem and physical DNA from the 2012 distribution kit.

References

• Derr P., Boder E. and Goulian M. Changing the specificity of a bacterial chemoreceptor. J. Mol. Biol. 2006. 355:923–932.
• Anderson promoters from the 2006 Berkeley group