Part:BBa_K2195002

Neomycin Phosphotransferase (Codon Optimized for E. Coli) with medium promoter and RBS

This part confers antibiotic resistance to kanamycin and neomycin and is regulated by the constitutive promoter/RBS in part BBa_K608006. This part was improved from BBa_K1021001 which is the coding sequence for Neomycin Phosphotransferase (nptII). Neomycin phosphotransferase is an enzyme that is known to confer resistance to several antibiotics such as neomycin, kanamycin, geneticin, and paramomycin. The original creators of the part intended to use it in the selection of the fungus, C. heterosporus, and the moss, P. Patens. There didn't seem to be a codon-optimized option for DH5α E. coli which is commonly used in biological research labs. In addition, kanamycin is a common antibiotic used for selection as well. In order to develop a better option for DH5α E. coli cells, we used the IDT codon optimizer to codon optimize the sequence from the original part (BBa_K1021001). After receiving the DNA, we selected a medium promoter and a medium RBS from a part (BBa_K608006) already in the registry. We ligated the two sequences together in a pSB1C3 plasmid and transformed it into DH5α E. coli cells, plating them both on a chloramphenicol plate and a kanamycin plate. Colonies appeared on both plates, confirming both the transformation and the efficacy of the gene. Below is a picture of the kanamycin plate.

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Sequence and Features

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]