Part:BBa_K2148002

aphA6

This part encodes the enzyme aminoglycoside phosphotransferase which confers antibiotic resistance to amikacin, neomycin, and kanamycin. The CDS is flanked by Phytobrick fusion sites which corresponds to a CDS/CTAG Phytobrick.

Usage and Biology

This part can be used with other level 0 Phytobrick to create a transcriptional unit to provide a selection pressure on transformations.

Selection conditions are more difficult for this antibiotic resistance gene. The construct should be functional in E. coli conferring kanamycin resistance @20µg/ml, observations indicate that the marker doesn't usually work well in the initial round of selection for positive transformants. The recommended strategy is to first select for E. coli transformants using the marker carried in the plasmid backbone (chloramphenicol for level 0 parts, kanamycin for level 1 part "alphas" and spectinomycin/streptinomycin for level 2 part "omegas"). Then the transformant colonies can be scored on 20µg/ml kanamycin plates.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 647
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 647
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 647
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 647
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 647
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Bateman J. M., Purton S. (2000). Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker. Mol. Gen. Genet. 263, 404–410. 10.1007/s004380051184