Part:BBa_P1014:Design

gentamicin resistance cassette aph(2)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

• PCR primers
  • gtttcttcgaattcgcggccgcttctagag gagataggttatgcaagat,P1014-F
  • gtttcttcctgcagcggccgctactagta gattccggattctaaaaaaagg,P1014-R2 (after terminator) [Bad, extra A]
  • gtttcttcctgcagcggccgctactagta ctatttaattttaatgcttctg,P1014-R3 (after stop codon)
  • gtttcttcctgcagcggccgctactagta gattccggattctaaaaaaggattgcta,P1014-R4
  • gtttcttcctgcagcggccgctactagta gattccggattctaaaaaagg,P1014-R5

• XbaI site at 660 removed by mutating c->t which also improves codon usage.
  • gatgatttttacagggatattttagagaacgaaatctac,P1014-XF
  • gatgatttttacagggatattttagagaacgaaatctact,P1014-XF2
  • gtagatttcgttctctaaaatatccctgtaaaaatcatc,P1014-XR

Source

• Novel gentamicin resistance gene
• Acquired by PCR from pTEX5500ts, Genbank DQ208936, kind gift of Barbara E. Murray.
• pTEX5500ts was, in turn was derived from Genbank AF016483.

References

• Nallapareddy SR, Singh KV, Murray BE. Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus. Appl Environ Microbiol. 2006 Jan;72(1):334-45 faecium strains. PMID 16391062

• Tsai SF, Zervos MJ, Clewell DB, Donabedian SM, Sahm DF, Chow JW. A new high-level gentamicin resistance gene, aph(2)-Id, in Enterococcus spp. Antimicrob Agents Chemother. 1998 May;42(5):1229-32. PMID 9593155