Difference between revisions of "Part:BBa P1014:Design"

(→‎Design Notes)
(→‎Design Notes)
 
(8 intermediate revisions by the same user not shown)
Line 8: Line 8:
 
===Design Notes===
 
===Design Notes===
 
*PCR primers
 
*PCR primers
−
**gtttcttcgaattcgcggccgcttctagaggagataggttatgcaagat,P1014-F
+
**gtttcttcgaattcgcggccgcttctagag gagataggttatgcaagat,P1014-F
−
**gtttcttcctgcagcggccgctactagtacctttttttagaatccggaatc,P1014-R
+
**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.
 
*XbaI site at 660 removed by mutating c->t which also improves codon usage.
 
**gatgatttttacagggatattttagagaacgaaatctac,P1014-XF
 
**gatgatttttacagggatattttagagaacgaaatctac,P1014-XF
 +
**gatgatttttacagggatattttagagaacgaaatctact,P1014-XF2
 
**gtagatttcgttctctaaaatatccctgtaaaaatcatc,P1014-XR
 
**gtagatttcgttctctaaaatatccctgtaaaaatcatc,P1014-XR
  
 
===Source===
 
===Source===
−
 
+
*Novel gentamicin resistance gene
−
DQ208936
+
*Acquired by PCR from pTEX5500ts, Genbank DQ208936, kind gift of Barbara E. Murray.
 +
*pTEX5500ts was, in turn was derived from Genbank AF016483.
  
 
===References===
 
===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

Latest revision as of 03:23, 25 July 2006


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