Difference between revisions of "Part:BBa K510013:Design"

 
(→‎Source)
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K510013 short</partinfo>
 
<partinfo>BBa_K510013 short</partinfo>
Line 7: Line 6:
  
 
===Design Notes===
 
===Design Notes===
−
The pUC18R6KT-mini-Tn7-Cm is a derivative of pUC18R6KT-mini-Tn7-Gm (BBa_K510012)
+
NcoI and SphI sites were added at the ends of the chloramphenicol resistance cassette to facilitate cloning
−
by replacing the gentamycin resistance cassette by a chloramphenicol resistance cassette amplified from pSB1C3 with these primers: tatGCATGCCCATGGaacttggtctgacagctcgag and tatGCATGCCCATGGtcgggcacgtaagaggttcc.
+
−
 
+
−
 
+
  
 
===Source===
 
===Source===
−
 
+
This construct is the result of replacing the gentamycin resistance cassette in pUC18R6KT-miniTn7BB-Gm (BBa_K510012) by a chloramphenicol resistance cassette obtained from pBS1C3 by PCR amplification with these primers: tatGCATGCCCATGGaacttggtctgacagctcgag and tatGCATGCCCATGGtcgggcacgtaagaggttcc.
−
The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg.
+
−
The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products. In order to change the resistance cassettes NcoI digestions were made.
+
  
 
===References===
 
===References===
 +
Kyoung-Hee Choi, Jared B Gaynor, Kimberly G White, Carolina Lopez, Catharine M Bosio, RoxAnn R Karkhoff-Schweizer & Herbert P Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.

Latest revision as of 19:49, 18 September 2011

pUC18R6KT-miniTn7BB-Cm


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4640
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4640
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 4646
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4640
    Illegal BglII site found at 3212
    Illegal BglII site found at 3483
    Illegal XhoI site found at 3569
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4640
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4640
    Illegal XbaI site found at 4655
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1854
    Illegal SapI site found at 518


Design Notes

NcoI and SphI sites were added at the ends of the chloramphenicol resistance cassette to facilitate cloning

Source

This construct is the result of replacing the gentamycin resistance cassette in pUC18R6KT-miniTn7BB-Gm (BBa_K510012) by a chloramphenicol resistance cassette obtained from pBS1C3 by PCR amplification with these primers: tatGCATGCCCATGGaacttggtctgacagctcgag and tatGCATGCCCATGGtcgggcacgtaagaggttcc.

References

Kyoung-Hee Choi, Jared B Gaynor, Kimberly G White, Carolina Lopez, Catharine M Bosio, RoxAnn R Karkhoff-Schweizer & Herbert P Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.