Difference between revisions of "Part:BBa K1172303:Design"

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===Design Notes===
 
===Design Notes===
 
we eliminated three illegal restriction sites: Therefore we substituted one base per illegal restriction site by using PCR. The designed PCR-primers generated homologue overlaps, thus enabling Gibson Assembly afterwards:
 
we eliminated three illegal restriction sites: Therefore we substituted one base per illegal restriction site by using PCR. The designed PCR-primers generated homologue overlaps, thus enabling Gibson Assembly afterwards:
−
* Pst1 at 234 bp (changing xxxxxx to xxyxxx)
+
* Pst1 at 234 bp (changing ctgca^g to ctgTag)
−
* Pst1 at 1644 bp (changing xxxxxx to xxyxxx)
+
* Pst1 at 1644 bp (changing ctgca^g to ctgTag)
−
* EcoR1 at 1840 bp (changing xxxxxx to xxyxxx)
+
* EcoR1 at 1840 bp (changing g^aattc to gaattT)
  
 
These Primers were used to eliminate the illegal restriction sites:
 
These Primers were used to eliminate the illegal restriction sites:

Revision as of 09:21, 26 October 2013

Riboflavin synthesis gene cluster from shewanella oneidensis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1114
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

we eliminated three illegal restriction sites: Therefore we substituted one base per illegal restriction site by using PCR. The designed PCR-primers generated homologue overlaps, thus enabling Gibson Assembly afterwards:

  • Pst1 at 234 bp (changing ctgca^g to ctgTag)
  • Pst1 at 1644 bp (changing ctgca^g to ctgTag)
  • EcoR1 at 1840 bp (changing g^aattc to gaattT)

These Primers were used to eliminate the illegal restriction sites:

  • pst1-234 fwd (38 bp)
    TGTCACCTTAGAACCCTGTAGCCATTATGGTCGTACGC
  • pst1-234 rev (32 bp)
    CGACCATAATGGCTACAGGGTTCTAAGGTGAC
  • pst1-1644 fwd (30 bp)
    GTGCCCCATACTGTAGGTGAAACCACGTTG
  • pst1-1644 rev (34 bp)
    AACGTGGTTTCACCTACAGTATGGGGCACAATCG
  • EcoR1 fwd (43 bp)
    GGCACTCAGTTCACTTAGGTATAGAATTTATAACAACAGTCAC
  • EcoR1 rev (43 bp)
    GTGACTGTTGTTATAAATTCTATACCTAAGTGAACTGAGTGCC

Source

Shewanella oneidensis MR-1 genomic sequence.

  • The strain was kindly provided by Dr. Johannes Gescher (Karlsruher Institut für Technologie)

References