Part:BBa_K300001:Design

BioBrick integrative base vector for S. cerevisiae

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a suffix.
  Illegal XbaI site found at 46
  Illegal SpeI site found at 2
  Illegal PstI site found at 261
  Illegal PstI site found at 1607
  Illegal PstI site found at 3663
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3727
  Illegal NheI site found at 1549
  Illegal NheI site found at 1600
  Illegal SpeI site found at 2
  Illegal PstI site found at 261
  Illegal PstI site found at 1607
  Illegal PstI site found at 3663
  Illegal NotI site found at 3733
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3727
  Illegal BglII site found at 51
  Illegal XhoI site found at 1499
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3727
  Plasmid lacks a suffix.
  Illegal XbaI site found at 46
  Illegal SpeI site found at 2
  Illegal PstI site found at 261
  Illegal PstI site found at 1607
  Illegal PstI site found at 3663
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3727
  Plasmid lacks a suffix.
  Illegal XbaI site found at 46
  Illegal XbaI site found at 3742
  Illegal SpeI site found at 2
  Illegal PstI site found at 261
  Illegal PstI site found at 1607
  Illegal PstI site found at 3663
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 2695
  

Design Notes

-

Source

1. BBa_K300980, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).
2. BBa_I763007, available in the Registry, was digested with EcoRI-PstI (Roche), gel-ran and its pSB1A2 vector was gel-extracted as described above.
3. Digested BBa_K300980 and pSB1A2 have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (BBa_V1009) which were then plated on Ampicillin (100 ug/ml) plates.
4. Positive transformants were identified by colony PCR with VF2 (BBa_G00100) and VR (BBa_G00101) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid had BBa_B0033 flanked by EcoRI and "nucleotide T"-SpeI.
5. The yielded plasmid was then digested with EcoRI-SpeI (Roche) and BBa_K300007 (digested with EcoRI-SpeI as well) was assembled in the vector, thus yielding BBa_K300001-BBa_K300007 (i.e. there is the BBa_K300007 part in the cloning site).