Part:BBa_K2350015

3NSII-ORI-5NSII

In order to finish double-crossover homologous gene recombination in S. elongatus PCC 7942, our vector contains 5’- and 3’-ends of the neutral site II (NSII). 5NSII is part of neutral site gene, which is near 5’-ends of nucleotide sequence, and 3NSII is part of neutral site gene, which is near 3’-ends of nucleotide sequence. Both of 5’- and 3’-ends of the neutral site II (NSII) gene sequence are retrieved from S. elongatus PCC 7942 genomic DNA. Additionally, in order to easily manipulate DNAs for gene cloning and plasmid preparation in E. coli DH5a, the replication origin (ORI) of pBR322 was also introduced to make our plasmid vector replicable in E. coli. We fused 5’- and 3’-ends of the neutral site II (NSII) with pBR322 replication origin (ORI) together using three pieces fusion PCR. To insert 3NSII-ORI-5NSII with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting sites in both 5NSII and 3NSII nucleotide sequence.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1866
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2428
  Illegal BamHI site found at 2725
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2646
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2328