Difference between revisions of "Part:BBa K2350005:Design"
 
Line 10: Line 10:
 
===Design Notes===
 
===Design Notes===
 
1. All Pst1 cutting sites in 3NSII were site-directed mutated.
 
1. All Pst1 cutting sites in 3NSII were site-directed mutated.
 +
 
Site-directed mutagenesis primers:
 
Site-directed mutagenesis primers:
 +
 
Forward: CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA
 
Forward: CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA
 +
 
Reverse: TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG
 
Reverse: TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG
  
 
2. PCR primers for S. elongatus PCC7942 genomic DNA
 
2. PCR primers for S. elongatus PCC7942 genomic DNA
 +
 
Forward: gatgcgCTCGAGATCAACAAGCACACCATCAC
 
Forward: gatgcgCTCGAGATCAACAAGCACACCATCAC
 +
 
Reverse: CTGAGTCGTGATGCCAATGG
 
Reverse: CTGAGTCGTGATGCCAATGG
  

Latest revision as of 08:33, 21 October 2017


3’-ends of the neutral site II (NSII)

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). 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.To insert 3NSII with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting site in 3NSII nucleotide sequence.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 198
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 760
    Illegal BamHI site found at 1057
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 978
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 660


Design Notes

1. All Pst1 cutting sites in 3NSII were site-directed mutated.

Site-directed mutagenesis primers:

Forward: CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA

Reverse: TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG

2. PCR primers for S. elongatus PCC7942 genomic DNA

Forward: gatgcgCTCGAGATCAACAAGCACACCATCAC

Reverse: CTGAGTCGTGATGCCAATGG


Source

Synechoccocus elongatus PCC7942 genomic DNA

References