Difference between revisions of "Part:BBa K2350005:Design"

 
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2350005 short</partinfo>
 
<partinfo>BBa_K2350005 short</partinfo>
 +
 +
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.
  
 
<partinfo>BBa_K2350005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2350005 SequenceAndFeatures</partinfo>
Line 7: Line 9:
  
 
===Design Notes===
 
===Design Notes===
−
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:
 +
Forward: CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA
 +
Reverse: TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG
 +
 
 +
2. PCR primers for S. elongatus PCC7942 genomic DNA
 +
Forward: gatgcgCTCGAGATCAACAAGCACACCATCAC
 +
Reverse: CTGAGTCGTGATGCCAATGG
  
  

Revision as of 08:32, 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