Difference between revisions of "Part:BBa K2350015:Design"
(→‎Design Notes)
 
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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.  
 
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.  
  
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primers Pst1 site directed mutagenesis of 3NSII
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Primers for Pst1 site directed mutagenesis in 3NSII
  
 
CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA  
 
CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA  
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(RC) TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG
 
(RC) TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG
  
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primers Pst1 site directed mutagenesis of 5NSII
+
Primers for Pst1 site directed mutagenesis in 5NSII
  
 
1. GGTATGGAAAACGCTTGCTGGAGCATCAGATCGATGCGAT
 
1. GGTATGGAAAACGCTTGCTGGAGCATCAGATCGATGCGAT
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2. TTTGGTGGCGGCGATGGCTGGAGAGGCCAGCATTGAAGAC  
 
2. TTTGGTGGCGGCGATGGCTGGAGAGGCCAGCATTGAAGAC  
  
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(RC) GTCTTCAATGCTGGCCTCTCCAGCCATCGCCGCCACCAAA  
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(RC) GTCTTCAATGCTGGCCTCTCCAGCCATCGCCGCCACCAAA
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+
−
 
+
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+
  
 
===Source===
 
===Source===

Latest revision as of 13:28, 21 October 2017


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


Design Notes

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.

Primers for Pst1 site directed mutagenesis in 3NSII

CGCGGATGGGGTTCGTTCTGGAGCAGTTCGTTCTGTTGGA

(RC) TCCAACAGAACGAACTGCTCCAGAACGAACCCCATCCGCG

Primers for Pst1 site directed mutagenesis in 5NSII

1. GGTATGGAAAACGCTTGCTGGAGCATCAGATCGATGCGAT

(RC) ATCGCATCGATCTGATGCTCCAGCAAGCGTTTTCCATACC

2. TTTGGTGGCGGCGATGGCTGGAGAGGCCAGCATTGAAGAC

(RC) GTCTTCAATGCTGGCCTCTCCAGCCATCGCCGCCACCAAA

Source

Replication origin is from pBR322 nucleotide sequence, and 5NSII and 3NSII are from Synechoccocus elongatus PCC7942 genomic DNA.

References