Difference between revisions of "Part:BBa K2350006:Design"
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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. 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 5NSII with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting site in 5NSII nucleotide sequence.  
 
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. 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 5NSII with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting site in 5NSII nucleotide sequence.  
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<partinfo>BBa_K2350006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2350006 SequenceAndFeatures</partinfo>
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===Design Notes===
 
===Design Notes===
−
All Pst1 cutting sites were site-directed mutated.
+
1. All Pst1 cutting sites were site-directed mutated.
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 +
(1)First site-directed mutagenesis primers:
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 +
Forward: GGTATGGAAAACGCTTGCTGGAGCATCAGATCGATGCGAT
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 +
Reverse: ATCGCATCGATCTGATGCTCCAGCAAGCGTTTTCCATACC
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 +
(2)Second site-directed mutagenesis primers:
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Forward: TTTGGTGGCGGCGATGGCTGGAGAGGCCAGCATTGAAGAC
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Reverse: GTCTTCAATGCTGGCCTCTCCAGCCATCGCCGCCACCAAA
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2. PCR primers for S. elongatus PCC7942 genomic DNA
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Forward: caggcaatcacgatgcgct
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 +
Reverse: ataataGAATTCTCATTGCACACCCATCCATG
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Revision as of 08:46, 21 October 2017


5’-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). 5NSII is part of neutral site gene, which is near 5’-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 5NSII with EcoR1 and Pst1 cutting sites, we use site-directed mutagenesis to remove Pst1 cutting site in 5NSII nucleotide sequence.



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

1. All Pst1 cutting sites were site-directed mutated.

(1)First site-directed mutagenesis primers:

Forward: GGTATGGAAAACGCTTGCTGGAGCATCAGATCGATGCGAT

Reverse: ATCGCATCGATCTGATGCTCCAGCAAGCGTTTTCCATACC

(2)Second site-directed mutagenesis primers:

Forward: TTTGGTGGCGGCGATGGCTGGAGAGGCCAGCATTGAAGAC

Reverse: GTCTTCAATGCTGGCCTCTCCAGCCATCGCCGCCACCAAA

2. PCR primers for S. elongatus PCC7942 genomic DNA

Forward: caggcaatcacgatgcgct

Reverse: ataataGAATTCTCATTGCACACCCATCCATG



Source

Synechoccocus elongatus PCC7942 genomic DNA

References