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> | ||
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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. |
+ | |||
+ | (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=== | ===Source=== |
Latest 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.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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