Template:SBB10 ConstructionFiles BenB sbb01
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Construction of sbb01: N15 Protelomerase
PCR BDBn15001F/BDBn15001R on Bca1455 (942 bp, gp=A)
PCR BDBn15002F/BDBn15002R on Bca1485 (1020 bp, gp=B)
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PCR BDBn15001F/BDBn15002R on A+B (1924 bp, EcoR1/BamH1)
Sub in pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L)
Product is pBjk2741-sbb01 {rbs.part!}
BDBn15001F Forward retrieval, RBS introduction and BioBricking on Bca1455
CCATAgaattcATGagatctTCAATCTCGGAGGAACGGTAAGGAAGGAAAACatgAGCAAGGTAAAAATCGGTGAG
BDBn15001R Reverse retrieval and SOE-site introduciton on Bca1455
CGCTTTTATCTTCACTGCGTTTTTTAGCTTGCCCTGAG
BDBn15002F Forward retrieval and SOE-site introduciton on Bca1485
CTCAGGGCAAGCTAAAAAACGCAGTGAAGATAAAAGCG
BDBn15002R Reverse retrieval and BioBricking on Bca1485
CTGATggatccGCTGTAGTACGTTTCCCATGCGG
JCA Notes
- got 974 for first pcr
- No stop codon on BDBn15002R
- Otherwise correct
- replaced first oligo with shorter, it's pressing your luck to use an oligo that long for soeing
- May need to alter this one based on PCR results
Construction of sbb01: N15 Protelomerase PCR BDBn15001F/CA1472 on Bca1455 1-3 (595bp, gp=A) PCR ca1461/BDBn15001R on Bca1455 1-1 (762bp, gp=B) PCR BDBn15002F/BDBn15002R on Bca1485 2-1 (1023 bp, gp=C) -------------------------------------------------------------- PCR BDBn15001F/BDBn15002R on A+B+C (1950 bp, EcoR1/BamH1) Sub in pBjk2741-Bca1144 (EcoRI/BamHI, 910+2170, L) Product is pBjk2741-sbb01 {rbs.part!} BDBn15001F Forward retrieval, RBS introduction and BioBricking on Bca1455 CCATAgaattcATGagatctGAGGAACGGTAAGGAAGGAAAACatgAGCAAGGTAAAAATCGG CA1461 GGAAAGGATTGCAGAAAAGAATAACCGCGAATACTTTTAACGCCTATATGA PCA assembly of protelomerasefiveprime (Bca1455) CA1472 TTGTTGGAATAACTTATAAAGATAGTCACGGCGCTCCTTCCAATCATCACT PCA assembly of protelomerasefiveprime (Bca1455) BDBn15001R Reverse retrieval and SOE-site introduciton on Bca1455 CGCTTTTATCTTCACTGCGTTTTTTAGCTTGCCCTGAG BDBn15002F Forward retrieval and SOE-site introduciton on Bca1485 CTCAGGGCAAGCTAAAAAACGCAGTGAAGATAAAAGCG BDBn15002R Reverse retrieval and BioBricking on Bca1485 CTGATggatccttaGCTGTAGTACGTTTCCCATGCGG part: gatctGAGGAACGGTAAGGAAGGAAAACatgAGCAAGGTAAAAATCGGTGAGTTGATCAACACGCTTGTGAATGAGGTAGAGGCCATTGATGCCTCAGACCGCCCACAAGGCGACAAAACGAAGAGAATTAAAGCCGCAGCCGCACGGTATAAGAACGCGTTATTTAATGATAAAAGAAAGTTCCGTGGGAAAGGATTGCAGAAAAGAATAACCGCGAATACTTTTAACGCCTATATGAGCAGGGCAAGAAAGCGGTTTGATGATAAATTACATCATAGCTTTGATAAAAATATTAATAAATTATCGGAAAAGTATCCTCTTTACAGCGAAGAATTATCTTCATGGCTTTCTATGCCTACGGCTAATATTCGCCAGCACATGTCATCGTTACAATCTAAATTGAAAGAAATAATGCCGCTTGCCGAAGAGTTATCAAATGTAAGAATAGGCTCTAAAGGCAGTGATGCAAAAATAGCAAGACTAATAAAAAAATATCCAGATTGGAGTTTTGCTCTTAGTGATTTAAACAGTGATGATTGGAAGGAGCGCCGTGACTATCTTTATAAGTTATTCCAACAAGGCTCTGCGTTGTTAGAAGAACTACACCAGCTCAAGGTCAACCATGAGGTTCTGTACCATCTCCAGCTAAGCCCTGCGGAGCGTACATCTATACAGCAACGATGGGCCGATGTTCTGCGCGAGAAGAAGCGTAATGTTGTGGTTATTGACTACCCAACATACATGCAGTCTATCTATGATATTTTGAATAATCCTGCGACTTTATTTAGTTTAAACACTCGTTCTGGAATGGCACCTTTGGCCTTTGCTCTGGCTGCGGTATCAGGGCGAAGAATGATTGAGATAATGTTTCAGGGTGAATTTGCCGTTTCAGGAAAGTATACGGTTAATTTCTCAGGGCAAGCTAAAAAACGCAGTGAAGATAAAAGCGTAACCAGAACGATTTATACTTTATGCGAAGCAAAATTATTCGTTGAATTATTAACAGAATTGCGTTCTTGCTCTGCTGCATCTGATTTCGATGAGGTTGTTAAAGGATATGGAAAGGATGATACAAGGTCTGAGAACGGCAGGATAAATGCTATTTTAGCAAAAGCATTTAACCCTTGGGTTAAATCATTTTTCGGCGATGACCGTCGTGTTTATAAAGATAGCCGCGCTATTTACGCTCGCATCGCTTATGAGATGTTCTTCCGCGTCGATCCACGGTGGAAAAACGTCGACGAGGATGTGTTCTTCATGGAGATTCTCGGACACGACGATGAGAACACCCAGCTGCACTATAAGCAGTTCAAGCTGGCCAACTTTTCCAGAACCTGGCGACCGGAAGTTGGGGATGAAAACACCAGGCTGGTGGCTCTCCAGAAACTGGACGATGAAATGCCAGGCTTTGCCAGAGGTGACGCTGGCGTCCGTCTCCATGAAACCGTTAAGCAGCTGGTGGAGCAGGACCCATCAGCAAAAATAACCAACAGCACTCTCCGGGCCTTTAAATTTAGCCCGACGATGATTAGCCGGTACCTGGAGTTTGCCGCTGATGCATTGGGGCAGTTCGTTGGCGAGAACGGGCAGTGGCAGCTCAAGATAGAGACACCTGCAATCGTCCTGCCTGATGAAGAATCCGTTGAGACCATCGACGAACCGGATGATGAGTCCCAAGACGACGAGCTGGATGAAGATGAAATTGAGCTCGACGAGGGTGGCGGCGATGAACCAACCGAAGAGGAAGGGCCAGAAGAACATCAGCCAACTGCTCTAAAACCCGTCTTCAAGCCTGCAAAAAATAACGGGGACGGAACGTACAAGATAGAGTTTGAATACGATGGAAAGCATTATGCCTGGTCCGGCCCCGCCGATAGCCCTATGGCCGCAATGCGATCCGCATGGGAAACGTACTACAGCTAAG
