Template:SBB10 ConstructionFiles BenB sbb01

Construction of sbb01: N15 Protelomerase

PCR BDBn15001F/BDBn15001R on Bca1455		(942 bp, gp=A) PCR BDBn15002F/BDBn15002R on Bca1485		(1020 bp, gp=B) -- 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
Construction of sbb01: N15 Protelomerase
 * 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

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