SBB09 Construct17

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Construction of {<ECOHLY>} Basic Part M10022 (Bdvs001) into pBca9495AK
PCR Odvs001F/Odvs002rR on VH              (379bp, gp = A)
PCR Odvs002rF/Odvs001R on VH              ( 89bp, gp = B)
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PCR Odvs001F/Odvs001R on A+B to make Bdvs001        (436bp, EcoRI/BamHI)
Sub M10022 into pBca9495KA-Bca1144#5                (EcoRI/BamHI, 3171+910, L)
Product is pBca9495AK-M10022   {<ECOHLY>}           (3491bp)
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Odvs0007F   Forward EcoRI/BglII for Bglbrick of ECOHLY   ccataGAATTCATGAGATCTtattccgtggaagaacttattgggacc
Odvs0007R   Reverse BamHI for Bglbrick of ECOHLY         ctgatGGATCCataaatatcattaccatatccacc
Odvs0008rF  Removing the BglII site in ECOHLY (Forward)  ggcagtgagggagcagaCctgcttgatggcgg
Odvs0008rR  Removing the BglII site in ECOHLY (Reverse)  ccgccatcaagcagGtctgctccctcactgcc

JCA: revised 1

Construction of beta roll intermediate
PCR Odvs0007F/Odvs0008rR on CFT073 gen.         (379bp, gp = A)
PCR Odvs0008rF/Odvs0007R on CFT073 gen.         ( 89bp, gp = B)
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PCR Odvs0007F/Odvs0007R on A+B to make Bdvs001        (436bp, EcoRI/BamHI)
TA kit, product is pCR-betaroll
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Odvs0007F   Forward EcoRI/BglII for Bglbrick of ECOHLY   ccataGAATTCATGAGATCTtattccgtggaagaacttattgggacc
Odvs0007R   Reverse BamHI for Bglbrick of ECOHLY         ctgatGGATCCataaatatcattaccatatccacc
Odvs0008rF  Removing the BglII site in ECOHLY (Forward)  ggcagtgagggagcagaCctgcttgatggcgg
Odvs0008rR  Removing the BglII site in ECOHLY (Reverse)  ccgccatcaagcagGtctgctccctcactgcc


Construction of {<ECOHLY>} Basic Part M10022 (Bdvs001) into pBca9495AK
PCR Odvs0007F/ca1383 on CFT073 gen.         (304bp, gp = A)
PCR ca1382/Odvs0007R on CFT073 gen.         (152bp, gp = B)
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PCR Odvs0007F/Odvs0007R on A+B to make Bdvs001        (436bp, EcoRI/BamHI)
Sub M10022 into pBca9495KA-Bca1144#5                (EcoRI/BamHI, 3171+910, L)
Product is pBca9495AK-M10022   {<Beta Roll>}           (3491bp)
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Odvs0007F   Forward EcoRI/BglII for Bglbrick of ECOHLY   ccataGAATTCATGAGATCTtattccgtggaagaacttattgggacc
Odvs0007R   Reverse BamHI for Bglbrick of ECOHLY         ctgatGGATCCataaatatcattaccatatccacc
ca1382	Forward oligo to remove ecoRI site in beta roll	GTTCAGGGcAATTCTCTTGC
ca1383	Reverse oligo to remove ecoRI site in beta roll	GCAAGAGAATTgCCCTGAAC

JCA: revised 2

PCR Odvs0007F/ca1383 on CFT073 gen.           (304bp, gp = A)
PCR ca1382/Odvs0008rR  on CFT073 gen.         (96bp, gp = B)
PCR Odvs0008rF/Odvs0007R on CFT073 gen.       (89 bp, gp = C)
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PCR Odvs0007F/Odvs0007R on A+B+C           (436bp, EcoRI/BamHI)
sub into pBca9495????? .....
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Odvs0007F   Forward EcoRI/BglII for Bglbrick of ECOHLY   ccataGAATTCATGAGATCTtattccgtggaagaacttattgggacc
Odvs0007R   Reverse BamHI for Bglbrick of ECOHLY         ctgatGGATCCataaatatcattaccatatccacc
Odvs0008rF  Removing the BglII site in ECOHLY (Forward)  ggcagtgagggagcagaCctgcttgatggcgg
Odvs0008rR  Removing the BglII site in ECOHLY (Reverse)  ccgccatcaagcagGtctgctccctcactgcc
ca1382	Forward oligo to remove ecoRI site in beta roll	GTTCAGGGcAATTCTCTTGC
ca1383	Reverse oligo to remove ecoRI site in beta roll	GCAAGAGAATTgCCCTGAAC
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