Template:Team:UC Berkeley/Notebook/MJV construction

patching attR2 site in assembly vector OriV entry vector

Construction of {} Basic Part K112101 PCR mv003/mv004 on pBca9145-Bca1010   (1060bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112101 --- mv003	Forward Biobricking of Cre> ccataGAATTCatgAGATCTatgtccaatttactgaccgtac mv004	Reverse Biobricking of Cre> cgttaGGATCCatcgccatcttccagcaggcg

Construction of {} Basic Part K112102 PCR mv001/mv004 on pBca9145-Bca1010   (1057bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112102 --- mv001	Forward Biobricking of  cgttaGGATCCatcgccatcttccagcaggcg

Construction of {r.BamHI!} Basic Part K112103 PCR mv005/mv006 on pBca9145-I716210   (673bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112103 --- mv005	Forward Biobricking of r.BamH1! ccataGAATTCatgAGATCTatggaagtagaaaaagagtttattactgatgaagc mv006	Reverse Biobricking of r.BamH1! cgttaGGATCCctatttgttttcaactttatctttcc

Construction of {r.BamHI>} Basic Part K112104 PCR mv005/mv007 on pBca9145-I716210   (670bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112104 --- mv005	Forward Biobricking of r.BamHI! ccataGAATTCatgAGATCTatggaagtagaaaaagagtttattactgatgaagc mv007	Reverse Biobricking of r.BamHI> cgttaGGATCCtttgttttcaactttatctttcc

Construction of {} Basic Part K112105 PCR mv008/mv007 on pBca9145-I716210   (667bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112105 --- mv008	Forward Biobricking of  ccataGAATTCatgAGATCTgaagtagaaaaagagtttattactgatg mv007	Reverse Biobricking of r.BamHI> cgttaGGATCCtttgttttcaactttatctttcc

Construction of {r.BglII!} Basic Part K112106 PCR mv009/mv010 on pBca9145-Bca1147   (703bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112106 --- mv009	Forward Biobricking of r.BglII! ccataGAATTCatgAGATCTatgaagattgatataacggactataaccatgc mv010	Reverse Biobricking of r.BglII! cgttaGGATCCttaatatgtcacgattgttcctc

Construction of {r.BglII>} Basic Part K112107 PCR mv009/mv011 on pBca9145-Bca1147   (700bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112107 --- mv009	Forward Biobricking of r.BglII! ccataGAATTCatgAGATCTatgaagattgatataacggactataaccatgc mv011	Reverse Biobricking of r.BglII> cgttaGGATCCatatgtcacgattgttcctc

Construction of {} Basic Part K112108 PCR mv012/mv011 on pBca9145-Bca1147   (697bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112108 --- mv012	Forward Biobricking of  ccataGAATTCatgAGATCTaagattgatataacggactataacc mv011	Reverse Biobricking of r.BglII> cgttaGGATCCatatgtcacgattgttcctc

Construction of {rbs.ligase!} Basic Part K112109 PCR mv013/mv016 on T4 DNA ligase   (269 bp, gp = A) PCR mv015/mv018 on T4 DNA ligase    (376 bp, gp = B) PCR mv017/mv020 on T4 DNA ligase    (191 bp, gp = C) PCR mv019/mv014 on T4 DNA ligase    (746 bp, gp = D) --- PCR mv013/mv018 on A+B             (622 bp, gp = E) PCR mv017/mv014 on C+D              (914 bp, gp = F) --- PCR mv013/mv014 on E+F             (1514bp, EcoRI/BamHI/DpnI) Sub into pBca1256                  (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112109 --- mv013	Forward Biobricking of rbs.ligase! ccataGAATTCatgAGATCTaaactctaaaggatgaacaatg mv014	Reverse Biobricking of ligase! cgttaGGATCCtcatagaccagttacctcatg mv015 Removing the 1st EcoRI site in T4 DNA ligase(F)  gacttcattgaGttcacattagc mv016 Removing the 1st EcoRI site in T4 DNA ligase(R)  gctaatgtgaaCtcaatgaagtc mv017 Removing the BglII site in T4 DNA ligase(F)  ctaggattagaCcttcttaagg mv018 Removing the BglII site in T4 DNA ligase(R)  ccttaagaagGtctaatcctag mv019 Removing the 2nd EcoRI site in T4 DNA ligase(F)  gctaaagaGttcgccgaagtagc mv020 Removing the 2nd EcoRI site in T4 DNA ligase(R)  gctacttcggcgaaCtctttagc

Construction of {ligase!} Basic Part K112110 PCR mv021/mv014 on pBca1256-K112109   (1495bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112110 --- mv021	Forward Biobricking of ligase! ccataGAATTCatgAGATCTatgattcttaaaattctgaacg mv014	Reverse Biobricking of ligase! cgttaGGATCCtcatagaccagttacctcatg

Construction of {} Basic Part K112112 PCR mv021/mv023 on pBca1256-K112109   (1492bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112112 --- mv021	Forward Biobricking of ligase! ccataGAATTCatgAGATCTatgattcttaaaattctgaacg mv023	Reverse Biobricking of ligase> cgttaGGATCCtagaccagttacctcatg

Construction of { } Basic Part K112113 PCR mv022/mv023 on pBca1256-K112109   (1489bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112113 --- mv022	Forward Biobricking of  cgttaGGATCCtagaccagttacctcatg

Construction of {a~ligase!} Basic Part K112114 PCR mv024/mv014 on pBca1256-K112109   (1499bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112114 --- mv024	Forward Biobricking of a~ligase! ccataGAATTCatgAGATCTaacaatgattcttaaaattctgaacg mv014	Reverse Biobricking of ligase! cgttaGGATCCtcatagaccagttacctcatg

Construction of {a~Cre!} Basic Part K112115 PCR mv025/mv002 on pBca9145-Bca1010   (1067bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112115 --- mv025	Forward Biobricking of a~Cre! ccataGAATTCatgAGATCTttaaatgtccaatttactgaccgtacacc mv002	Reverse Biobricking of <Cre! cgttaGGATCCttaatcgccatcttccagc

Construction of {a~r.BamHI!} Basic Part K112116 PCR mv026/mv006 on pBca9145-I716210   (677bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112116 --- mv026	Forward Biobricking of a~r.BamH1! ccataGAATTCatgAGATCTtagaatggaagtagaaaaagagtttattactgatgaagc mv006	Reverse Biobricking of r.BamH1! cgttaGGATCCctatttgttttcaactttatctttcc

Construction of {a~r.BglII!} Basic Part K112117 PCR mv027/mv010 on pBca9145-Bca1147   (707bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112117 --- mv027	Forward Biobricking of a~r.BglII! ccataGAATTCatgAGATCTcataatgaagattgatataacggactataaccatgc mv010	Reverse Biobricking of r.BglII! cgttaGGATCCttaatatgtcacgattgttcctc

Construction of rrnB P1 promoter Basic Part K112118 PCR mv028/mv029 on rrnB native sequence  (721bp, EcoRI/BamHI/DpnI) Sub into pBca1256                        (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112118 --- mv028	Forward Biobricking of rrnB P1 promoter cgataGAATTCatgAGATCTatgcactaaaacgtatcctacgcccg mv029  Reverse Biobricking of rrnB P1 promoter cgttaGGATCCcgccttcccgctacagagtcaagc

Construction of {rbs.Cre!} Basic Part K112119 PCR mv030/mv002 on pBca9145-Bca1010   (1082bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112119 --- mv030	Forward Biobricking of rbs.Cre! cgataGAATTCatgAGATCTaccattatctgagtgttaaatgtccaatttactgaccgtacacc mv002	Reverse Biobricking of <Cre! cgttaGGATCCttaatcgccatcttccagc

Construction of {rbs.r.BamHI!} Basic Part K112120 PCR mv031/mv006 on pBca9145-I716210   (692bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112120 --- mv031	Forward Biobricking of rbs.r.BamH1! cgataGAATTCatgAGATCTtaagtgggggcaaattagaatggaagtagaaaaagagtttattactgatgaagc mv006	Reverse Biobricking of r.BamH1! cgttaGGATCCctatttgttttcaactttatctttcc

Construction of {rbs.r.BglII!} Basic Part K112121 PCR mv032/mv010 on pBca9145-Bca1147   (722bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112121 --- mv032	Forward Biobricking of rbs.r.BglII! cgataGAATTCatgAGATCTtggaaggagacactcataatgaagattgatataacggactataaccatgc mv010	Reverse Biobricking of r.BglII! cgttaGGATCCttaatatgtcacgattgttcctc

Construction of {Cre!} Basic Part K112122 PCR mv003/mv002 on pBca9145-Bca1010   (1063bp, EcoRI/BamHI/DpnI) Sub into pBca1256                     (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112122 --- mv003	Forward Biobricking of Cre> ccataGAATTCatgAGATCTatgtccaatttactgaccgtac mv002	Reverse Biobricking of  ccataGAATTCatgAGATCTaagattgatataacggactataacc mv010	Reverse Biobricking of r.BglII! cgttaGGATCCttaatatgtcacgattgttcctc

Construction of {rbs.trfA!} Basic Part K112124 PCR JH102F/JH102R on RP4 genome     (416bp, EcoRI/BamHI/DpnI) Sub into pBjh1601KC                 (EcoRI/BamHI, 3132+778, L) Product is pBjh1601KC-K112124 --- JH102F	 Forward Biobricking of {rbs.trfA!} ccatgGAATTCatgAGATCTgcgaggggcggcttccgctAtgtacaaccag JH102R	 Reverse Biobricking of {rbs.trfA!} cgttaGGATCCtcagagccttccacgcagc

Construction of {OriV} Basic Part K112125 PCR mv037/mv038 on pSMART Fosmid Blunt Vector (Lucigen)   (677bp, EcoRI/BamHI/DpnI) Sub into pBca1256                       (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112125 --- mv037 Forward biobricking of OriV from pSMART cgataGAATTCatgAGATCTtgcttcctcgctcactcgac mv038 Reverse biobricking of OriV form pSMART gctagGGATCCaagccctcggctggttgc

'''Construction of {temp. sensitive promoter} Basic Part K112126''' PCR JH115F/JH115R on pAH57 sub into pBca1256 Product is pBca1256-K112126 --- JH115F Forward biobricking of cI, Pr cgataGAATTCatgAGATCTgaagattcttgctcaattgttatcagc JH115R Reverse biobricking of cI, Pr gctagGGATCCagaattgatacaacctccttagtacatgc

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