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Template:Team:UC Berkeley/Notebook/MJV construction
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| + | [[Template:Team:UC Berkeley/Notebook/MJV construction/corrected assembly vector | patching attR2 site in assembly vector]]<br> | ||
| + | [[Template:Team:UC Berkeley/Notebook/MJV construction/OriV entry vector | OriV entry vector]] | ||
| + | |||
| + | |||
'''Construction of {<Cre!} Basic Part K112100''' | '''Construction of {<Cre!} Basic Part K112100''' | ||
<pre> | <pre> | ||
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mv038 Reverse biobricking of OriV form pSMART | mv038 Reverse biobricking of OriV form pSMART | ||
gctagGGATCCaagccctcggctggttgc | gctagGGATCCaagccctcggctggttgc | ||
| + | </pre> | ||
| + | |||
| + | '''Construction of {temp. sensitive promoter} Basic Part K112126''' | ||
| + | <pre> | ||
| + | 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 | ||
</pre> | </pre> | ||
Latest revision as of 23:25, 22 September 2008
patching attR2 site in assembly vector
OriV entry vector
Construction of {<Cre!} Basic Part K112100
PCR mv001/mv002 on pBca9145-Bca1010 (1060bp, gp, EcoRI/BamHI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112100 ----------------------------------------------- mv001 Forward Biobricking of <Cre! ccataGAATTCatgAGATCTtccaatttactgaccgtacacc mv002 Reverse Biobricking of <Cre! cgttaGGATCCttaatcgccatcttccagc
Construction of {Cre>} 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 {<Cre>} 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 <Cre! ccataGAATTCatgAGATCTtccaatttactgaccgtacacc mv004 Reverse Biobricking of Cre> 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 {<r.BamHI>} 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 <r.BamHI> 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 {<r.BglII>} 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 <r.BglII> 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 {<ligase!} Basic Part K112111
PCR mv022/mv014 on pBca1256-K112109 (1492bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112111 ----------------------------------------------- mv022 Forward Biobricking of <ligase! ccataGAATTCatgAGATCTattcttaaaattctgaacg mv014 Reverse Biobricking of ligase! cgttaGGATCCtcatagaccagttacctcatg
Construction of {ligase>} 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 {<ligase>} 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 <ligase! ccataGAATTCatgAGATCTattcttaaaattctgaacg mv023 Reverse Biobricking of ligase> 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 <Cre! cgttaGGATCCttaatcgccatcttccagc
Construction of {<r.BglII!} Basic Part K112123
PCR mv012/mv010 on pBca9145-Bca1147 (700bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112123 ----------------------------------------------- mv012 Forward Biobricking of <r.BglII> 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
