User:Allancrossman/Gibson

From 2011.igem.org

(Difference between revisions)
(→Plac-RBS-LacZ-RBS-INP[spacer]-Endoglucanase)
(→Plac-RBS-LacZ-RBS-INP[spacer]-malS)
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** INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating spacer3fs and spacer3rt
** INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating spacer3fs and spacer3rt
** malS = <partinfo>BBa_K523001</partinfo>
** malS = <partinfo>BBa_K523001</partinfo>
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===Sequences===
 
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Only the relevant parts of the sequence are shown; any flanking regions have not been shown, since they are irrelevant to the planning of primers. Since Gibson is scarless, the concatenation of the sequences is the final sequence.
 
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====Vector pSB1C3====
 
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<pre style="font-size:10pt">
 
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tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt
 
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ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct
 
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gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt
 
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atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc
 
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caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga
 
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gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata
 
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ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac
 
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cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg
 
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taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc
 
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cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag
 
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acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt
 
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aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt
 
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atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg
 
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atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac
 
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gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca
 
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gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac
 
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ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac
 
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ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag
 
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cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc
 
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aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt
 
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cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca
 
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gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga
 
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agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg
 
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ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt
 
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aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac
 
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tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac
 
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tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca
 
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tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg
 
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tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg
 
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actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc
 
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cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa
 
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atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga
 
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tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa
 
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aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat
 
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gatttctggaattcgcggccgcttctagag
 
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</pre>
 
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====Plac-RBS-LacZ-RBS====
 
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(Three bases added at end to give RBS room to work)
 
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<pre style="font-size:10pt">
 
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ctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcg
 
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gtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcat
 
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taatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttac
 
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actttatgcttccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcg
 
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ttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccg
 
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caccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga
 
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tactagagctcaaggaggTAA
 
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</pre>
 
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====INP[spacer]====
 
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(Three bases added at start to give RBS room to work)
 
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<pre style="font-size:10pt">
 
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ATAatgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgtggcctgatctggcctgcttctggtacagttgaaagc
 
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cgctattggcagtctacacgtcgtcacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgttcatgccgatgctcgtt
 
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ggattgtttgtgaggttgctgttgccgacatcattagcctggaagaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtat
 
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tagcgcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctctaccagtacactgacaccaatgccaaccgctatccct
 
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actcctatgccggctgtagcctctgttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtctgctgctgctgctcctg
 
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ttaacactctgcctgttaccacgcctcaaaatctgcaaacggcgacctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatgg
 
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ctctaacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggcaggtgatcaatctcgtctgaccgctggcaaaaattcc
 
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gttctgaccgctggggcacgttctaaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatctttcgtctgtgggatggta
 
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aacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtggaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccgga
 
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cgaagatgatgattggatcgaagtgaaaggtactagcccgacgaccagccccacgccgaccccgacg
 
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</pre>
 
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====malS [mature peptide only, aa 18 onwards]====
 
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<pre style="font-size:10pt">
 
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gccagctggacttctc
 
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cggggtttcccgcctttagcgaacaggggacaggaacatttgtcagccacgcgcagttgcccaaaggtacgcgtccactaacgctaaattttgaccaaca
 
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gtgctggcagcctgcggatgcgataaaactcaatcagatgctttccctgcaaccttgtagcaacacgccgcctcaatggcgattgttcagggacggcgaa
 
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tatacgctgcaaatagacacccgctccggtacgccaacattgatgatttccatccagaacgccgccgaaccggtagcaagcctggtccgtgaatgcccga
 
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aatgggatggattaccgctcacagtggatgtcagcgccactttcccggaaggagccgccgtacgggattattacagccagcaaattgcgatagtgaagaa
 
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cggtcaaataatgttacaacccgctgccaccagcaacggtttactcctgctggaacgggcagaaactgacacatccgcccctttcgactggcataacgcc
 
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acggtttactttgtgctgacagatcgtttcgaaaacggcgatcccagtaatgaccagagttacggacgtcataaagacggtatggcggaaattggcactt
 
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ttcacggcggcgatttacgcggcctgaccaacaaactggattacctccagcagttgggcgttaatgctttatggataagcgccccatttgagcaaattca
 
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cggctgggtcggcggcggtacaaaaggcgatttcccgcattatgcctaccacggttattacacacaggactggacgaatcttgatgccaatatgggcaac
 
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gaagccgatctacggacgctggttgatagcgcacatcagcgcggtattcgtattctctttgatgtcgtgatgaaccacaccggctatgccacgctggcgg
 
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atatgcaggagtatcagtttggcgcgttatatctttctggtgacgaagtgaaaaaatcgctgggtgaacgctggagcgactggaaacctgccgccgggca
 
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aacctggcatagctttaacgattacattaatttcagcgacaaaacaggctgggataaatggtggggaaaaaactggatcagaacggatatcggcgattac
 
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gacaatcctggattcgacgatctcactatgtcgctagcctttttgccggatatcaaaaccgaatcaactaccgcttctggtctgccggtgttctataaaa
 
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acaaaatggatacccacgccaaagccattgacggctatacgccgcgcgattacttaacccactggttaagtcagtgggtccgcgactatgggattgatgg
 
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ttttcgggtcgataccgccaaacatgttgagttgcccgcctggcagcaactgaaaaccgaagccagcgccgcgcttcgcgaatggaaaaaagctaacccc
 
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gacaaagcattagatgacaaacctttctggatgaccggtgaagcctggggccacggcgtgatgcaaagtgactactatcgccacggcttcgatgcgatga
 
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tcaatttcgattatcaggagcaggcggcgaaagcagtcgactgtctggcgcagatggatacgacctggcagcaaatggcggagaaattgcagggtttcaa
 
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cgtgttgagctacctctcgtcgcatgatacccgcctgttccgtgaagggggcgacaaagcagcagagttattactattagcgccaggcgcggtacaaatc
 
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ttttatggtgatgaatcctcgcgtccgttcggtcctacaggttctgatccgctgcaaggtacacgttcggatatgaactggcaggatgttagcggtaaat
 
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ctgccgccagcgtcgcgcactggcagaaaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttttgctgaagcaggg
 
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ctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaa
 
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</pre>
 
===Primers===
===Primers===

Revision as of 14:28, 5 August 2011

In the event that a straight-up Gibson assembly becomes desirable...

Primer strategy: to create homology between part A and part B, use >= 20 bases from the end of A and the start of B; gene A's reverse primer is the reverse complement of gene B's forward primer. It is possible to add bases in the junction. If a stop codon is needed, I only use one stop codon in case we need to use a template that completely lacks stops; in that case one stop codon will be added by the PCR.

Contents

Plac-RBS-LacZ-RBS-INP[spacer]-malS

  • Templates:
    • Vector = pSB1C3
    • Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
    • INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating spacer3fs and spacer3rt
    • malS = <partinfo>BBa_K523001</partinfo>

Primers

Vector

  • Forward: gtctgggcagggcaacagtaa tactagtagcggccgctgcag
  • Reverse: gttaacggcgggatataacatgag ctctagaagcggccgcgaattc

Plac-RBS-LacZ-RBS

  • Forward: gaattcgcggccgcttctagag ctcatgttatatcccgccgttaac
  • Reverse: gctttatccagggtcatTAT TTAcctccttgagctctagtatc

INP[spacer]

  • Forward: gatactagagctcaaggaggTAA ATAatgaccctggataaagc
  • Reverse: cccggagaagtccagctggc cgtcggggtcggcgtggggc

malS

  • Forward: gccccacgccgaccccgacg gccagctggacttctccggg
  • Reverse: ctgcagcggccgctactagta ttactgttgccctgcccagac

Plac-RBS-LacZ-RBS-INP[spacer]-Endoglucanase

  • Templates:
    • Vector = pSB1C3
    • Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
    • INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating spacer3fs and spacer3rt
    • Endoglucanase = <partinfo>BBa_K118023</partinfo>

Sequences

Endoglucanase [mature peptide only, aa 32 onwards]

gctcccg
gctgccgcgtcgactacgccgtcaccaaccagtggcccggcggcttcggcgccaacgtcacgatcaccaacctcggcgaccccgtctcgtcgtggaagct
cgactggacctacaccgcaggccagcggatccagcagctgtggaacggcaccgcgtcgaccaacggcggccaggtctccgtcaccagcctgccctggaac
ggcagcatcccgaccggcggcacggcgtcgttcgggttcaacggctcgtgggccgggtccaacccgacgccggcgtcgttctcgctcaacggcaccacct
gcacgggcaccgtgccgacgaccagccccacgccgaccccgacgccgacgacccccacgccgacgccgaccccgacccccacccccacgccgacggtcac
gccgcagccgaccagcggcttctacgtcgacccgacgacgcagggctaccgcgcgtggcaggccgcgtccggcacggacaaggcgctgctcgagaagatc
gcgctcaccccgcaggcgtactgggtcggcaactgggccgacgcgtcgcacgcgcaggccgaggtcgccgactacaccggccgcgccgtcgcggccggga
agacgccgatgctcgtcgtctacgcgatcccgggccgcgactgcggctcgcactccggcggtggtgtgtccgagtccgagtacgcgcgctgggtcgacac
cgtcgcgcagggcatcaagggcaacccgatcgtgatcctcgagcccgacgcgctcgcgcagctcggcgactgctccggccagggtgaccgcgtcggcttc
ctcaagtacgccgccaagtcgctcaccctcaagggcgcgcgcgtctacatcgacgcgggccacgcgaagtggctgtcggtcgacacgccggtgaaccgcc
tcaaccaggtcggcttcgagtacgcggtgggcttcgcgctcaacacgtcgaactaccagacgacggcggacagcaaggcgtacggccagcagatctcgca
gcggctgggcggcaagaagttcgtcatcgacacctcgcgcaacggcaacggctcgaacggcgagtggtgcaacccgcgcggccgcgcgctcggcgaacgc
ccggtcgcggtgaacgacggctccggcctggacgcgctcctgtgggtcaagctgcccggcgagtccgacggcgcgtgcaacggcggcccggccgccggcc
agtggtggcaggagatcgccctggagatggcgcgcaacgccaggtggtaa

Vector

  • Forward: cgcgcaacgccaggtggtaa tactagtagcggccgctgcag
  • Reverse: [as for malS]

Plac-RBS-LacZ-RBS

  • Forward: [as for malS]
  • Reverse: [as for malS]

INP[spacer]

  • Forward: [as for malS]
  • Reverse: gtcgacgcggcagccgggagc cgtcggggtcggcgtggggc

Endoglucanase

  • Forward: gccccacgccgaccccgacg gctcccggctgccgcgtcgac
  • Reverse: ctgcagcggccgctactagta ttaccacctggcgttgcgcg
Retrieved from "http://2011.igem.org/User:Allancrossman/Gibson"