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User:Allancrossman/Gibson
From 2011.igem.org
(Difference between revisions)
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In the event that a straight-up Gibson assembly becomes desirable... | In the event that a straight-up Gibson assembly becomes desirable... | ||
| - | '''Primer strategy:''' use >= 20 bases | + | '''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. |
==Plac-RBS-LacZ-RBS-INP[spacer]-malS== | ==Plac-RBS-LacZ-RBS-INP[spacer]-malS== | ||
Revision as of 14:04, 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.
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>
Sequences
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 sequence is the final sequence. In all cases I only use one stop codon in case we need to use a template that completely lacks it.
Vector pSB1C3
tactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgcccttt ttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgct gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggc caggaaccgtaaaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagata ccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctg taggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaag acacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattac gcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac ttggtctgacagctcgaggcttggattctcaccaataaaaaacgcccggcggcaaccgag cgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcc aagcgagctcgatatcaaattacgccccgccctgccactcatcgcagtactgttgtaatt cattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgcca gcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcga agaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattgg ctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgt aacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcac tccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacac tatcccatatcaccagctcaccgtctttcattgccatacgaaattccggatgagcattca tcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacgg tctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactg actgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatc cagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaa atacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgcccga tcaactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaa aataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggat gatttctggaattcgcggccgcttctagag
Plac-RBS-LacZ-RBS
(Three bases added at end to give RBS room to work)
ctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcg gtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcat taatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttac actttatgcttccggctcgtatgttgtgtgaaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcg ttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccg caccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtga tactagagctcaaggaggTAA
INP[spacer]
(Three bases added at start to give RBS room to work)
ATAatgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgtggcctgatctggcctgcttctggtacagttgaaagc cgctattggcagtctacacgtcgtcacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgttcatgccgatgctcgtt ggattgtttgtgaggttgctgttgccgacatcattagcctggaagaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtat tagcgcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctctaccagtacactgacaccaatgccaaccgctatccct actcctatgccggctgtagcctctgttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtctgctgctgctgctcctg ttaacactctgcctgttaccacgcctcaaaatctgcaaacggcgacctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatgg ctctaacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggcaggtgatcaatctcgtctgaccgctggcaaaaattcc gttctgaccgctggggcacgttctaaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatctttcgtctgtgggatggta aacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtggaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccgga cgaagatgatgattggatcgaagtgaaaggtactagcccgacgaccagccccacgccgaccccgacg
malS [mature peptide only, aa 18 onwards]
gccagctggacttctc cggggtttcccgcctttagcgaacaggggacaggaacatttgtcagccacgcgcagttgcccaaaggtacgcgtccactaacgctaaattttgaccaaca gtgctggcagcctgcggatgcgataaaactcaatcagatgctttccctgcaaccttgtagcaacacgccgcctcaatggcgattgttcagggacggcgaa tatacgctgcaaatagacacccgctccggtacgccaacattgatgatttccatccagaacgccgccgaaccggtagcaagcctggtccgtgaatgcccga aatgggatggattaccgctcacagtggatgtcagcgccactttcccggaaggagccgccgtacgggattattacagccagcaaattgcgatagtgaagaa cggtcaaataatgttacaacccgctgccaccagcaacggtttactcctgctggaacgggcagaaactgacacatccgcccctttcgactggcataacgcc acggtttactttgtgctgacagatcgtttcgaaaacggcgatcccagtaatgaccagagttacggacgtcataaagacggtatggcggaaattggcactt ttcacggcggcgatttacgcggcctgaccaacaaactggattacctccagcagttgggcgttaatgctttatggataagcgccccatttgagcaaattca cggctgggtcggcggcggtacaaaaggcgatttcccgcattatgcctaccacggttattacacacaggactggacgaatcttgatgccaatatgggcaac gaagccgatctacggacgctggttgatagcgcacatcagcgcggtattcgtattctctttgatgtcgtgatgaaccacaccggctatgccacgctggcgg atatgcaggagtatcagtttggcgcgttatatctttctggtgacgaagtgaaaaaatcgctgggtgaacgctggagcgactggaaacctgccgccgggca aacctggcatagctttaacgattacattaatttcagcgacaaaacaggctgggataaatggtggggaaaaaactggatcagaacggatatcggcgattac gacaatcctggattcgacgatctcactatgtcgctagcctttttgccggatatcaaaaccgaatcaactaccgcttctggtctgccggtgttctataaaa acaaaatggatacccacgccaaagccattgacggctatacgccgcgcgattacttaacccactggttaagtcagtgggtccgcgactatgggattgatgg ttttcgggtcgataccgccaaacatgttgagttgcccgcctggcagcaactgaaaaccgaagccagcgccgcgcttcgcgaatggaaaaaagctaacccc gacaaagcattagatgacaaacctttctggatgaccggtgaagcctggggccacggcgtgatgcaaagtgactactatcgccacggcttcgatgcgatga tcaatttcgattatcaggagcaggcggcgaaagcagtcgactgtctggcgcagatggatacgacctggcagcaaatggcggagaaattgcagggtttcaa cgtgttgagctacctctcgtcgcatgatacccgcctgttccgtgaagggggcgacaaagcagcagagttattactattagcgccaggcgcggtacaaatc ttttatggtgatgaatcctcgcgtccgttcggtcctacaggttctgatccgctgcaaggtacacgttcggatatgaactggcaggatgttagcggtaaat ctgccgccagcgtcgcgcactggcagaaaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttttgctgaagcaggg ctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaataa
Primers
Vector
- Forward:
- Reverse:
Plac-RBS-LacZ-RBS
- Forward:
- Reverse:
INP[spacer]
- Forward:
- Reverse:
malS
- Forward:
- Reverse:
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 agtggtggcaggagatcgccctggagatggcgcgcaacgccaggtggtaataa
