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From 2011.igem.org

In the event that a Gibson assembly becomes desirable...

Contents 1 Plac-RBS-LacZ-RBS-INP[spacer]-malS 1.1 Sequences 1.1.1 Plac-RBS-LacZ-RBS 1.1.2 INP[spacer] 1.1.3 malS [mature peptide only] 1.2 Primers

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.

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]

gccagctggacttctc
cggggtttcccgcctttagcgaacaggggacaggaacatttgtcagccacgcgcagttgcccaaaggtacgcgtccactaacgctaaattttgaccaaca
gtgctggcagcctgcggatgcgataaaactcaatcagatgctttccctgcaaccttgtagcaacacgccgcctcaatggcgattgttcagggacggcgaa
tatacgctgcaaatagacacccgctccggtacgccaacattgatgatttccatccagaacgccgccgaaccggtagcaagcctggtccgtgaatgcccga
aatgggatggattaccgctcacagtggatgtcagcgccactttcccggaaggagccgccgtacgggattattacagccagcaaattgcgatagtgaagaa
cggtcaaataatgttacaacccgctgccaccagcaacggtttactcctgctggaacgggcagaaactgacacatccgcccctttcgactggcataacgcc
acggtttactttgtgctgacagatcgtttcgaaaacggcgatcccagtaatgaccagagttacggacgtcataaagacggtatggcggaaattggcactt
ttcacggcggcgatttacgcggcctgaccaacaaactggattacctccagcagttgggcgttaatgctttatggataagcgccccatttgagcaaattca
cggctgggtcggcggcggtacaaaaggcgatttcccgcattatgcctaccacggttattacacacaggactggacgaatcttgatgccaatatgggcaac
gaagccgatctacggacgctggttgatagcgcacatcagcgcggtattcgtattctctttgatgtcgtgatgaaccacaccggctatgccacgctggcgg
atatgcaggagtatcagtttggcgcgttatatctttctggtgacgaagtgaaaaaatcgctgggtgaacgctggagcgactggaaacctgccgccgggca
aacctggcatagctttaacgattacattaatttcagcgacaaaacaggctgggataaatggtggggaaaaaactggatcagaacggatatcggcgattac
gacaatcctggattcgacgatctcactatgtcgctagcctttttgccggatatcaaaaccgaatcaactaccgcttctggtctgccggtgttctataaaa
acaaaatggatacccacgccaaagccattgacggctatacgccgcgcgattacttaacccactggttaagtcagtgggtccgcgactatgggattgatgg
ttttcgggtcgataccgccaaacatgttgagttgcccgcctggcagcaactgaaaaccgaagccagcgccgcgcttcgcgaatggaaaaaagctaacccc
gacaaagcattagatgacaaacctttctggatgaccggtgaagcctggggccacggcgtgatgcaaagtgactactatcgccacggcttcgatgcgatga
tcaatttcgattatcaggagcaggcggcgaaagcagtcgactgtctggcgcagatggatacgacctggcagcaaatggcggagaaattgcagggtttcaa
cgtgttgagctacctctcgtcgcatgatacccgcctgttccgtgaagggggcgacaaagcagcagagttattactattagcgccaggcgcggtacaaatc
ttttatggtgatgaatcctcgcgtccgttcggtcctacaggttctgatccgctgcaaggtacacgttcggatatgaactggcaggatgttagcggtaaat
ctgccgccagcgtcgcgcactggcagaaaatcagccagttccgcgcccgccatcccgcaattggcgcgggcaaacaaacgacacttttgctgaagcaggg
ctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaataa

Primers