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From 2011.igem.org

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 1 Plac-RBS-LacZ-RBS-INP[spacer]-malS 1.1 Primers 1.1.1 Vector 1.1.2 Plac-RBS-LacZ-RBS 1.1.3 INP[spacer] 1.1.4 malS 2 Plac-RBS-LacZ-RBS-INP[spacer]-Endoglucanase 2.1 Sequences 2.1.1 Endoglucanase [mature peptide only, aa 32 onwards] 2.1.2 Vector 2.1.3 Plac-RBS-LacZ-RBS 2.1.4 INP[spacer] 2.1.5 Endoglucanase

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