"

From 2011.igem.org

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

Primer strategy: use >= 20 bases in each direction; gene A's reverse primer is the reverse complement of gene B's forward primer.

Contents 1 Plac-RBS-LacZ-RBS-INP[spacer]-malS 1.1 Sequences 1.1.1 Vector pSB1C3 1.1.2 Plac-RBS-LacZ-RBS 1.1.3 INP[spacer] 1.1.4 malS [mature peptide only, aa 18 onwards] 1.2 Primers 1.2.1 Vector 1.2.2 Plac-RBS-LacZ-RBS 1.2.3 INP[spacer] 1.2.4 malS 2 Plac-RBS-LacZ-RBS-INP[spacer]-Endoglucanase 2.1 Sequences 2.1.1 Endoglucanase [mature peptide only, aa 32 onwards]

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