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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.

Note that (before any PCR) the INP part has to be created by attaching linker2fs and linker2rt to <partinfo>BBa_K523008</partinfo>. Note that we must use the new, serine-rich linker to avoid certain issues.

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 Primers 2.1.1 Vector 2.1.2 Plac-RBS-LacZ-RBS 2.1.3 INP[spacer] 2.1.4 Endoglucanase 3 Plac-RBS-LacZ-RBS-INP[spacer]-Exoglucanase 3.1 Primers 3.1.1 Vector 3.1.2 Plac-RBS-LacZ-RBS 3.1.3 INP[spacer] 3.1.4 Exoglucanase 4 Plac-RBS-LacZ-RBS-INP[spacer]-B.Glucosidase 4.1 Primers 4.1.1 Vector 4.1.2 Plac-RBS-LacZ-RBS 4.1.3 INP[spacer] 4.1.4 B-Glucosidase 5 Appendix: Sequences 5.1 Vector pSB1C3 5.2 Plac-RBS-LacZ-RBS 5.3 INP[with serine-rich linker] 5.4 malS [mature peptide only, aa 18 onwards] 5.5 Endoglucanase BBa_K118023 [mature peptide only, aa 32 onwards] 5.6 Exoglucanase BBa_K118022 [mature peptide only, aa 43 onwards] 5.7 B-glucosidase BBa_K392008 (or whatever we have)

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 linker2fs and linker2rt
  • 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 ccgccacgctgctcgggctg

malS

• Forward: cagcccgagcagcgtggcgg 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 linker2fs and linker2rt
  • Endoglucanase - use <partinfo>BBa_K118023</partinfo>. Don't use anything else; in particular, our previous primers to PCR this altered some codons and the product doesn't match the new primers below. So use K118023.

Primers

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 ccgccacgctgctcgggctg

Endoglucanase

• Forward: cagcccgagcagcgtggcgg gctcccggctgccgcgtcgac
• Reverse: ctgcagcggccgctactagta ttaccacctggcgttgcgcg

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

• Templates:
  • Vector = pSB1C3
  • Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
  • INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating linker2fs and linker2rt
  • Exoglucanase = <partinfo>BBa_K118022</partinfo>

Primers

Vector

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

Plac-RBS-LacZ-RBS

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

INP[spacer]

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

Exoglucanase

• Forward: cagcccgagcagcgtggcgg gcgaccacgctcaaggaggc
• Reverse: ctgcagcggccgctactagta ttagccgaccgtgcagggcg

Plac-RBS-LacZ-RBS-INP[spacer]-B.Glucosidase

• Templates:
  • Vector = pSB1C3
  • Plac-RBS-LacZ-RBS = <partinfo>BBa_K523005</partinfo>
  • INP[spacer] - make by cutting <partinfo>BBa_K523008</partinfo> with SpeI and ligating linker2fs and linker2rt
  • B-glucosidase = <partinfo>BBa_K392008</partinfo>

Primers

Vector

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

Plac-RBS-LacZ-RBS

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

INP[spacer]

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

B-Glucosidase

• Forward: cagcccgagcagcgtggcgg accaccacgcgcccctcggg
• Reverse: ctgcagcggccgctactagta tcagggctggtaggtcgcgg

Appendix: 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 sequences involved is the final sequence.

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[with serine-rich linker]

(Three bases added at start to give RBS room to work)

ATAatgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattgtggcctgatctggcctgcttctggtacagttgaaagc
cgctattggcagtctacacgtcgtcacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgttcatgccgatgctcgtt
ggattgtttgtgaggttgctgttgccgacatcattagcctggaagaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtat
tagcgcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctctaccagtacactgacaccaatgccaaccgctatccct
actcctatgccggctgtagcctctgttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtctgctgctgctgctcctg
ttaacactctgcctgttaccacgcctcaaaatctgcaaacggcgacctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatgg
ctctaacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggcaggtgatcaatctcgtctgaccgctggcaaaaattcc
gttctgaccgctggggcacgttctaaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatctttcgtctgtgggatggta
aacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgtggaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccgga
cgaagatgatgattggatcgaagtgaaaggtactagcagcagcattgcgagcagcagcccgagcagcgtggcgg

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
ctacggctttgttcgtgagcatggcgacgataaagtgctggtcgtctgggcagggcaacagtaa

Endoglucanase BBa_K118023 [mature peptide only, aa 32 onwards]

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

Exoglucanase BBa_K118022 [mature peptide only, aa 43 onwards]

gcgaccacgctcaaggaggccgccgacggcgccggccgggacttcggcttcgcgctcgaccccaaccggctctc
ggaggcgcagtacaaggcgatcgccgacagcgagttcaacctcgtcgtcgccgagaacgcgatgaagtgggacgccaccgagccctcgcagaacagcttc
tccttcggcgcgggcgaccgcgtcgcgagctacgccgccgacaccggcaaggagctgtacggccacacgctcgtatggcactcgcagctgcccgactggg
cgaagaacctcaacggctccgcgttcgagagcgcgatggtcaaccacgtgacgaaggtcgccgaccacttcgagggcaaggtcgcgtcgtgggacgtcgt
caacgaggcgttcgccgacggcggcggccgccggcaggactcggcgttccagcagaagctcggcaacggctacatcgagaccgcgttccgggcggcacgt
gcggcggacccgaccgccaagctgtgcatcaacgactacaacgtcgagggcatcaacgcgaagagcaactcgctctacgacctcgtcaaggacttcaagg
cgcgcggcgtcccgctcgactgcgtcgggttccagtcgcacctcatcgtcggccaggtgccgggcgacttccggcagaacctgcaacggttcgcggacct
gggcgtggacgtgcgcatcaccgagctcgacatccgcatgcggacgccctccgacgcgaccaagctcgcgacccaggcggccgactacaagaaggtcgtg
caggcctgcatgcaggtgacccgctgccagggcgtgaccgtctggggcatcaccgacaagtactcgtgggtgccggacgtcttcccgggcgagggggccg
cgctggtgtgggacgcgagctacgccaagaagccggcctacgccgccgtgatggaggccttcggcgcgagcccgacgccgacgcccaccacgccgacccc
gacgcccacgacgccgacgccgaccccgacgtccggtccggccgggtgccaggtgctgtggggcgtcaaccagtggaacaccggcttcaccgcgaacgtc
accgtgaagaacacgtcctccgctccggtcgacggctggacgctcacgttcagcttcccgtccggccagcaggtcacccaggcgtggagctcgacggtca
cgcagtccggctcggccgtgacggtccgcaacgccccgtggaacggctcgatcccggcgggcggcaccgcgcagttcggcttcaacggctcgcacacggg
caccaacgccgcgccgacggcgttctcgctcaacggcacgccctgcacggtcggctaa

B-glucosidase BBa_K392008 (or whatever we have)

accaccacgcgcccctcgggccgccagttctccgacgacttcctgtggggctcggccacggcctcctaccagatcgag
ggcgcgcacgacgagggcggtcgcggcccgtcgatctgggacacgttctcccgcaccccgggcaaggtcctcaacggcgacaccggcgacgtcgcggtcg
accactaccaccgcgtccccgaggacgtcgagatcatgaagagcctcggcctccaggcctaccggttctcgatcgcctggccccgcatccagccgaccgg
ctcgggcgagttcaaccaggccgggctcgacttctactccgacctcgtcgaccggctcatcgccgccggcatcaagcccgtcgcgacgctgtaccactgg
gacctcccgcagcccctggaggacgagggcggctgggcgaaccgcgcgacggcgtaccggttcgtcgagtacgcgcgcaagctcgccgaggtcctcggca
agcgcgtcgacctgtggaccacgctcaacgagccgtggtgctccgcgttcctcggctacgcctcaggcgtgcacgccccggcgtcaccgacccggtcaag
gccctgcgcgccgtccaccacctcaacctcgcgcacggcctcgcgggccgcgcgatccgcgaggtcctcggcgagaacgcgcccgtctcgatcacgtgaa
cctgcacgtgacgcgcgccgccgacgactccgccgactccgtcgaggccaagcgccgcatcgacaccatcgcgaacgaggtcttcctcggcccgctgctc
gacggcgagtaccccaaggaggtcttcgcggacaccgcgcaccacaccgactggtccttcgtcgagccgggcgacctcgagctcatccgcatcccgctca
ccgtgctcggcgtcaactactactcgaccggccgcgtgaagaagggcagcgcgcgggcgccgacccgggcaagccgggccccgacggccaccgcgcctcc
gagcactcctcgtgggtcggcgccgacgaggtcgagtggctcccgcagcccggcccgcacaccgcgatgggctggaacatcgagcccgacggcctcgtcg
acctgctgctcgagctgcgcgaccgttaccccagccagcccctggcgatcacggagaacggtgccgcgttctacgacacggtcagcgaggacggccgcgt
gcacgaccccgagcgcgtcggctacctgcacgaccacgtcgacgccgtcggcgaggccatcgacaagggcgccgacgtgcgcgcctacttcgtgtggtcg
ctgctcgacaacttcgagtggcgctacggctacgaccgccggttcggcatcgtgcgcgtcgactacgacacgcacgagcgcatcgtgaaggactcgggcc
tctggtaccgcgagctcgtgcgcacccgcaccatcgctccggccgaggacgccgcgacctaccagccctga