"

From 2011.igem.org

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 seems 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. Some primers have had bases added or deleted from the 3' end to make a sane Tm.

Need to check these with Gibthon, and also check I have enough bases in the non-complementary regions...

Contents 1 Primers 1.1 Vector - malS 1.2 Vector - Endo 1.3 Vector - Exo 1.4 Vector - β-Glu 2 Appendix: sequences 2.1 Vector with PlacLacZ-INP (~3700 bp) 2.2 malS [mature peptide only, aa 18 onwards] 2.3 Endoglucanase BBa_K118023 [mature peptide only, aa 32 onwards] 2.4 Exoglucanase BBa_K118022 [mature peptide only, aa 43 onwards] 2.5 B-glucosidase BBa_K392008 (or whatever we have)

Primers

Vector - malS

Vector

• tctgggcagggcaacagtaa gtcacactggctcaccttcg
  • Forward. Complementary Tm = 66.1
• cccggagaagtccagctggc gctggtcgtcgggctagta [avoid dimer]
  • Reverse. Complementary Tm = 65.3

malS

• gtactagcccgacgaccagc gccagctggacttctccggg
  • Forward. Complementary Tm = 72.5
• cgaaggtgagccagtgtgac ttactgttgccctgcccagac
  • Reverse. Complementary Tm = 68.4

Vector - Endo

Vector

• cgcgcaacgccaggtggtaa gtcacactggctcaccttcg
  • Forward. Complementary Tm = 66.1
• tcgacgcggcagccgggagc gctggtcgtcgggctagta [avoid dimer]
  • Reverse. Complementary Tm = 65.3

Endo

• gtactagcccgacgaccagc gctcccggctgccgcgt
  • Forward. Complementary Tm = 76.8
• cgaaggtgagccagtgtgac ttaccacctggcgttgcg [avoid dimer]
  • Reverse. Complementary Tm = 68.3

Vector - Exo

Vector

• cgccctgcacggtcggctaa gtcacactggctcaccttcg
  • Forward. Complementary Tm = 66.1
• gcctccttgagcgtggtcgc gctggtcgtcgggctagta [avoid dimer]
  • Reverse. Complementary Tm = 65.3

Exo

• gtactagcccgacgaccagc gcgaccacgctcaaggag
  • Forward. Complementary Tm = 67.4
• cgaaggtgagccagtgtgac ttagccgaccgtgcagggc
  • Reverse. Complementary Tm = 72.7

Vector - β-Glu

Vector

• ccgcgacctaccagccctga gtcacactggctcaccttcg
  • Forward. Complementary Tm = 66.1
• cccgaggggcgcgtggtggt gctggtcgtcgggctagta [avoid dimer]
  • Reverse. Complementary Tm = 65.3

β-Glu

• gtactagcccgacgaccagc accaccacgcgcccctc
  • Forward. Complementary Tm = 72.4
• cgaaggtgagccagtgtgac tcagggctggtaggtcgc
  • Reverse. Complementary Tm = 66.5

Appendix: sequences

Vector with PlacLacZ-INP (~3700 bp)

• Some of the "stuffing" is included just to get nice forward primer Tm
• Note that the end of this sequence has homology with part of Endoglucanase - problem?

gtcacactggctcaccttcgggtgggcctttctgcgtttata
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 gatttctggaattcgcggccgcttctagagttaatacgactcactatagggagaggaggt acgatctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcgg tgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgccca atacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacagg tttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcat taggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgaaattgtgagc ggataacaatttcacacaggaaacagctatgaccatgattacggattcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcaca tccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaaca gttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgcc ggaaagctggctggagtgatactagctgatatttcgcgtaaggaaatccattatgGGATC Tatgaccctggataaagcgctggtgctgcgcacttgtgccaataatatggccgaccattg tggcctgatctggcctgcttctggtacagttgaaagccgctattggcagtctacacgtcg tcacgaaaacgggctggttgggctgctgtggggtgccggaacatccgcctttctgagcgt tcatgccgatgctcgttggattgtttgtgaggttgctgttgccgacatcattagcctgga agaaccgggtatggtgaaatttcctcgtgctgaagtggtccatgttggtgatcgtattag cgcctcccatttcatttcagctcgtcaggctgatccagcaagcacatcaacaagcacctc taccagtacactgacaccaatgccaaccgctatccctactcctatgccggctgtagcctc tgttactctgcctgtggccgaacaagctcgccacgaagtctttgacgttgcctctgtgtc tgctgctgctgctcctgttaacactctgcctgttaccacgcctcaaaatctgcaaacggc gacctatggctcaacactgagtggtgataaccattctcgtctgattgccgggtatggctc taacgaaaccgccggaaaccattctgatctgatcggtggtcacgattgtacgctgatggc aggtgatcaatctcgtctgaccgctggcaaaaattccgttctgaccgctggggcacgttc taaactgatcgggagcgaaggctctactctgtccgccggagaagattctaccctgatctt tcgtctgtgggatggtaaacgttatcgtcagctggtcgctcgtacaggtgaaaatggtgt ggaagccgacattccgtattatgtgaatgaggacgatgatattgtggacaaaccggacga
agatgatgattggatcgaagtgaaaggtactagcccgacgaccagc

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)

accaccacgcgcccctcgggccgccagttctccgacgacttcctgtggggctcggccacggcctcctaccagatc
gagggcgcgcacgacgagggcggtcgcggcccgtcgatctgggacacgttctcccgcaccccgggcaaggtcctcaacggcgacaccggcgacgtcgcgg
tcgaccactaccaccgcgtccccgaggacgtcgagatcatgaagagcctcggcctccaggcctaccggttctcgatcgcctggccccgcatccagccgac
cggctcgggcgagttcaaccaggccgggctcgacttctactccgacctcgtcgaccggctcatcgccgccggcatcaagcccgtcgcgacgctgtaccac
tgggacctcccgcagcccctggaggacgagggcggctgggcgaaccgcgcgacggcgtaccggttcgccgagtacgcgcgcaagctcgccgaggtcctcg
gcaagcgcgtcgacctgtggaccacgctcaacgagccgtggtgctccgcgttcctcggctacgcctcaggcgtgcacgcccccggcgtcaccgacccggt
caaggccctgcgcgccgtccaccacctcaacctcgcgcacggcctcgcgggccgcgcgatccgcgaggtcctcggcgagaacgcgcccgtctcgatcacg
ctcaacctgcacgtgacgcgcgccgccgacgactccgccgactccgtcgaggccaagcgccgcatcgacaccatcgcgaacgaggtcttcctcggcccgc
tgctcgacggcgagtaccccaaggaggtcttcgcggacaccgcgcacctcaccgactggtccttcgtcgagccgggcgacctcgagctcatccgcatccc
gctcaccgtgctcggcgtcaactactactcgaccggccgcgtgaagaagggcagcgccgcgggcgccgacccgggcaagccgggccccgacggccaccgc
gcctccgagcactcctcgtgggtcggcgccgacgaggtcgagtggctcccgcagcccggcccgcacaccgcgatgggctggaacatcgagcccgacggcc
tcgtcgacctgctgctcgagctgcgcgaccgttaccccagccagcccctggcgatcacggagaacggtgccgcgttctacgacacggtcagcgaggacgg
ccgcgtgcacgaccccgagcgcgtcggctacctgcacgaccacgtcgacgccgtcggcgaggccatcgacaagggcgccgacgtgcgcgcctacttcgtg
tggtcgctgctcgacaacttcgagtgggcctacggctacgaccgccggttcggcatcgtgcgcgtcgactacgacacgcacgagcgcatcgtgaaggact
cgggcctctggtaccgcgagctcgtgcgcacccgcaccatcgctccggccgaggacgccgcgacctaccagccctga