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User:Allancrossman/Gibson2
From 2011.igem.org
(→Primers) |
(→Vector - Endo) |
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* gtactagcccgacgaccagc gctcccggctgccgcgt <!-- Gibthon: gtactagcccgacgaccagc GCTCCCGGCTGCCGCGTCGA --> | * gtactagcccgacgaccagc gctcccggctgccgcgt <!-- Gibthon: gtactagcccgacgaccagc GCTCCCGGCTGCCGCGTCGA --> | ||
** Forward. Complementary Tm = 76.8 | ** Forward. Complementary Tm = 76.8 | ||
| - | * cgaaggtgagccagtgtgac ttaccacctggcgttgcg <!-- Gibthon: CGAAGGTGAGCCAGTGTGAC ttaccacctggcgttgcgcg --> | + | * cgaaggtgagccagtgtgac ttaccacctggcgttgcg [avoid dimer] <!-- Gibthon: CGAAGGTGAGCCAGTGTGAC ttaccacctggcgttgcgcg --> |
** Reverse. Complementary Tm = 68.3 | ** Reverse. Complementary Tm = 68.3 | ||
Latest revision as of 14:51, 2 September 2011
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 |
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
