Wiki/Team:Warsaw/primers

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<h2>Primers</h2>
 
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<a name="Z_a_Z_o"><h3>Purification of His_Z_alpha and His_Z_omega</h3></a>
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<br>
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<p>Culture <i>E. coli</i> producer strain in 3 ml of liquid LB medium + kanamycin for 8 hours. Then use it to inoculate 200 ml of liquid LB medium
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<table class=month width=100%>
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<tr>
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<td width=50%><a href=javascript:history.back()><img src="https://static.igem.org/mediawiki/2008/4/45/Previous_page.png" alt="Back to previous page"></a></td>
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<td><a href=https://2008.igem.org/wiki/Team:Warsaw/igem_notebook.htm><img src="https://static.igem.org/mediawiki/2008/d/da/Up.png" alt="Notebook view"></a></td>
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</tr></table>
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<h1>Primers</h1>
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<h2>'One-armed bandit' strain preparation</h2>
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<h4>AID and T7 cloning</h4>
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<pre>
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<a name="T7lLinkB">T7lLinkB</a>   5' CGGATCCGGCGGTGGCGGTAGTGGAGGAGGTGGGTCTGGAGGCGGAGGGAACACGATTAACATCGCTAAGAAC  3'
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<a name="T7lRBSHi">T7lRBSHi</a>  5' CACAAGCTTAGGAGGTGTACTAGATGAACACGATTAACATCGCTAAG  3'
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<a name="T7pXbSal">T7pXbSal</a>  5' TCTGTCGACTCTAGATTACGCGAACGCGAAGTCC  3'
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<a name="AIDlNrH">AIDlNrH</a>    5' CGTAAGCTTAGGAGGTGTAGACCATGGACAGCCTCTTGATG  3'
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<a name="AIDpLinB">AIDpLinB</a>  5' CCACTACCGCCACCGCCGGATCCGCCACCTCCGCTGCCTCCACCACCAAGTCCCAAAGTACGAAATGC  3'
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</pre>
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<h4>Sequencing of pMPM-T5</h4>
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<pre>
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<a name="pMPMT5seqF">pMPMT5seqF</a> 5' CTACTGTTTCTCCATACCCG  3'
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<a name="pMPMT5seqR">pMPMT5seqR</a> 5' GAAAATCTTCTCTCATCCGC 3'
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<a name="AIDseqP">AIDseqP</a>     5' GCTTCGGCGCATCCTTTTG 3'
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</pre>
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<h4>Sequencing of β-galactosidase from pZC320</h4>
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<pre>
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<a name="pZCseqL">pZCseqL</a>    5' CTTGACCCGCAGTTGCAAAC 3'
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<a name="pZCseqR">pZCseqR</a>    5' CCCAACTGATCTTCAGCATC 3'
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</pre>
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<h4>Sequencing of the T7 polymerase gene</h4>
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<pre>
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<a name="T7-2seq">T7-2seq</a>  5' AGGTGCGCTGGCTGGCATC 3'
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<a name="T7-3seq">T7-3seq</a>  5' AAGGAAGGTTACTACTGGCT 3'
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<a name="T7-4seq">T7-4seq</a>  5' CAAAGAGTTCGGCTTCCGTC 3'
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<a name="T7Rseq">T7Rseq</a>    5' GCTTCAACCGCAGCTACCAC 3'
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</pre>
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<h2>'Hunter and prey' strain preparation</h2>
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+ kanamycin supplemented with 0,5 mM IPTG and grow it overnight. In the morning spin down the culture (5000 RPM, 10 min, 4 deg. C).
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<h4>Fusion of protein A and ompA and alpha or omega part of beta-lactamase</h4>
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<pre>
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<a name="AlphaL_link">alphaL_link</a>  5' CAGTGGTGGAAGCGGTGGCGGAGGATCTGGAGGAGGTGGTTCTGGAGGTGGAGGTCACCCAGAAACGCTGGTGAAAG 3'
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<a name="AlphaP_XB">alphaP_XB</a>    5' GG GGATCCGGTCTAGATTATTCGCCAGTTAATAGTTTGCG 3'
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<a name="OmegaL_link">omegaL_link</a>  5' CAGTGGTGGAAGCGGTGGCGGAGGATCTGGAGGAGGTGGTTCTGGAGGTGGAGGTCTACTTACTCTAGCTTCCCGG 3'
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<a name="OmegaP_EPB">omegaP_EPB</a>  5' AAGGATCCAACTGCAGAAGAATTCTTACCAATGCTTAATCAGTG 3'
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<a name="OmpaL_N">ompaL_N</a>      5' CATATGAAAGCTACTAAACTGGTACTGGG 3'
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<a name="OmpaP_link">ompaP_link</a>  5' GCCACCGCTTCCACCACTGCGGCCGCCAGAGCTCCCTCCTCCAGAACCTCCGCCGTTGTCCGGACGAGTGCCGATG 3'
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</pre>
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Resuspend the pellet in PBS buffer and disrupt cells by sonication. Spin down sonication mixture (13200 RPM, 10 min, 4 deg. C) and discard
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<h4>Cloning of A and/or Z protein to expression vector (pET15b/pET24a)</h4>
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<pre>
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<a name="AlphaP+link+Sac">AlphaP+link+Sac</a>  5' GG GAGCTC CC ACCACCCCCACCCCCGCTACCTC  3'
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<a name="AlphaL+Nde">AlphaL+Nde</a>        5' CGGG CATATG CACCCAGAAACGCTGGTGAAAG  3'
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</pre>
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supernatant – purified protein is present in sonication debris. Resuspend it in sterile ice cold ddH2O and store at 4 deg. C.</p>
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<h4>Mutagenesis of the A protein</h4>
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<pre>
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<a name="ADelL+KpnI">ADelL+KpnI</a>  5' AGGTACCCATGTAAGATCTCATAGAACGCG  3'
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<a name="ADelP">AdelP</a>      5' AACTTAAACGAAGAACAACGAAACGCC  3'
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<a name="AmutL+KpnI">AmutL+KpnI</a>  5' TCGTTTAAGTTAGGTACCGGTGAGATCTCAGAGGACGCGTTTTGTTGTTCTTTGTTG  3'
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<a name="AmutP">AmutP</a>      5' AGAACAAGGAAACGCCTTCGCCCAAAGTTTAGCAGATGACCCAAGCCAAAGCGC  3'
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</pre>
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<h4>Fusion of the A protein with the OmpaA protein and the alpha/omega part of beta-lactamase protein</h4>
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<pre>
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<a name="AL+link10+homo2">AL+link10+homo2</a>    5' TCTGGAGGTGGAGGTAGCGGGGGTGGGGGTGGTTCGGGTGGAGGTGGTAAAACCGCGGCTCTTGCGC  3'
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<a name="AlphaP+link10+homo2">AlphaP+link10+homo2</a> 5' CCGCTACCTCCACCTCCAGAACCACCTCCTCCGCTTCCTCCGCCACCTTCGCCAGTTAATAGTTTGCG  3'
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<a name="OmegaP+link10+homo2">OmegaP+link10+homo2</a> 5' CCGCTACCTCCACCTCCAGAACCACCTCCTCCGCTTCCTCCGCCACCCCAATGCTTAATCAGTGAGGC  3'
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<a name="AL+SacI">AL+SacI</a>            5' GG GAGCTCTAAAACCGCGGCTCTTGCGCAAC  3'
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<a name="AP+NotI">AP+NotI</a>            5' AA GCGGCCGCCGGTTGACTTCCCCGCGGAATTC  3'
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<a name="AlphaL+SacI">AlphaL+SacI</a>        5' GG GAGCTCTCACCCAGAAACGCTGGTGAAAG  3'
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<a name="OmegaL+SacI">OmegaL+SacI</a>        5' GGGAGCTCGCTACTTACTCTAGCTTCCCGG  3'
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<a name="AlphaPlinkSac">AlphaPlinkSac</a>      5' TTGAGCTCCCTCCACCTCCGCTACCTCCACCAGAACCACCTCCTCCGCTTCCGGTACCTTCGCCAGTTAATAGTTTGC  3'
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</pre>
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<a name="A_a"><h3>Purification of His_A_alpha</h3></a>
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<h2>Preparation of the BioBricks Standard Parts</h2>
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<p>Culture, induce and disrupt <i>E. coli</i> in the same way as to purify His_Z_alpha. The protein is present in supernatant (about 10% of total protein) and can be added to selection medium without further purification.</p>
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<pre>
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<a name="AIDpSNP">AIDP_SpeNotPst</a>       5' AAACTGCAGCGGCCGCTACTAGTATCAAAGTCCCAAAGTACGAAATG  3'
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<a name="AL_BNXNE">AL_BNXNE</a>            5' TAGAATTCGCGGCCGCTTCTAGAGGGCATATGGGATCCAAAACCGCGGCTCTTGCGCAAC  3'
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<a name="AlphaPSpe">AlphaPSpe</a>           5' GCACTAGTATTATTCGCCAGTTAATAGTTTGCG  3'
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<a name="APSacSpe">APSacSpe</a>             5' GGACTAGTAGAGCTCCCGTCTACTTTCGGCGCCTGAGC  3'
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<a name="LinLSXNE">LinLSXNE</a>             5' ATGAATTCGCGGCCGCTTCTAGAGGGAGCTCTGGTGGAAGCGGTGGCGGAGGATC  3'
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<a name="LinP_BS">LinP_BS</a>              5' CCACTAGTACCGGATCCCGAACCACCCCCACCCCCGCTAC  3'
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<a name="LinP_BS2">LinP_BS2</a>            5' ATACTAGTACCGGATCCAGAACCTCCTCCACCGCTACCTCCACCTCCAGAAC  3'
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<a name="LinPBSNP">LinPBSNP</a>            5' AGCCTGCAGCGGCCGCTACTAGTACCGGATCCCGAACCACCCCCACCCCCGCTACC  3'
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<a name="OmegPSpe">OmegPSpe</a>            5' GCACTAGTATTACCAATGCTTAATCAGTGAG  3'
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<a name="OmegLNde">OmegLNde</a>            5' CAGCCATATGCTACTTACTCTAGCTTCCCGG  3'
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<a name="OmpLNXNE">OmpLNXNE</a>            5' TAGAATTCGCGGCCGCTTCTAGAGGGCATATGAAAGCTACTAAACTGGTAC  3'
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<a name="OmLNXNEB">OmLNXNEB</a>            5' ATTAGATCTGAATTCGCGGCCGCTTCTAGAGGGCATATGAAAGCTACTAAACTGGTAC  3'
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<a name="PETt7L_XNE">pETt7L_XNE</a>          5' ATGAATTCGCGGCCGCTTCTAGAGATCCCGCGAAATTAATACG  3'
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<a name="PlacL_XNE">PlacL_XNE</a>            5' TAGAATTCGCGGCCGCTTCTAGAGCTGGCACGACAGGTTTCCC  3'
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<a name="AP+XhoI+SpeI">AP+0XhoI+SpeI</a>        5' AAACTAGTCTCGAGGGTTGACTTCCCCGCGGAATTC  3'
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<a name=AIDl>AIDl</a>                5' TATCTAGAACCATGGACAGCCTCTTG 3'
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<a name=AraCl>AraCl</a>                5' GCTCTAGATTATGACAACTTGACGGC 3'
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</pre>
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<br><br>
</tr></table>
</tr></table>
</html>
</html>

Latest revision as of 21:45, 29 October 2008

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Primers

'One-armed bandit' strain preparation

AID and T7 cloning

T7lLinkB   5' CGGATCCGGCGGTGGCGGTAGTGGAGGAGGTGGGTCTGGAGGCGGAGGGAACACGATTAACATCGCTAAGAAC  3'
T7lRBSHi   5' CACAAGCTTAGGAGGTGTACTAGATGAACACGATTAACATCGCTAAG  3'
T7pXbSal   5' TCTGTCGACTCTAGATTACGCGAACGCGAAGTCC  3'
AIDlNrH    5' CGTAAGCTTAGGAGGTGTAGACCATGGACAGCCTCTTGATG  3'
AIDpLinB   5' CCACTACCGCCACCGCCGGATCCGCCACCTCCGCTGCCTCCACCACCAAGTCCCAAAGTACGAAATGC  3'

Sequencing of pMPM-T5

pMPMT5seqF  5' CTACTGTTTCTCCATACCCG  3'
pMPMT5seqR  5' GAAAATCTTCTCTCATCCGC 3'
AIDseqP     5' GCTTCGGCGCATCCTTTTG 3'

Sequencing of β-galactosidase from pZC320

pZCseqL     5' CTTGACCCGCAGTTGCAAAC 3'
pZCseqR     5' CCCAACTGATCTTCAGCATC 3'

Sequencing of the T7 polymerase gene

T7-2seq   5' AGGTGCGCTGGCTGGCATC 3'
T7-3seq   5' AAGGAAGGTTACTACTGGCT 3'
T7-4seq   5' CAAAGAGTTCGGCTTCCGTC 3'
T7Rseq    5' GCTTCAACCGCAGCTACCAC 3'

'Hunter and prey' strain preparation

Fusion of protein A and ompA and alpha or omega part of beta-lactamase

alphaL_link  5' CAGTGGTGGAAGCGGTGGCGGAGGATCTGGAGGAGGTGGTTCTGGAGGTGGAGGTCACCCAGAAACGCTGGTGAAAG 3'
alphaP_XB    5' GG GGATCCGGTCTAGATTATTCGCCAGTTAATAGTTTGCG 3'
omegaL_link  5' CAGTGGTGGAAGCGGTGGCGGAGGATCTGGAGGAGGTGGTTCTGGAGGTGGAGGTCTACTTACTCTAGCTTCCCGG 3'
omegaP_EPB   5' AAGGATCCAACTGCAGAAGAATTCTTACCAATGCTTAATCAGTG 3'
ompaL_N      5' CATATGAAAGCTACTAAACTGGTACTGGG 3'
ompaP_link   5' GCCACCGCTTCCACCACTGCGGCCGCCAGAGCTCCCTCCTCCAGAACCTCCGCCGTTGTCCGGACGAGTGCCGATG 3'

Cloning of A and/or Z protein to expression vector (pET15b/pET24a)

AlphaP+link+Sac   5' GG GAGCTC CC ACCACCCCCACCCCCGCTACCTC  3'
AlphaL+Nde        5' CGGG CATATG CACCCAGAAACGCTGGTGAAAG  3'

Mutagenesis of the A protein

ADelL+KpnI  5' AGGTACCCATGTAAGATCTCATAGAACGCG  3'
AdelP       5' AACTTAAACGAAGAACAACGAAACGCC  3'
AmutL+KpnI  5' TCGTTTAAGTTAGGTACCGGTGAGATCTCAGAGGACGCGTTTTGTTGTTCTTTGTTG  3'
AmutP       5' AGAACAAGGAAACGCCTTCGCCCAAAGTTTAGCAGATGACCCAAGCCAAAGCGC  3'

Fusion of the A protein with the OmpaA protein and the alpha/omega part of beta-lactamase protein

 
AL+link10+homo2     5' TCTGGAGGTGGAGGTAGCGGGGGTGGGGGTGGTTCGGGTGGAGGTGGTAAAACCGCGGCTCTTGCGC  3'
AlphaP+link10+homo2 5' CCGCTACCTCCACCTCCAGAACCACCTCCTCCGCTTCCTCCGCCACCTTCGCCAGTTAATAGTTTGCG  3'
OmegaP+link10+homo2 5' CCGCTACCTCCACCTCCAGAACCACCTCCTCCGCTTCCTCCGCCACCCCAATGCTTAATCAGTGAGGC  3'
AL+SacI             5' GG GAGCTCTAAAACCGCGGCTCTTGCGCAAC  3'
AP+NotI             5' AA GCGGCCGCCGGTTGACTTCCCCGCGGAATTC  3'
AlphaL+SacI         5' GG GAGCTCTCACCCAGAAACGCTGGTGAAAG  3'
OmegaL+SacI         5' GGGAGCTCGCTACTTACTCTAGCTTCCCGG  3'
AlphaPlinkSac       5' TTGAGCTCCCTCCACCTCCGCTACCTCCACCAGAACCACCTCCTCCGCTTCCGGTACCTTCGCCAGTTAATAGTTTGC  3'

Preparation of the BioBricks Standard Parts

AIDP_SpeNotPst       5' AAACTGCAGCGGCCGCTACTAGTATCAAAGTCCCAAAGTACGAAATG  3'
AL_BNXNE             5' TAGAATTCGCGGCCGCTTCTAGAGGGCATATGGGATCCAAAACCGCGGCTCTTGCGCAAC  3'
AlphaPSpe            5' GCACTAGTATTATTCGCCAGTTAATAGTTTGCG  3'
APSacSpe             5' GGACTAGTAGAGCTCCCGTCTACTTTCGGCGCCTGAGC  3'
LinLSXNE             5' ATGAATTCGCGGCCGCTTCTAGAGGGAGCTCTGGTGGAAGCGGTGGCGGAGGATC  3'
LinP_BS              5' CCACTAGTACCGGATCCCGAACCACCCCCACCCCCGCTAC  3'
LinP_BS2             5' ATACTAGTACCGGATCCAGAACCTCCTCCACCGCTACCTCCACCTCCAGAAC  3'
LinPBSNP             5' AGCCTGCAGCGGCCGCTACTAGTACCGGATCCCGAACCACCCCCACCCCCGCTACC  3'
OmegPSpe             5' GCACTAGTATTACCAATGCTTAATCAGTGAG  3'
OmegLNde             5' CAGCCATATGCTACTTACTCTAGCTTCCCGG  3'
OmpLNXNE             5' TAGAATTCGCGGCCGCTTCTAGAGGGCATATGAAAGCTACTAAACTGGTAC  3'
OmLNXNEB             5' ATTAGATCTGAATTCGCGGCCGCTTCTAGAGGGCATATGAAAGCTACTAAACTGGTAC  3'
pETt7L_XNE           5' ATGAATTCGCGGCCGCTTCTAGAGATCCCGCGAAATTAATACG  3'
PlacL_XNE            5' TAGAATTCGCGGCCGCTTCTAGAGCTGGCACGACAGGTTTCCC  3'
AP+0XhoI+SpeI        5' AAACTAGTCTCGAGGGTTGACTTCCCCGCGGAATTC  3'
AIDl                 5' TATCTAGAACCATGGACAGCCTCTTG 3'
AraCl                5' GCTCTAGATTATGACAACTTGACGGC 3'


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