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- | =Introduction=
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- | The Ribozyme Project aims to establish a novel way to make fusion proteins, therefore, it is important to ensure that the intron components will effectively remove an interrupting sequence and effectively re-ligate together, ensuring a functional protein product. Consequently, an easily detectable protein product must be interrupted with a sequence that is definite to either stop translation or alter the protein enough to render it ineffective. In our design, GFP is interrupted with a stop codon-containing sequence, which is flanked by the two intron parts. The parts submitted for this design were sequence and subsequently ligated into PSB1C3.
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- | =Making the Construct with <nowiki>RFC53</nowiki>=
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- | [[File:rfc53overview.png|none|thumb|500px|'''Figure 6''': Making the Construct]]
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- | '''Figure 6''' is a flow chart of the general work-flow involved in the construction of our experimental plasmid, as per <nowiki>RFC53</nowiki> conventions. The enzymes used are SacI, EarI and SapI, however, Bg1II could have been used if ligation were done in the opposite direction.
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- | *'''(1)''' The insert is isolated through a series of enzyme digestions. As shown, the plasmid is initially cut by SacI. One Intron (in blue) is shown here as a representation. Note that SacI is the first enzyme to cut in the meta-prefix, instead of EarI because of the TCGA complementary overhand needed for ligation in '''(3)'''. Next, EarI cuts at the meta-suffix to produce the CCA overhang also necessary for the step '''(3)''' ligation. The desired intert (containing the intron) is isolated for subsequent use.
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- | *'''(2)''' Similarly, the PSB1C3 vector is isolated through enzyme digestion. Note that "N" indicates that this is the vector portion. SapI cuts in the meta-prefix region to produce the GGT overhang (complimentary to CCA in '''(1)'''). Next, SacI further cuts in the meta-prefix to produce the other sticky end (AGCT). The vector is also isolated for the ligation step.
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- | *'''(3)''' The two components (insert and vector) are ligated together to produce the final construct.
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- | *'''(4)''' According to the experimental design, the final construct will contain self-excising ribozymes, which in the last step result in a non-disruptive ligation scar and, therefore, the expression of GFP.
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- | ==<ins>Sequences:</ins>==
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- | *'''''Promoter-RBS-GFP1-metasuffix''''' (588 nt)
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- | <table border="0.5">
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- | <tr>
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- | <td>GAATTCGCGG</td>
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- | <td>CCGCTTCTAG</td>
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- | <td>AGTTTACAGC</td>
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- | <td>TAGCTCAGTC</td>
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- | <td>CTAGGTATTA</td>
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- | <td>TGCTAGCTAC</td>
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- | </tr>
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- | <tr>
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- | <td>TAGAGAAAGA</td>
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- | <td>GGAGAAATAC</td>
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- | <td>TAGATGCGTA</td>
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- | <td>AAGGAGAAGA</td>
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- | <td>ACTTTTCACT</td>
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- | <td>GGAGTTGTCC</td>
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- | </tr>
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- | <tr>
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- | <td>CAATTCTTGT</td>
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- | <td>TGAATTAGAT</td>
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- | <td>GGTGATGTTA</td>
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- | <td>ATGGGCACAA</td>
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- | <td>ATTTTCTGTC</td>
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- | <td>AGTGGAGAGG</td>
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- | </tr>
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- | <tr>
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- | <td>GTGAAGGTGA</td>
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- | <td>TGCAACATAC</td>
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- | <td>GGAAAACTTA</td>
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- | <td>CCCTTAAATT</td>
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- | <td>TATTTGCACT</td>
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- | <td>ACTGGAAAAC</td>
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- | </tr>
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- | <tr>
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- | <td>TACCTGTTCC</td>
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- | <td>ATGGCCAACA</td>
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- | <td>CTTGTCACTA</td>
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- | <td>CTTTCGGTTA</td>
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- | <td>TGGTGTTCAA</td>
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- | <td>TGCTTTGCGA</td>
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- | </tr>
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- | <tr>
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- | <td>GATACCCAGA</td>
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- | <td>TCATATGAAA</td>
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- | <td>CAGCATGACT</td>
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- | <td>TTTTCAAGAG</td>
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- | <td>TGCCATGCCC</td>
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- | <td>GAAGGTTATG</td>
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- | </tr>
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- | <tr>
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- | <td>TACAGGAAAG</td>
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- | <td>AACTATATTT</td>
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- | <td>TTCAAAGATG</td>
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- | <td>ACGGGAACTA</td>
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- | <td>CAAGACACGT</td>
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- | <td>GCTGAAGTCA</td>
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- | </tr>
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- | <tr>
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- | <td>AGTTTGAAGG</td>
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- | <td>TGATACCCTT</td>
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- | <td>GTTAATAGAA</td>
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- | <td>TCGAGTTAAA</td>
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- | <td>AGGTATTGAT</td>
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- | <td>TTTAAAGAAG</td>
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- | </tr>
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- | <tr>
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- | <td>ATGGAAACAT</td>
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- | <td>TCTTGGACAC</td>
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- | <td>AAATTGGAAT</td>
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- | <td>ACAACTATAA</td>
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- | <td>CTCACACAAT</td>
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- | <td>GTATACATCA</td>
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- | </tr>
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- | <tr>
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- | <td>TGGCAGACAA</td>
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- | <td>ACAAGGTTGA</td>
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- | <td>AGAGATCTAC</td>
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- | <td>TAGTAGCGGC</td>
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- | <td>CGCTGCAG</td>
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- | </tr>
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- | </table>
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- | *'''''IN1''''' (172 nt)
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- | <table border="0.5">
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- | <tr>
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- | <td>GAATTCGCGG</td>
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- | <td>CCGCTTCTAG</td>
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- | <td>AGGAGCTCTT</td>
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- | <td>CAGGTAAATA</td>
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- | <td>ATTGCCTCTT</td>
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- | <td>TATACAGTAA</td>
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- | </tr>
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- | <tr>
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- | <td>TGTATATCGA</td>
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- | <td>AAAATCCTCT</td>
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- | <td>AATTCAGGGA</td>
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- | <td>ACACCTAAAC</td>
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- | <td>AAACTAAGAT</td>
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- | <td>GTAGGCAATC</td>
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- | </tr>
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- | <tr>
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- | <td>CTGAGCTAAG</td>
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- | <td>CTCTTAGTAT</td>
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- | <td>GTGAAGAGAT</td>
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- | <td>CTACTAGTAG</td>
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- | <td>CGGCCGCTGC</td>
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- | <td>AG</td>
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- | </tr>
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- | </table>
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- | *'''''IN2''''' (215 nt)
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- | <table border="0.5">
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- | <tr>
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- | <td>GAATTCGCGG</td>
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- | <td>CCGCTTCTAG</td>
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- | <td>AGGAGCTCTT</td>
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- | <td>CAGGTAATAA</td>
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- | <td>GAGAAAGTGC</td>
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- | <td>AACGACTATT</td>
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- | </tr>
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- | <tr>
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- | <td>CCGATAGGAA</td>
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- | <td>GTAGGGTCAA</td>
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- | <td>GTGACTCGAA</td>
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- | <td>ATGGGGATTA</td>
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- | <td>CCCTTCTAGG</td>
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- | <td>GTAGTGATAT</td>
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- | </tr>
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- | <tr>
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- | <td>AGTCTGATCA</td>
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- | <td>TATATGGAAA</td>
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- | <td>CATATAGAAG</td>
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- | <td>GATAGGAGTA</td>
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- | <td>ACGAACCTAT</td>
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- | <td>TCGTAACATA</td>
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- | </tr>
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- | <tr>
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- | <td>AATGTGAAGA</td>
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- | <td>GATCTACTAG</td>
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- | <td>TAGCGGCCGC</td>
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- | <td>TGCAG</td>
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- | </tr>
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- | </table>
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- | *'''''metaprefix-GFP2-TT''''' (442 nt)
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- | <table border="0.5">
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- | <tr>
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- | <td>GAATTCGCGG</td>
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- | <td>CCGCTTCTAG</td>
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- | <td>AGGAGCTCTT</td>
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- | <td>CAATGAAGAA</td>
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- | <td>TGGAATCAAA</td>
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- | <td>GTTAACTTCA</td>
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- | </tr>
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- | <tr>
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- | <td>AAATTAGACA</td>
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- | <td>CAACATTGAA</td>
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- | <td>GATGGAAGCG</td>
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- | <td>TTCAACTAGC</td>
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- | <td>AGACCATTAT</td>
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- | <td>CAACAAAATA</td>
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- | </tr>
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- | <tr>
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- | <td>CTCCAATTGG</td>
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- | <td>CGATGGCCCT</td>
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- | <td>GTCCTTTTAC</td>
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- | <td>CAGACAACCA</td>
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- | <td>TTACCTGTCC</td>
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- | <td>ACACAATCTG</td>
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- | </tr>
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- | <tr>
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- | <td>CCCTTTCGAA</td>
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- | <td>AGATCCCAAC</td>
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- | <td>GAAAAGAGAG</td>
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- | <td>ACCACATGGT</td>
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- | <td>CCTTCTTGAG</td>
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- | <td>TTTGTAACAG</td>
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- | </tr>
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- | <tr>
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- | <td>CTGCTGGGAT</td>
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- | <td>TACACATGGC</td>
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- | <td>ATGGATGAAC</td>
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- | <td>TATACAAATA</td>
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- | <td>ATAATACTAG</td>
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- | <td>AGCCAGGCAT</td>
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- | </tr>
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- | <tr>
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- | <td>CAAATAAAAC</td>
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- | <td>GAAAGGCTCA</td>
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- | <td>GTCGAAAGAC</td>
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- | <td>TGGGCCTTTC</td>
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- | <td>GTTTTATCTG</td>
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- | <td>TTGTTTGTCG</td>
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- | </tr>
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- | <tr>
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- | <td>GTGAACGCTC</td>
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- | <td>TCTACTAGAG</td>
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- | <td>TCACACTGGC</td>
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- | <td>TCACCTTCGG</td>
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- | <td>GTGGGCCTTT</td>
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- | <td>CTGCGTTTAT</td>
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- | </tr>
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- | <tr>
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- | <td>ATACTAGTAG</td>
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- | <td>CGGCCGCTGC</td>
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- | <td>AG</td>
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- | </tr>
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- | </table>
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