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<p>The whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3</p>
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<div class="highlight decoration_background">
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<span class="highlightYellow">GAATTC</span>
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TGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA
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<span class="highlightGreen"> ATGGCGGAAGGTGAAGAGGCCAAGAAGAAGAATTTACAGACAAGTTACTTCGACGTCGTTGGAATCTGCTGTACATCGGAGGTTTCTATCG TCGGTGACGTTCTCCGTCCACTTGACGGCGTCAAA </span>
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<span class="highlightDarkBlue">GA</span>
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<span class="highlightRed">G</span>
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<span class="highlightDarkBlue">TTC</span>
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<span class="highlightGreen"> TCCGTTATCGTCCCTTCTAGAACCGTCATCGTTGTCCATGACACTTTCTTGATTTCTCCGCTTCAAATCGTCAAGGCTCTGAATCAAGCAAGACTAGAAGCAAGTGTGAGACCATACGGAGAAACAAGCTTGAAGAGTCAATGGCCAAGTCCTTTTGCAATACTTTCTGGGGTATTTCTTGCTCTCTCCTTCTTCAAATACTTTTATAGTCTGCTTGAATGGCTCGCTGTTGTTGCCGTGGTGGCCGGGATTTTCCCCATCCTTGCTAAAGCTGTTGCTTCGGTCACAAGGTTCAGACTTGATATCAACGCTCTCACTTTTATTGCTGTGATAGCAACACTATGTATGCAGAATTTCACAGAAGCTGCCACAATTGTGTTTCTATTCTCAGTTGCAGATTGGCTAGAGTCTAGTGCTGCTCATAAGGCAAGCACAGTAATGTCATCACTGATGAGCTTAGCGCCACGAAAGGCAGTGATAGCGGAAACTGGACACGAAGTCGATGTAGATGAGGTTAGGATCAACACAATTGTTTCAGTGAAAGCTGGAGAAAGTATACCGATTGATGGAGTTGTGGTGGATGGAAGCTGTGATGTGGATGAGAAAACATTGACAGGAGAGTCATTCCCTGTCTCCAAACAGAGAGATTCAACTGTTTTGGCTGCAACCATAAATCTTAATGGTTATATAAAGGTGAAAACTACAGCTCTAGCCCGGGACTGCGTAGTCGCGAAAATGACTAAGCTTGTAGAAGAAGCTCAAAAAAGCCAAACCAAAACTCAAAGGTTTATAGATAAATGTTCTCGCTACTACACTCCAGCTGTTGTCGTGTTAGCAGCATGTTTTGCGGTGATCCCGGTATTGTTAAAGCTTCAGGACCTTAGCCATTGGTTTCACTTAGCACTTGTAGTGTTAGTAAGTGGTTGTCCATGTGGTCTTATCTTATCCACACCTATTGCTACCTTTTGTGCTCTCACTAAGGCAGCCATGTCGGGGTTTCTGATCAAAACTGGTGATTGTCTAGAGACTCTTGCAAAGATCAAGATTGTTGCTTTTGACAAAACAGGAACTATTACAAAGGCTGAGTTCATGGTCTCGGATTTTAGGTCTCTTTCTCACAATATCAATCTGCACAACTTGCTTTACTGGGTCTCGAGCATTGAGAGCAAGTCAAGTCATCCGATGGCAGCGGCGCTTATTGACTATGCAAGATCAGTTTCTGTTGAGCCTAAGCCTGATCTCGTTGAGAACTTTCAAAACTTTCCAGGAGAAGGAGTTTATGGGAGAATAGATGGTCAAGATATCTACATTGGAAACAAAAGAATTGCACAGAGAGCTGGATGCTTAACAGTTCCGGATATGGAAGCTAATATGAAGCGAGGTAAGACCATTGGTTACATATACATTGGAGCAAAACTGTCCGGAAGTTTCAACCTTATTGACAGTTGTCGATATGGGGTTGCTCAAGCTCTCAAGGAGCTCAAGTCTTTAGGAATCAAAACTGCAATGCTCACAGGAGATAACCGAGACGCAGCCCTGTCTACTCAAGAACAGTTAGAGAATGCTTTGGATATTGTTCACTCTGAACTCCTTCCACAAGACAAAGCAAGAATCATCGAT</span>
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<span class="highlightDarkBlue">GA</span>
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<span class="highlightRed">G</span>
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<span class="highlightDarkBlue">TTC</span>
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<span class="highlightGreen"> AAGATCCAAGGGCCTACAATGATGGTAGGAGACGGGCTTAACGATGCACCGGCTTTAGCGAAAGCAGACATTGGCCTTTCAATGGGGATCTCAGGGTCAGCACTTGCAACAGAGACAGGAGACATCATTCTTATGTCAAACGATATAAGGAAGATCCCGAAAGGGATGAGACTAGCGAAGAGAAGTCATAAGAAAGTGATTGAGAATGTTGTTTTGTCTGTGAGCATAAAAGGAGCAATCATGGTTCTTGCTTTTGTAGGTTACCCTCTGGTTTGGGCAGCTGTACTTGCAGATGCAGGAACTTGTTTGCTTGTGATACTCAATAGTATGATGCTTCTACGCGATGAGCGTGAAGCCGTGTCTACATGTTACCGTGCTTCTTCTTCGCCGGTGAAACTTGAGGAGGATGAAGCAGAGGATCTAGAAGTTGGCTTGTTGCAGAAGAGTGAGGAGACAAATAAAAAGAGTTGTTGCTCTGGTTCTTGTAGTGGCCCTAAGGACAATCAACAAAAGTGA</span>
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<span class="highlightLightBlue">CTGCAG</span>
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</p>
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Revision as of 13:16, 10 October 2018

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Design

The HMA3 Sequence is with 2619 bp quite big which make it a little bit difficult for us to actually isolate this sequence out of the A. halleri. That is the reason why we decided to get it synthesized by IDT.

First option was to use the whole sequence (2631 bp). A CaMV 35S (universal promoter, Cauliflower Mosaic Virus) is used as a promoter with a length of 345 bp.

For our Biobrick we decided to use the Vector called pSB1C3 with a chloramphenicol resistance. By including the sequence of the restriction sites of EcoRI at the beginning and PstI at the end of our sequence, which are the same restriction sites for the plasmide, we can insert our sequence into our targeted vector. In our specific case EcoRI would cut about two times into our Gene HMA3 for example. To make sure the restriction enzyme will not cut several times into our sequence we had to change some codons in the original sequence of our Gene HMA3 (you can see the changes of the codons below in red).

sequence length [bp] color
CaMV 35S promoter 245 black
AhHMA3 cDNA 2.274 green
restriction site of EcoRI (G/AATTC) 6 yellow
restriction site of PstI (CTGCA/G) 6 light blue
whole sequence 2.613

The whole promoter and gene sequence including the restriction sites to ligate into the vector pSB1C3

GAATTC TGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGA ATGGCGGAAGGTGAAGAGGCCAAGAAGAAGAATTTACAGACAAGTTACTTCGACGTCGTTGGAATCTGCTGTACATCGGAGGTTTCTATCG TCGGTGACGTTCTCCGTCCACTTGACGGCGTCAAA GA G TTC TCCGTTATCGTCCCTTCTAGAACCGTCATCGTTGTCCATGACACTTTCTTGATTTCTCCGCTTCAAATCGTCAAGGCTCTGAATCAAGCAAGACTAGAAGCAAGTGTGAGACCATACGGAGAAACAAGCTTGAAGAGTCAATGGCCAAGTCCTTTTGCAATACTTTCTGGGGTATTTCTTGCTCTCTCCTTCTTCAAATACTTTTATAGTCTGCTTGAATGGCTCGCTGTTGTTGCCGTGGTGGCCGGGATTTTCCCCATCCTTGCTAAAGCTGTTGCTTCGGTCACAAGGTTCAGACTTGATATCAACGCTCTCACTTTTATTGCTGTGATAGCAACACTATGTATGCAGAATTTCACAGAAGCTGCCACAATTGTGTTTCTATTCTCAGTTGCAGATTGGCTAGAGTCTAGTGCTGCTCATAAGGCAAGCACAGTAATGTCATCACTGATGAGCTTAGCGCCACGAAAGGCAGTGATAGCGGAAACTGGACACGAAGTCGATGTAGATGAGGTTAGGATCAACACAATTGTTTCAGTGAAAGCTGGAGAAAGTATACCGATTGATGGAGTTGTGGTGGATGGAAGCTGTGATGTGGATGAGAAAACATTGACAGGAGAGTCATTCCCTGTCTCCAAACAGAGAGATTCAACTGTTTTGGCTGCAACCATAAATCTTAATGGTTATATAAAGGTGAAAACTACAGCTCTAGCCCGGGACTGCGTAGTCGCGAAAATGACTAAGCTTGTAGAAGAAGCTCAAAAAAGCCAAACCAAAACTCAAAGGTTTATAGATAAATGTTCTCGCTACTACACTCCAGCTGTTGTCGTGTTAGCAGCATGTTTTGCGGTGATCCCGGTATTGTTAAAGCTTCAGGACCTTAGCCATTGGTTTCACTTAGCACTTGTAGTGTTAGTAAGTGGTTGTCCATGTGGTCTTATCTTATCCACACCTATTGCTACCTTTTGTGCTCTCACTAAGGCAGCCATGTCGGGGTTTCTGATCAAAACTGGTGATTGTCTAGAGACTCTTGCAAAGATCAAGATTGTTGCTTTTGACAAAACAGGAACTATTACAAAGGCTGAGTTCATGGTCTCGGATTTTAGGTCTCTTTCTCACAATATCAATCTGCACAACTTGCTTTACTGGGTCTCGAGCATTGAGAGCAAGTCAAGTCATCCGATGGCAGCGGCGCTTATTGACTATGCAAGATCAGTTTCTGTTGAGCCTAAGCCTGATCTCGTTGAGAACTTTCAAAACTTTCCAGGAGAAGGAGTTTATGGGAGAATAGATGGTCAAGATATCTACATTGGAAACAAAAGAATTGCACAGAGAGCTGGATGCTTAACAGTTCCGGATATGGAAGCTAATATGAAGCGAGGTAAGACCATTGGTTACATATACATTGGAGCAAAACTGTCCGGAAGTTTCAACCTTATTGACAGTTGTCGATATGGGGTTGCTCAAGCTCTCAAGGAGCTCAAGTCTTTAGGAATCAAAACTGCAATGCTCACAGGAGATAACCGAGACGCAGCCCTGTCTACTCAAGAACAGTTAGAGAATGCTTTGGATATTGTTCACTCTGAACTCCTTCCACAAGACAAAGCAAGAATCATCGAT GA G TTC AAGATCCAAGGGCCTACAATGATGGTAGGAGACGGGCTTAACGATGCACCGGCTTTAGCGAAAGCAGACATTGGCCTTTCAATGGGGATCTCAGGGTCAGCACTTGCAACAGAGACAGGAGACATCATTCTTATGTCAAACGATATAAGGAAGATCCCGAAAGGGATGAGACTAGCGAAGAGAAGTCATAAGAAAGTGATTGAGAATGTTGTTTTGTCTGTGAGCATAAAAGGAGCAATCATGGTTCTTGCTTTTGTAGGTTACCCTCTGGTTTGGGCAGCTGTACTTGCAGATGCAGGAACTTGTTTGCTTGTGATACTCAATAGTATGATGCTTCTACGCGATGAGCGTGAAGCCGTGTCTACATGTTACCGTGCTTCTTCTTCGCCGGTGAAACTTGAGGAGGATGAAGCAGAGGATCTAGAAGTTGGCTTGTTGCAGAAGAGTGAGGAGACAAATAAAAAGAGTTGTTGCTCTGGTTCTTGTAGTGGCCCTAAGGACAATCAACAAAAGTGA CTGCAG

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