Team:Jilin China/NOTEBOOK

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<h3>Welcome!  <br>Team Jilin China</h3>
 
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    <center><h3>Acknowledgement
 
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      <p>We would like to express our deepest thanks for individuals and institutes generously giving help to us.</p>
 
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      <p>Laboratory supports:<br/>
 
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      </p>
 
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      <p>Greatest thanks to National Engineering Laboratory AIDS Vaccine (NELAV),
 
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        Translational Cancer Medicine Laboratory, Bio pharmaceutical laboratory.Our experiments is totally supported by them. </p>
 
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      <p>Material support:<br/>
 
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      </p>
 
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      <p>Great thanks to Prof. Wu Yongge, Assoc Prof. Gao Chaohui and Assoc Prof. Jiang Yuqun and all the seniors and postgraduates in their labs.Without their selfless help and endless materials, we couldn not accomplish the whole program.</p>
 
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      <p>Wiki support:<br/>
 
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      </p>
 
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      <p>Great thanks to two postgraduates Zhangjing, Wang Guichao,Zhu Jinchao, they generously assisted us to accomplish Wiki page.</p>
 
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      <p>Cooperation support:<br/>
 
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      </p>
 
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      <p>We would like to appreciate the iGEM team from Nanjing University[1], they generously provided Plasmid MlrD.</p>
 
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      <p>Project suggestions support:<br/>
 
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      </p>
 
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      <p>Our project is developed by each piece of suggestions and advices given by following people:Wu Hui,  junior Wan Zhuo, Lu Xiaotong, Liu Yuting, Yin Mengya,  sophomore Zhou Jun, Zhang Zhaoyu.</p>
 
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      <p>sponsor supports:<br/>
 
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      </p>
 
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      <p>Our project is supported by School of Life Sciences,Jilin University[2],Teaching Affairs Office of Jilin University[3] and Teaching Center for Undergraduate Honors Program in Science , Jilin University.</p>
 
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      <p>Special:<br/>
 
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      </p>
 
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      <p>Thanks to Affiliated middle school to Jilin University, they arranged for students to have a lecture of us, which got them a clear idea of iGEM.</p>
 
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<br/><br/>
 
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    <div class="sim-connect"><a href="http://www.nju.edu.cn" >[1]http://www.nju.edu.cn/</a><br/> <a href="http://www.life.jlu.edu.cn" >[2]http://www.life.jlu.edu.cn/</a> <br/><a href="http://www.nju.edu.cn" >[3]http://www.nju.edu.cn/</a></br> </div>
 
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<h1 align="center">Mlr启动子合成与验证 </h1>
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<h1 align="center">Synthesis and Characterisation of the Mlr Promoter</h1>
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<h2>Research background</h2>
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<p>A  novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin  microcystin LR was identified in a newly isolated <em>Sphingomonas sp.</em> (Bourne et al. 1996 Appl. Environ. Microbiol. 62:  4086–4094). And the gene cluster involved in bacterial degradation of the  cyanobacterial<br />
 +
  toxin  microcystin LR were be reported by David G. Bourne in 2001. The cloning and  molecular characterisation of four genes from this <em>Sphingomonas sp. </em>that exist on a 5.8-kb genomic fragment and encode  the three hydrolytic enzymes involved in this pathway together with a putative  oligopeptide transporter. Situated immediately downstream of <em>mlrA </em>with  the same direction of transcription is a gene <em>mlrD</em>, whose conceptual  translation (MlrD, 442residues) shows significant sequence identity and similar  potential transmembrane spanning regions to the PTR2family of oligopeptide  transporters. A gene <em>mlrB </em>is situated downstream of the <em>mlrA </em>and <em>mlrD </em>genes, but transcribed in the opposite direction. The gene encodes the  enzyme MlrB (402residues) which cleaves linear microcystin LR to a tetrapeptide  degradation product. This enzyme belongs to the “penicillin-binding  enzyme”  family of active site serine hydrolases. The final gene in the cluster <em>mlrC</em>,  is located upstream of the <em>mlrA </em>gene and is transcribed in the opposite  direction. It codes for MlrC (507 residues) which mediates further peptidolytic  degradation of the tetrapeptide. This protein shows significant sequence  identity to a hypothetical protein from <em>Streptomyces coelicolor</em>. It is  suspected to be a metallopeptidase based on inhibition by metal chelators. It  is postulated on the basis of comparison with other microorganisms that the  genes in this cluster may all be involved in cell wall peptidoglycan cycling  and subsequently act fortuitously in hydrolysis of microcystin LR.<br />
 +
  In  this study, we  synthesized a RFP-promoter-GFP  sequence to detect the relationship between MLR promoter and microcystin LR.  This synthesized sequence using the promoter sequence between mlrA and mlrC, adding  the red fluorescent protein sequence in the upstream to instead mlrC gene, and adding  green fluorescent protein sequence in the downstream to instead mlrA gene. <img src="file:///C|/Users/Administrator/AppData/Roaming/Macromedia/Dreamweaver 8/OfficeImageTemp/clip_image001.gif" alt="cvv" width="579" height="119" /> </p>
 +
<p>&nbsp;</p>
 +
<p align="center">Fig 1 Restriction  map of the 5.8 kb of sequence data and localisation of genes involved in  microcystin LR degradation.</p>
 +
<h2>methods</h2>
 +
<h3>Synthetic  sequence</h3>
 +
<p>    BamHI<br />
 +
  GCCTGCAGctataaaaataaatgatgtctaccttctgttctttcatattgttcaacaattgtataatcttcattatgtgatgtaatatctaacttagcatctacataataatatcctggtaattgaactggttttttagccatataaattgatttaaattcaactaaataatgtcctccgtcctttaattttagagctttatgtgtttctccctttaaaaccccgtctcttggatataatctttctgtactagcttcccatcccattgtttttttttgcataactggtccgtctgatggaaaattaaccccaataaatttaactttataaataaaacaaccatcttgtaatgatgaatcttgtgtaactgtagcaactccaccatcttcaaaattcataactctttcccatttaaatccttctggaaaagataattttttataatctggaatatcagctggatgtttaacgtaaacctttgatccatattgaaattgtggtgataaaatatcccacgcaaatggtaatggtcctccttttgtaacctttaatttaactgtattatgtccttcataCggtctaccttctccttctccctcaatttcaaattcatgtccattaactgtaccttccattctaactttaaatctcataaattctgtaataacattttctgatgaagccatAacacgagtcttcggtttgctgttgtttgcaccagctgctgcatcttcaccccataaatcaaccgaacggacagtcaattcttgttgaccatcctgggcctatctgaaatgttctgctgacagaacgggagaattgaaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcaCatgaaacagcatgactttttcaagagtgccatgcctgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaGAATTCCG <br />
 +
  EcoRI</p>
 +
<h3>Sequence analysis </h3>
 +
<p align="left">&nbsp;_Base Count : 1551 bp (489 A, 496 T, 314  C, 252 G)<br />
 +
  &nbsp;_Composition : 36% GC, 64% AT<br />
 +
  <br />
 +
  <strong>Absent Sites</strong><br />
 +
  AarI AatII Acc65I AciI AclI AfeI AflII AgeI  AhdI AleI ApaI ApaLI AscI AseI AsiSI AvaI BamHI BanI BanII BbvCI BceAI BciVI  BclI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BsaBI BsaHI BsaWI BseMII  BseRI BsgI BsiEI BsiHKAI BsiWI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI  BsrDI BsrFI BssHII BstEII BstUI Bsu36I BtrI BtsI Cac8I ClaI DraIII EagI EarI  EciI Eco53KI EcoNI EcoO109I EcoRV FauI FseI FspAI FspI HaeII HgaI HhaI HinP1I  HindIII HpaII Hpy99I KasI KpnI MluI MmeI NaeI NarI NciI NdeI NgoMIV NheI NlaIV  NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PfoI PmeI PpuMI PshAI  PspOMI PspXI PstI PvuI RsrII SacI SacII SalI SanDI SbfI ScaI SexAI SfiI SfoI  SgrAI SmaI SnaBI SpeI SphI SrfI SspI StuI TauI TspMI Tth111I XbaI XcmI XhoI  XmaI XmnI ZraI<br />
 +
  <strong>Unique Sites</strong> <br />
 +
  <strong>Unique Sites</strong><br />
 +
  BamHI(3)<br />
 +
  DdeI (92)<br />
 +
  Esp3I (223)<br />
 +
  SfcI (367)<br />
 +
  BstXI (512) MslI (513)<br />
 +
  BssSI (688) PleI (691)  BbsI (693)<br />
 +
  MwoI (714)<br />
 +
  BpmI (851)<br />
 +
  BsmFI (859)<br />
 +
  Bsp1286I (895)<br />
 +
  BtgI (991) MscI (994)  Tsp45I (1006)<br />
 +
  BsrGI (1101)<br />
 +
  AflIII (1147) BsaAI  (1148)<br />
 +
  BstZ17I (1273)<br />
 +
  HpaI (1312)<br />
 +
  BtgZI (1391)<br />
 +
  AsuII (1446) BstYI (1452)  BsaI (1469) DrdI (1471)<br />
 +
  SmlI (1486) BseYI (1504)<br />
 +
  EcoRI (1543)</p>
 +
<h3>Building  Block Design</h3>
 +
<p align="left">The maximum allowable assembly oligo length is  equal to the target assembly oligo length (60). This may cause some weird  behavior, especially in terms of overlap melting temperature.<strong> </strong></p>
 +
<p align="left">2 building blocks were generated.<br />
 +
  Building Block  mlrAC.1&nbsp;&nbsp;&nbsp;789bp&nbsp;&nbsp;&nbsp;1..789&nbsp;<br />
 +
  Left&nbsp; - 5' GCCCTAGGCTATAAAAATAAATGATG  3'<br />
 +
  Rght&nbsp; - 5'&nbsp;<strong>A</strong><u>TAGGCCCAGGA</u><strong>T</strong>GGTCAA  3'<br />
 +
  RghtU - 5'&nbsp;<strong>A</strong><u>TAGGCCCAGGA</u><strong>U</strong>GGTCAA 3'<br />
 +
  Sequence:<br />
 +
  Assembly Oligos: average overlap Tm is 48°;average oligo length is 56bp.</p>
 +
GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAATTGTATAATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAATTGAACTGGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCCTTTAATTTTAGAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTACTAGCTTCCCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCCCAATAAATTTAACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCAACTCCACCATCTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAATCTGGAATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAAATATCCCACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTCATACGGTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTTCCATTCTAACTTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGAGTCTTCGGTTTGCTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGACAGTCAATTCTTGTTGACCATCCTGGGCCTAT<br />
 +
GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT&nbsp;&nbsp;&nbsp;&nbsp;AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTG&nbsp;&nbsp;&nbsp;&nbsp;GTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATT&nbsp;&nbsp;&nbsp;&nbsp;TTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGG&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACG&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACAT&nbsp;&nbsp;&nbsp;&nbsp;ACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGG&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGC&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;TAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGG&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGG&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;AGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGT&nbsp;&nbsp;&nbsp;&nbsp;GACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACG&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;GGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA<br />
 +
CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGAT
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Revision as of 13:55, 17 October 2014

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Mlr启动子合成与验证

Synthesis and Characterisation of the Mlr Promoter

Research background

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086–4094). And the gene cluster involved in bacterial degradation of the cyanobacterial
toxin microcystin LR were be reported by David G. Bourne in 2001. The cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the “penicillin-binding enzyme” family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.
In this study, we  synthesized a RFP-promoter-GFP sequence to detect the relationship between MLR promoter and microcystin LR. This synthesized sequence using the promoter sequence between mlrA and mlrC, adding the red fluorescent protein sequence in the upstream to instead mlrC gene, and adding green fluorescent protein sequence in the downstream to instead mlrA gene. cvv

 

Fig 1 Restriction map of the 5.8 kb of sequence data and localisation of genes involved in microcystin LR degradation.

methods

Synthetic sequence

    BamHI
GCCTGCAGctataaaaataaatgatgtctaccttctgttctttcatattgttcaacaattgtataatcttcattatgtgatgtaatatctaacttagcatctacataataatatcctggtaattgaactggttttttagccatataaattgatttaaattcaactaaataatgtcctccgtcctttaattttagagctttatgtgtttctccctttaaaaccccgtctcttggatataatctttctgtactagcttcccatcccattgtttttttttgcataactggtccgtctgatggaaaattaaccccaataaatttaactttataaataaaacaaccatcttgtaatgatgaatcttgtgtaactgtagcaactccaccatcttcaaaattcataactctttcccatttaaatccttctggaaaagataattttttataatctggaatatcagctggatgtttaacgtaaacctttgatccatattgaaattgtggtgataaaatatcccacgcaaatggtaatggtcctccttttgtaacctttaatttaactgtattatgtccttcataCggtctaccttctccttctccctcaatttcaaattcatgtccattaactgtaccttccattctaactttaaatctcataaattctgtaataacattttctgatgaagccatAacacgagtcttcggtttgctgttgtttgcaccagctgctgcatcttcaccccataaatcaaccgaacggacagtcaattcttgttgaccatcctgggcctatctgaaatgttctgctgacagaacgggagaattgaaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcaCatgaaacagcatgactttttcaagagtgccatgcctgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaGAATTCCG
EcoRI

Sequence analysis

 _Base Count : 1551 bp (489 A, 496 T, 314 C, 252 G)
 _Composition : 36% GC, 64% AT

Absent Sites
AarI AatII Acc65I AciI AclI AfeI AflII AgeI AhdI AleI ApaI ApaLI AscI AseI AsiSI AvaI BamHI BanI BanII BbvCI BceAI BciVI BclI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BsaBI BsaHI BsaWI BseMII BseRI BsgI BsiEI BsiHKAI BsiWI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI BsrDI BsrFI BssHII BstEII BstUI Bsu36I BtrI BtsI Cac8I ClaI DraIII EagI EarI EciI Eco53KI EcoNI EcoO109I EcoRV FauI FseI FspAI FspI HaeII HgaI HhaI HinP1I HindIII HpaII Hpy99I KasI KpnI MluI MmeI NaeI NarI NciI NdeI NgoMIV NheI NlaIV NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PfoI PmeI PpuMI PshAI PspOMI PspXI PstI PvuI RsrII SacI SacII SalI SanDI SbfI ScaI SexAI SfiI SfoI SgrAI SmaI SnaBI SpeI SphI SrfI SspI StuI TauI TspMI Tth111I XbaI XcmI XhoI XmaI XmnI ZraI
Unique Sites
Unique Sites
BamHI(3)
DdeI (92)
Esp3I (223)
SfcI (367)
BstXI (512) MslI (513)
BssSI (688) PleI (691) BbsI (693)
MwoI (714)
BpmI (851)
BsmFI (859)
Bsp1286I (895)
BtgI (991) MscI (994) Tsp45I (1006)
BsrGI (1101)
AflIII (1147) BsaAI (1148)
BstZ17I (1273)
HpaI (1312)
BtgZI (1391)
AsuII (1446) BstYI (1452) BsaI (1469) DrdI (1471)
SmlI (1486) BseYI (1504)
EcoRI (1543)

Building Block Design

The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature.

2 building blocks were generated.
Building Block mlrAC.1   789bp   1..789 
Left  - 5' GCCCTAGGCTATAAAAATAAATGATG 3'
Rght  - 5' ATAGGCCCAGGATGGTCAA 3'
RghtU - 5' ATAGGCCCAGGAUGGTCAA 3'
Sequence:
Assembly Oligos: average overlap Tm is 48°;average oligo length is 56bp.

GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAATTGTATAATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAATTGAACTGGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCCTTTAATTTTAGAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTACTAGCTTCCCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCCCAATAAATTTAACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCAACTCCACCATCTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAATCTGGAATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAAATATCCCACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTCATACGGTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTTCCATTCTAACTTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGAGTCTTCGGTTTGCTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGACAGTCAATTCTTGTTGACCATCCTGGGCCTAT
GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT      ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT      GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC          GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC       CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC           ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA          CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT    AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA      CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC     GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT          TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA            CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA                                
                                 AGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTG    GTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATT    TTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGG            AGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACG          GCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACAT    ACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGG        GGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGC          TAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGG            GAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGG            AGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGT    GACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACG            GGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA
CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGAT