Difference between revisions of "Part:BBa K2560011:Design"

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===Design Notes===
 
===Design Notes===
 
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The design of “neutral DNA” started with generating 300 bp of random DNA which do not possess recognition sites of BsaI, BsmBI and any of the enzymes used in Biobrick Assembly, using a self-made Matlab script. These sequences were analyzed with the reverse mode of the R2oDNA designer (Casini et al. 2014). The R2oDNA designer can take a DNA sequence as input and quantifies the extent of secondary structures, repeats and forbidden sequence motifs, such as promoter or RBS motifs. Because the genome sequence of V. natriegens is not included in the tool, a BLAST search was performed manually with the sequences to check for homologies with the genome of V. natriegens. The top hit was considered as a fourth score to quantify the quality of a spacer sequence. At the end of this process, four scores were assigned to every sequence, each describing a different characteristic. We decided that spacer sequences with decent scores in each category are better suited as insulators than sequences with a superior score in one and inferior scores in other categories.
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This connectors were designed to enable flexible cloning of LVL 2 plasmids. For more informations feel free to visit our <a href="http://2018.igem.org/Team:Marburg/Design">Design Page</a>.
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We developed a Matlab script to find the “best” spacer sequences by increasing quantiles and picking spacers that fall into the lowest quantiles for all four categories.
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The sequence was synthesized <i> in silico </i> by Daniel Stukenberg and ordered from <a href="https://eu.idtdna.com/pages">Integrated DNA Technologies</a>(IDT).  
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===Source===
 
===Source===
 
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The part was created by annealing single stranded oligonucleotides and subsequent integration into the part entry vector <a href="https://parts.igem.org/Part:BBa_K2560002">BBa_K2560002</a> using Golden Gate assembly. If you stuggle with <i> de novo </i> synthesis we recomended this
 
The part was created by annealing single stranded oligonucleotides and subsequent integration into the part entry vector <a href="https://parts.igem.org/Part:BBa_K2560002">BBa_K2560002</a> using Golden Gate assembly. If you stuggle with <i> de novo </i> synthesis we recomended this
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<a href="https://parts.igem.org/Help:Promoters/Construction">site</a>.
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<a href="https://parts.igem.org/Help:Promoters/Construction">site</a>.  
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<b> Reverse Oligo:</b>
 
<b> Reverse Oligo:</b>
 
CTCACTCCCTCTAGAAGCGGCCGCGAATTCTGTT
 
CTCACTCCCTCTAGAAGCGGCCGCGAATTCTGTT
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===References===
 

Latest revision as of 00:02, 17 October 2018


Phytobrick version of 5'Connector Dummy


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal NotI site found at 7
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal XbaI site found at 16
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

This connectors were designed to enable flexible cloning of LVL 2 plasmids. For more informations feel free to visit our Design Page.

Source

The part was created by annealing single stranded oligonucleotides and subsequent integration into the part entry vector BBa_K2560002 using Golden Gate assembly. If you stuggle with de novo synthesis we recomended this site.

Forward Oligo: CTCGAACAGAATTCGCGGCCGCTTCTAGAGGGAG

Reverse Oligo: CTCACTCCCTCTAGAAGCGGCCGCGAATTCTGTT