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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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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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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.
[[File:T--Marburg--Connector_Construct.png|500px|thumb|left|'''Figure 1''': <b> Design of our insulating connectors. </b> <br> 300 bp neutral DNA are flanked by two strong terminators. A BsmBI site for level 2 cloning is incorporated at the 5'end.]]
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===Source===
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The selected sequences shown in Figure 1 were flanked by synthetic transcriptional terminators that were developed in the lab of Christopher A. Voigt <a href="https://www.nature.com/articles/nmeth.2515"><abbr title ="Ying-Ja Chen, Peng Liu, Alec A K Nielsen, Jennifer A N Brophy, Kevin Clancy, Todd Peterson, Christopher A Voigt, Characterization of 582 natural and synthetic terminators and quantification of their design constraint (2013) 659–664" >( Chen <i>et al.</i>2013)</abbr></a>.
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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
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Our priority was to prevent transcription into the respective transcription unit from the upstream sequence, so
each spacer sequence was equipped with a strong terminator at the 3’ and 5’ end of the 5’ connector in forward and reverse orientation, respectively.
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===Source===
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<b> Forward Oligo:</b>
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CTCGCGCTTACTGGAGACGAGCT
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More information coming soon!
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<b> Reverse Oligo:</b>
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CTCAAGCTCGTCTCCAGTAAGCG
===References===
===References===
Latest revision as of 23:54, 16 October 2018
Phytobrick version of 3'Con1_L1 Connector
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
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.
