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| − | This is the Phytobrick version of the Connector 5'Connector Dummy and was build as a part of the Marburg Collection. Instructions of how to use the Marburg Collection are provided at the bottom of the page. | + | |
| | + | This part is contained in the green expansion, a range of parts from iGEM Marburg 2020 that enables user of the Marburg Collection 2.0 to design MoClo compatible vectors for cyanobacteria as well as to engineer the genome of several cyanobacterial species. |
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| | </p> | | </p> |
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| − | One novel key feature of our toolbox are the connectors. They were designed in order to function as insulators to prevent crosstalk between neighboring transcription units <a href="http://2018.igem.org/Team:Marburg/Design">(Design of the Marburg Collection)</a>. Therefore a perfectly insulating connector would prevent the readthrough from backbone sequences that most probably caused the notably high expression that was measured in the promoter experiment for the dummy promoter <a href="https://parts.igem.org/Part:BBa_K2560007#Characterization">(Promoter experiment)</a>. In addition to blocking transcriptional readthrough, a good connector must not possess any cryptic promoter activity.
| + | The green expansion in the Marburg Collection 2.0 features the world's first MoClo compatible shuttle vector for cyanobacteria (BBa_K3228069). This composite part contains the origins of replication ColE1 (for cloning) and the ori of panS (for maintenance in cyanobacteria) as well as a spectinomycin cassette. It can be used in the Marburg Collection like any other composite part of the type 7+8 (antibiotic cassette + origin of replication). |
| | <br> | | <br> |
| | <br> | | <br> |
| − | In already existing toolboxes, the combinatorics that come with choosing oris and resistances would be multiplied by the number of required positional vectors which provide the fusion sites for LVL2 assembly. A toolbox with five possible positions, four resistances and three oris would require 60 LVL1 destination plasmids. To enable inversion of transcription units, a built-in feature in our toolbox, the previous number would be multiplied by two, resulting in 120 required LVL1 destination plasmids. Unsurprisingly, this theoretical toolbox would not be convenient to be built and used.
| + | This expansion also contains all the parts needed for the genomic integration of one or multiple genes in cyanobacteria. It convinces with a striking flexibility and a very intuitive workflow for the de novo assembly of your plasmid of choice. It encompasses 5 different neutral integration sites to choose from: three conventional sites frequently used in the cyanobacterial community (NSI to NSIII) as well as our own rationally designed artificial neutral integration site options (a.N.S.o. 1 and 2). These sites show no transcriptional activity from neighboring regions and are therefore completely orthogonal. Additionally we offer 4 different antibiotic markers to use (chloramphenicol, gentamycin, spectinomycin and kanamycin). In theory with this setup up to 20 genes could be introduced into a cyanobacterial strain. |
| | + | Thanks to the flexible design this expansion can also be used for the genomic modification of any chassis after the introduction of new species specific LVL 0 integration sites to our Marburg Collection 2.0. As the workflow to build new homologies is a bit more intricate compared to the one pot on step assembly of our other parts due to the internal BsmbI cutting site, we described the workflow for that in our design section (See Design: Design of the A.N.S.O.s). |
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| | <br> | | <br> |
| − | <br>
| + | The green expansion proves a valuable addition to our Marburg Collection 2.0 and to the iGEM Registry of Parts. It services users of our chassis and other cyanobacterial strains with a useful tool for genomic modifications but it also contributes a shell that can be used to modify any other model organism as well. |
| − | The Marburg Collection presents a novel approach that achieves maximum flexibility with a minimum of required components (figure 1). | + | |
| − | </html>
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| − | <div style="width:100%;display:flex;flex-direction:row;flex-wrap: wrap; justify-content:space-evenly; align-items:center;">
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| − | [[File:T--Marburg--Default_Connectors.png|500px|thumb|left|'''Figure 1''': <b> Default setup of connectors for building a LVL2 plasmid consisting of 5 transcription units. </b> <br> The connectors match in the following pattern: 5'Con(N) connects to 3'Con(N-1) .]]
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| − | </div style>
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| − | We provide two sets of connectors. The first set, the “short connectors” solely provide the fusion sites for subsequent assembly. The fusion sites are identical to the ones that are used in building LVL1 plasmids to avoid having to design a complete new set of fusion sites. This also enables using LVL0 ori and resistance parts in LVL2 plasmids. Inversion of individual transcription units was also achieved by designing fusion sites that match in reverse order.
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| − | <div style="width:100%;display:flex;flex-direction:row;flex-wrap: wrap; justify-content:space-evenly; align-items:center;">
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| − | [[File:T--Marburg--Connector_inversed.png|600px|thumb|left|'''Figure 2''': <b>Inversion of transcription units is feasible with our connectors. </b> <br> To achieve inversion, the respective transcription unit has to be built with connectors that match the neighboring transcription units. The function by following pattern: 3'Con(N)inv connects to 3'Con(N-1) and 5'Con(N)inv connects to 5'Con(N+1)]]
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| − | </div style>
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| − | <html>
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| − | The 5’ and 3’ connectors can be chosen independently to build a LVL1 plasmid. This allows the user to combine any number of LVL1 plasmids between one and five into a LVL2 plasmid without end linkers that are required in other toolboxes <a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189892"><abbr title="Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly">(Andreou and Nakayama, 2018.)</abbr> </a> An unusual example is shown in figure 3 to demonstrate the number of possibilities. The only logic that has to be followed for designing multigene constructs is to follow the formula <b> 5'Con(N) connects to 3'Con(N-1)</b>. In the default set up TU(N) is built with 5'Con(N) and 3'Con(N) This will result in a LVL2 plasmid harboring five transcription units.
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| − | However, any other combination that follows the previously given rule will result in a functionally assembled LVL2 plasmid.
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| − | </html>
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| − | <div style="width:100%;display:flex;flex-direction:row;flex-wrap: wrap; justify-content:space-evenly; align-items:center;">
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| − | [[File:T--Marburg--Connector_examples.png|700px|thumb|left|'''Figure 3''': <b>Unusual examples are displayed to show the range of possibilities when using the Marburg Collection </b>]]
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| − | </div style>
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| − | | + | |
| − | ===Characterization===
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| − | <html>
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| − | <p align="justify">
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| − | We focused on characterizing the 5’ Connector because we expect the stronger influence on signal strengths. For characterizing our connector parts, we created 20 test plasmids with the <a href="https://parts.igem.org/Part:BBa_K2560051"><i>lux</i> operon</a> as the reporter. <br>
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| − | In our toolbox we provide five short connectors, which solely possess the fusion sites for LVL2 cloning, and five long connectors which additionally harbor self-designed insulators. Each of these ten connectors were cloned with the constitutive promoter <a href="https://parts.igem.org/Part:BBa_K2560007">K2560007 </a> (J23100), to check for effects on an active promoter, and with the Promoter Dummy to quantify the extent of transcriptional activity that reaches the Promoter Dummy. <br>
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| − | <figure>
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| − | <div class="imageContainer1x2">
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| − | <div><img src="https://static.igem.org/mediawiki/2018/f/ff/T--Marburg--Connectoren%2BLux.png">A</div>
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| − | <div><img src="https://static.igem.org/mediawiki/2018/d/d5/T--Marburg--Connectoren%2BDLux.png">B</div>
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| − | </div>
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| − | <figcaption><b>Figure 4: <br> Results of Connector measurmenet</b> <br> A) Connector constructs built with J23100 as promoter part <br> B) Connector constructs built with the Dummy Promoter as promoter part
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| − | </figure>
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| − | </p>
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| − | | + | |
| − | </html>
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| − | | + | |
| − | ===Result===
| + | |
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| − | <html>
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| − | <p align="justify">
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| − | The acquired data are shown in figure 4. The data were normalized over the test construct J23100, that was used in the promoter experiment and constructed with the connector dummies. For the five constructs with the active promoter and the long connectors we observed extremely varying signals. We measured a range from 0.2 to 2 fold change compared to the reference construct. It has been shown that the sequence directly upstream of small synthetic promoters can greatly impact the transcription efficiency <a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176013"><abbr title =" Swati B. Carr, Jacob Beal, Douglas M. Densmore , Reducing DNA context dependence in bacterial promoters (2017)" >( Carr <i>et al.</i>2017)</abbr></a>. In case of the long connectors, the sequence upstream of the promoter forms the terminator and could affect the efficiency of RNA-polymerase binding to the -35 and -10 regions. For the constructs built with small connectors, we also observed varying signals but to a lesser extent compared to the long connectors.
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| − | For all ten connectors that are provided in our toolbox, we show a tenfold range in the measured luminescence/OD600 signal. As a conclusion, we recommend to carefully consider the combination of promoter and 5’ Connector for rationally designing constructs. <br><br>
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| − | | + | |
| − | Taking a look at the constructs that were built with the Promoter Dummy, we also see a huge difference in the expression signals. For the long connectors we expected a negligibly low reporter expression which we observed for two out of five long 5’ Connectors resulting in a 14 fold signal reduction compared to the “Promoter Dummy” reference. The remarkably strong signal observed for the remaining three connectors could be due to inefficient terminators or cryptic promoters in the pretended “neutral sequence”. <br><br>
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| − | | + | |
| − | For the remaining five constructs possessing the five short 5’ connectors we observed a range from 0.3 to 5.5 fold compared to the “Promoter Dummy” reference. We are not able to give an experimental explanation for this observation but we could imagine that the LVL2 fusion sites, the only four bases that differ in these constructs, could constitute a weak promoter together with surrounding sequences.<br>
| + | |
| − | Summarizing the connector characterization, we found that sequences upstream of short synthetic promoters greatly affect reporter expression, which is in accordance with literature <a href="https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176013"><abbr title =" Swati B. Carr, Jacob Beal, Douglas M. Densmore , Reducing DNA context dependence in bacterial promoters (2017)" >( Carr <i>et al.</i>2017)</abbr></a>. Moreover, we demonstrated that two of our five self-designed connectors efficiently reduce the signal resulting from other sources than the actual promoter.
| + | |
| − | We additionally conclude that algorithms that predict the “neutrality” of sequences alone are not sufficient to create well functioning insulators.
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| − | </p>
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </article>
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| − | </html>
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| − | <!-- Add more about the biology of this part here
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| − | ===Usage and Biology===
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| − | | + | |
| − | <!-- -->
| + | |
| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K2560011 SequenceAndFeatures</partinfo>
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| − | | + | |
| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
| + | |
| − | <partinfo>BBa_K2560011 parameters</partinfo>
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| − | <!-- -->
| + | |
| − | | + | |
| − | ===Marburg Toolbox===
| + | |
| − | <html>
| + | |
| − | <p align="justify">We proudly present the Marburg Collection, a novel golden-gate-based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility. We overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete <i> de novo </i> assembly of plasmids.
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| − | <br>
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| − | <br>
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| − | 36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units. Additionally, our connectors function as insulators to avoid undesired crosstalk.
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| − | <br>
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| − | <br>
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| − | The Marburg Collection contains 123 parts in total, including:
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| − | <br>
| + | |
| − | inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools. To increase the value of the Marburg Collection, we additionally provide detailed experimental characterization for <i> V. natriegens </i> and a supportive software. We aspire availability of our toolbox for future iGEM teams to empower accelerated progression in their ambitious projects.</p>
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| − | </html>
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| − | <div style="width:100%;display:flex;flex-direction:row;flex-wrap: wrap; justify-content:space-evenly; align-items:center;">
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| − | [[File:T--Marburg--Cloning_Overview.png|800px|thumb|left|'''Figure 5''': <b> Hierarchical cloning is facilitated by subsequent Golden Gate reactions. </b> <br> Basic building blocks like promoters or terminators are stored in level 0 plasmids. Parts from each category of our collection can be chosen to built level 1 plasmids harboring a single transcription unit. Up to five transcription units can be assembled into a level 2 plasmid.]]
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| − | [[File:T--Marburg--Overview_Marburg_Toolbox.png|750px|thumb|middle|'''Figure 6''': <b> Additional bases and fusion sites ensure correct spacing and allow tags. </b> <br> Between some parts, additional base pairs were integrated to ensure correct spacing and to maintain the triplet code. We expanded our toolbox by providing N- and C- terminal tags by creating novel fusions and splitting the CDS and terminator part, respectively.]]
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| − | </div>
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| − | | + | |
| − | ===Parts of the Marburg Toolbox===
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| − | <div class="PCListIcon" style="display:flex;flex-direction:row; flex-wrap: nowrap; justify-content:space-evenly; align-items:flex-start;font-size:82%;">
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| − | [[File:T--Marburg--5'Con.png|90px|thumb|none|
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| − | <html>
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| − | <ul>
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| − | <b>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560011">K2560011 </a> (5'Connector Dummy) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560055">K2560055 </a> <br>(1-6 <br> Connector) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560065">K2560065 </a> (5'Con1) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560066">K2560066 </a> (5'Con2) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560067">K2560067 </a> (5'Con3) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560068">K2560068 </a> (5'Con4) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560069">K2560069 </a> (5'Con5) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560075">K2560075 </a> (5'Con1 <br> Short Res) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560076">K2560076 </a> (5'Con2 <br> Short) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560077">K2560077 </a> (5'Con3 <br> Short) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560078">K2560078 </a> (5'Con4 <br> Short) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560079">K2560079 </a> (5'Con5 <br> Short) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560095">K2560095 </a> (5'Con1 inv) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560096">K2560096 </a> (5'Con2 inv) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560097">K2560097 </a> (5'Con3 inv) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560098">K2560098 </a> (5'Con4 inv) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560099">K2560099 </a> (5'Con5 inv) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560105">K2560105 </a> (5'Con5 inv <br> Ori) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560107">K2560107 </a> (5'Con1 <br> Res) </li>
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| − | </b>
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| − | </ul>
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| − | </html>]]
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| − | [[File:T--Marburg--Promotor.png|90px|thumb|none|
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| − | <html>
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| − | <ul>
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| − | <b>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560007">K2560007 </a> (J23100) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560009">K2560009 </a> (J23104) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560014">K2560014 </a> (J23106) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560015">K2560015 </a> (J23115) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560017">K2560017 </a> (J23101) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560018">K2560018 </a> (J23102) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560019">K2560019 </a> (J23103) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560020">K2560020 </a> (J23105) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560021">K2560021 </a> (J23107) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560022">K2560022 </a> (J23108) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560023">K2560023 </a> (J23109) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560024">K2560024 </a> (J23110) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560025">K2560025 </a> (J23111) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560026">K2560026 </a> (J23113) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560027">K2560027 </a> (J23114) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560028">K2560028 </a> (J23116) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560029">K2560029 </a> (J23117) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560030">K2560030 </a> (J23118) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560031">K2560031 </a> (J23119) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560123">K2560123 </a> <br>(pTet) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560124">K2560124 </a> (pTrc) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560131">K2560131 </a> (Promoter Dummy) </li>
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| − | </ul>
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| − | </b>
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| − | </html>]]
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| − | [[File:T--Marburg--RBS.png|90px|thumb|none|
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| − | <html>
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| − | <ul>
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| − | <b>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560008">K2560008 </a> (B0034) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560010">K2560010 </a> (B0032) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560013">K2560013 </a> (B0031) </li>
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| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560016">K2560016 </a> (B0030) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560084">K2560084 </a> <br>(RBS Dummy) </li>
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| − | </b>
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| − | </ul>
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| − | </html>]]
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| − | | + | |
| − | [[File:T--Marburg--CDS.png|90px|thumb|none|
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| − | <html>
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| − | <ul>
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| − | <b>
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| − | | + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560033">K2560033 </a> (E1010) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560041">K2560041 </a> (E0030) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560042">K2560042 </a> (sfGFP) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560043">K2560043 </a> (sfGFP Vn) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560047">K2560047 </a> (Cas9) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560051">K2560051 </a> <br>(Lux Operon) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560054">K2560054 </a> (dCas9) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560082">K2560082 </a> <br>(CDS Dummy) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560114">K2560114 </a> (LacZ Alpha) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560128">K2560128 </a> (K660004) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560135">K2560135 </a> <br>(SXT Beta) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560268">K2560268 </a> <br>(tfox Vc) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560269">K2560269 </a> <br>(tfox Vn) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560270">K2560270 </a> <br>(SXT) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560271">K2560272 </a> <br>(flp recombinase) </li>
| + | |
| − |
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
| − | | + | |
| − | | + | |
| − | [[File:T--Marburg--Terminator.png|90px|thumb|none|
| + | |
| − | | + | |
| − | <html>
| + | |
| − | <ul>
| + | |
| − | <b>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560034">K2560034 </a> (B0010) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560035">K2560035 </a> (B0015) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560081">K2560081 </a> (Terminator Dummy) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560089">K2560089 </a> (B1002) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560091">K2560091 </a> (B1003) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560093">K2560093 </a> (B1006) </li>
| + | |
| − | | + | |
| − | | + | |
| − | </b>
| + | |
| − | </ul>
| + | |
| − | </html>]]
| + | |
| − | | + | |
| − | | + | |
| − | [[File:T--Marburg--3'Con.png|90px|thumb|none|<html>
| + | |
| − | <ul> <b>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560012">K2560012 </a> (3'Connector Dummy) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560070">K2560070 </a> (3'Con1) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560071">K2560071 </a> (3'Con2) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560072">K2560072 </a> (3'Con3) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560073">K2560073 </a> (3'Con4) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560080">K2560080 </a> (3'Con5 Ori) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560100">K2560100 </a> (3'Con1 inv <br>Short) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560101">K2560101 </a> (3'Con2 inv <br>Short) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560102">K2560102 </a> (3'Con3 inv <br>Short) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560103">K2560103 </a> (3'Con4 inv <br>Short) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560104">K2560104 </a> (3'Con5 inv <br>Short) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560106">K2560106 </a> (3'Con1 inv <br>Short Res) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560108">K2560108 </a> (3'Con1 inv) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560109">K2560109 </a> (3'Con1 inv <br>Res) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560110">K2560110 </a> (3'Con2 inv) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560111">K2560111 </a> (3'Con3 inv) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560112">K2560112 </a> (3'Con4 inv) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560113">K2560113 </a> (3'Con5 inv) </li>
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
| − | | + | |
| − | | + | |
| − | [[File:T--Marburg--ORI.png|90px|thumb|none|<html>
| + | |
| − | <ul> <b>
| + | |
| − | | + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560036">K2560036 </a> (ColE1) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560037">K2560037 </a> (pMB1) </li>
| + | |
| − | <li> <a href="https://parts.igem.org/Part:BBa_K2560046">K2560046 </a> (p15A) </li>
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
| − |
| + | |
| − | [[File:T--Marburg--Antibiotic_Resistance.png|90px|thumb|none|
| + | |
| − | <html>
| + | |
| − | <ul>
| + | |
| − | <b>
| + | |
| − | | + | |
| − | | + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560048">K2560048 </a> (Cam. Res. RFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560056">K2560056 </a> <br> (Kan. Res. (pSB3K3) RFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560057">K2560057 </a> <br> (Kan. Res. (pSB3K3) GFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560058">K2560058 </a> <br> (Tet. Res. (pSB3T5) RFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560059">K2560059 </a> <br> (Tet. Res. (pSB3T5) GFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560125">K2560125 </a> (Carb. Res. RFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560126">K2560126 </a> (Carb. Res. GFP) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560127">K2560127 </a> (Carb. Res. into BBa_K2560002) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560132">K2560132 </a> (Cam. Res. into BBa_K2560002) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560133">K2560133 </a> <br> (Kan. Res. into BBa_K2560002) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560134">K2560134 </a> <br> (Tet. Res. into BBa_K2560002) </li>
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
| − | </div>
| + | |
| − | | + | |
| − | ===Tags and Entry Vectors===
| + | |
| − | <div class="PCListIcon" style="display:flex;flex-direction:row; flex-wrap: nowrap; align-items:flex-start; font-size:82%;">
| + | |
| − | | + | |
| − | | + | |
| − | [[File:T--Marburg--N-Tag.png|90px|thumb|middle|
| + | |
| − | <html>
| + | |
| − | <ul>
| + | |
| − | <b>
| + | |
| − | | + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560060">K2560060 </a> (E1010 4x) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560063">K2560063 </a> (sfGFP 4x) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560085">K2560085 </a> <br> (His-Tag 4x) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560087">K2560087 </a> (FLAG-Tag 4x) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560129">K2560129 </a> (K660004 4x) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560257">K2560257 </a> (Streptag 4x) </li>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
| − | | + | |
| − | | + | |
| − | [[File:T--Marburg--C-Tag.png|90px|thumb|middle|
| + | |
| − | <html>
| + | |
| − | <ul>
| + | |
| − | <b>
| + | |
| − | | + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560061">K2560061 </a> (E1010 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560062">K2560062 </a> (B0015 5b) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560064">K2560064 </a> (sfGFP 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560086">K2560086 </a> <br> (His-Tag 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560088">K2560088 </a> (FLAG-Tag 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560090">K2560090 </a> (B1002 5b) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560092">K2560092 </a> (B1003 5b) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560094">K2560094 </a> (B1006 5b) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560117">K2560117 </a> (I11012 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560118">K2560118 </a> (M0050 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560119">K2560119 </a> (M0051 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560130">K2560130 </a> (K660004 5a) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560258">K2560258 </a> (Streptag 5a) </li>
| + | |
| − | | + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
| − | | + | |
| − | | + | |
| − | [[File:T--Marburg--Entry_Vector.png|90px|thumb|middle|
| + | |
| − | <html>
| + | |
| − | <ul>
| + | |
| − | <b>
| + | |
| − | | + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560001">K2560001 </a> (Entry Vector with RFP dropout) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560002">K2560002 </a> (Entry Vector with GFP dropout) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560005">K2560005 </a> (Resistance Entry Vector with RFP Dropout) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560006">K2560006 </a> (Resistance Entry Vector with GFP Dropout) </li>
| + | |
| − | <li><a href="https://parts.igem.org/Part:BBa_K2560305">K2560305 </a> (gRNA Entry Vector with GFP Dropout) </li>
| + | |
| − | | + | |
| − | | + | |
| − | </ul>
| + | |
| − | </b>
| + | |
| − | </html>]]
| + | |
