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<h1 class="header">Split Fluorescent Proteins for <em>C. elegans</em></h1>
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<p class="header">This project is maintained by <a class="header name" href="https://github.com/marimar128">Maria Ingaramo</a> in the <a class="header name" href="http://andrewgyork.github.io/">York lab</a>, and is funded by <a class="header name" href="https://www.calicolabs.com/">Calico Life Sciences LLC</a></p>
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<section>
<h1>Supplementary Text and Figures</h1>
<h2 id="supplementary_text">Supplementary Materials and Methods</h2>
<h3>Mammalian cell culture.</h3>
<p>HEK293T cells (ATCC # CRL-3216) were cultured in high-glucose DMEM supplemented with 10% FBS, 1 mM glutamine and 100 µg/mL penicillin/streptomycin (Gibco). A split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> cDNA codon-optimized for mammalian expression was fused to the C-terminus of eGFP and cloned into a pCDH lentiviral expression vector (SFFV GFP-split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>). Lentivirus was prepared using standard protocols
[<a class="citation" href="https://doi.org/10.1038/ncomms11046" title="Kamiyama, D. et al." Versatile protein tagging in cells with split fluorescent protein. " Nature Communications 7, 11046–9 ">Kamiyama 2016</a>]
and used to infect HEK293T cells. A polyclonal population of GFP-mScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> positive cells was isolated by FACS (using GFP fluorescence) and served as parental cell line for further experiments. For CLTA-N CRISPR engineering, S. pyogenes Cas9/sgRNA ribonucleoprotein complexes were prepared as in
[<a class="citation" href="https://doi.org/10.1073/pnas.1606731113" title="Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S. & Huang, B." A scalable strategy for high-throughput GFP tagging of endogenous human proteins." Natl. Acad. Sci. U.S.A. 113, E3501–8 (2016). ">Leonetti 2016</a>]
, mixed with HDR donor templates and electroporated into of GFP-mScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> cells by nucleofection.</p>
<h3>CLTA-N split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> donor library.</h3>
<p> A cDNA pool of degenerate split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> sequences was generated by oligonucleotide synthesis (GeneScript) and homology arms for HDR-mediated insertion at CLTA N-terminus were appended by PCR (Supplementary Material – <a href="#TableS7">Table S7</a> for sequences). Library diversity was verified by Illumina MiSeq deep-sequencing.</p>
<h2>Supplementary Results</h2>
<h3>Split mScarlet screening in mammalian cells</h3>
<p>We tested the applicability of the split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> system for mammalian cell engineering but were surprisingly unsuccessful at detecting fluorescence. We designed a human codon-optimized split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> cDNA and expressed it as a C-terminal GFP fusion in HEK293T cells by lentiviral transduction. Expression of GFP verified the successful expression of the fusion protein (<a href="#Figure_S10">Figure S10A</a>). However, subsequent expression of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> fragments did not give rise to detectable red fluorescence despite numerous attempts. We reasoned that the split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> amino-acid sequence might be sub-optimal for complementation in human cells and synthesized a library of degenerate split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> sequences covering any possible single and double amino-acid mutants. Using an established assay for CRISPR-based knock-in of sequences at the CLTA N-terminus (a highly expressed gene in HEK293T cells
[<a class="citation" href="https://doi.org/10.1073/pnas.1606731113" title="Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S. & Huang, B." A scalable strategy for high-throughput GFP tagging of endogenous human proteins." Natl. Acad. Sci. U.S.A. 113, E3501–8 (2016). ">Leonetti 2016</a>]
, neither our original split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> sequence nor its mutant library enabled detectable complementation (<a href="#Figure_S10">Figure S10B, left panels</a>). By contrast, a control experiment using the GFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>/GFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> system showed a high level of knock-in and complementation in HEK293T (<a href="#Figure_S10">Figure S10B, right panels</a>). It is possible that split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> is expressed in a non-functional form in human cells, or that its binding to split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> is occluded by competing interactions (with cellular chaperones, for example). In addition, we did not attempt complementation on primary non-transformed cell lines, like WI-38 cells, whose different proteostasis network and chaperones could aid split mScarlet folding. At this point, more experiments will be required to fully test the portability of split-wrmScarlet to mammalian systems.</p>
<h3>Experiments to investigate whether split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> functions as a degron in <em>C. elegans</em></h3>
<p>After finalizing our experiments, a paper that shows that C-terminal gly-gly sequences might function as degrons in mammalian cells was brought to our attention
[<a class="citation" href="https://doi.org/10.1016/j.cell.2018.04.028" title="Koren, I., Timms, R.T., Kula, T., Xu, Q., Li, M.Z. & Elledge, S.J."The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons." Cell 173, 1622-1635 (2018).">Koren 2018</a>].
Since we were unable to obtain non-sterile positive clones of TOMM-20::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> that did not have a mutation on the last glycine, and we were also unable to obtain EAT-6 homozygotes, we were concerned that our split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> might be recognized as a degron. To investigate this, we first labeled HIS-3, EAT-6, and TOMM-20 with the 24 a.a. split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub>, which adds the sequence MDELYK to the C-terminus of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>. These worms were fertile, and at least as bright as those labeled with split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> (<a href="#Figure_S6">Figure S6</a>). However, increased fluorescence could be due to increased molecular brightness rather than increased abundance. To address this, we compared the abundance of nuclear HIS-3, HIS-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>, and HIS-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> by western blot, and were unable to detect a significant change in abundance (<a href="#Figure_S11">Figure S11</a>). We also could not detect differences in abundance in <em>S. cerevisiae</em>, using a p416-GPD plasmid expressing a mTagBFP-mScarlet fusion or the same fusion truncated so that it ends with gly-gly (<a href="#Figure_S12">Figure S12</a>). However, because HIS-3 is a nuclear protein, and expression in yeast was done from an overexpressing plasmid, we cannot exclude that a protein ending with two glycines might be recognized as a degron in other cellular compartments, or at different expression levels, nor that there is no DesCEND degron pathway in yeast and worms. For these reasons, we recommend using the 24 a.a. split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> when labeling proteins at their C-termini.</p>
<h3 id="Figure_S1">Figure S1. Split-wrmScarlet sequence comparison to mScarlet.</h3>
<figure>
<img src="./images/figure_S1/figure_S1.png"
alt="Figure S1" id="Figure_S1_image">
<figcaption><a href="#Figure_S1" target="_self"><strong>Figure S1</strong></a><strong>: Split-wrmScarlet sequence comparison to mScarlet.</strong> (A) Protein sequence alignment of the full-length mScarlet and split-wrmScarlet. The ten amino acid substitutions are highlighted in red, the sequence corresponding to split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> in light gray and the sequence of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> in dark gray. Note that gly-gly sequences at the C-termini of proteins can be degrons in mammalian cells, so when labeling on the C-terminus, we recommend using the 24 a.a. split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub>.
(B) Protein structure from split-wrmScarlet generated with Phyre2 and PyMOL. Mutations of split-wrmScarlet relative to mScarlet are highlighted with surface rendering, and the split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> strand is colored in black.
</figcaption>
</figure><p></p>
<h3 id="Figure_S2">Figure S2. Split-wrmScarlet brightness in <em>S. cerevisiae</em>.</h3>
<figure>
<img src="./images/figure_S2/figure_S2.png"
alt="Figure S2" id="Figure_S2_image">
<figcaption><a href="#Figure_S2" target="_self"><strong>Figure S2</strong></a><strong>: Split-wrmScarlet brightness in <em>S. cerevisiae</em>.</strong> (A) Composite display of red and blue channels for membrane-localized mTagBFP-mScarlet (wild-type) fusion or split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> plus membrane localized mTagBFP-split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> in yeast. Images were acquired and are displayed under identical conditions. Note that the heterogeneity inherent to expression from plasmids is large, but split-wrmScarlet is capable of brightness levels similar to the parent protein. A schematic of the plasmids transformed is presented above each image.
(B) Histograms displaying the per-pixel ratio of red to blue fluorescence for background corrected, masked images. mScarlet/mTagBFP ratios are displayed in blue, and split-wrmScarlet/mTagBFP in orange. The inset displays the average red/blue ratio.
</figcaption>
</figure><p></p>
<h3 id="Figure_S3">Figure S3. Developmental toxicity in worms expressing split-sfCherry3 in somatic nuclei.</h3>
<figure>
<img src="./images/figure_S3/figure_S3.png"
alt="Figure S3" id="Figure_S3_image">
<figcaption><a href="#Figure_S3" target="_self"><strong>Figure S3</strong></a><strong>: Developmental toxicity in worms expressing split-sfCherry3 in somatic nuclei.</strong> (A) Schematic of the plasmids encoding split-wrmScarlet and split-sfCherry3 used for comparison. Each plasmid consists of a large FP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> fused to mNeonGreen, and the corresponding small FP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> fused to mTagBFP2, preceded with three SV40 nuclear localization sequences (NLS). The T2A sequence ensures the separation of NLS::mTagBFP2::FP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> and the corresponding mNeonGreen::FP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>.
(B) Quantification of mNeonGreen-positive animals into one of three classes: dead eggs, larvae or adults.
</figcaption>
</figure><p></p>
<h3 id="Figure_S4">Figure S4. Split-wrmScarlet and split-sfCherry3 comparison.</h3>
<figure>
<img src="./images/figure_S4/figure_S4.png"
alt="Figure S4" id="Figure_S4_image">
<figcaption><a href="#Figure_S4" target="_self"><strong>Figure S4</strong></a><strong>: Split-wrmScarlet and split-sfCherry3 comparison.</strong> Individual channels and overlays corresponding to the images displayed in Figure 1B.
</figcaption>
</figure><p></p>
<h3 id="Figure_S5">Figure S5. Brood size and lifespan of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> lines.</h3>
<figure>
<img src="./images/figure_S5/figure_S5.png"
alt="Figure S5" id="Figure_S5_image">
<figcaption><a href="#Figure_S5" target="_self"><strong>Figure S5</strong></a><strong>: Brood size and lifespan of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> lines.</strong> Split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and split-sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> lines produced wild-type numbers of progeny (A) and a wild-type lifespan (B). Genotypes: N2E (wild-type), CF4582 (muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III), CF4587 (muIs253[(Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III), CF4588 (muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)], muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III), CF4610
(muIs257[Pmyo-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR] I) and WBM1126 (wbmIs61[myo-3p::3XFLAG::dpy-10 crRNA::unc-54 3'UTR] I). Supplementary table S6 show survival statistics for all lifespan experiments.
</figcaption>
</figure><p></p>
<h3 id="Figure_S6">Figure S6. Proteins tagged at their C-terminus with the 24 a.a. split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub>.</h3>
<figure>
<img src="./images/figure_S6/figure_S6.png"
alt="Figure S5" id="Figure_S6_image">
<figcaption><a href="#Figure_S6" target="_self"><strong>Figure S6</strong></a><strong>: Proteins tagged at their C-terminus with the 24 a.a. split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub></strong>
Endogenous proteins tagged with split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> in animals expressing split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> in somatic tissues. (A-C) Confocal images of worms expressing somatic split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and (A) HIS-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> (nuclei), (B) TOMM-20::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> (mitochondria), or (C) EAT-6::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> (plasma membrane). (A-B) Maximum intensity projections of 3D stacks shown. (C) Single slice shown. Scale bars, 50 µm.
</figcaption>
</figure><p></p>
<h3 id="Figure_S7">Figure S7. Tissue-specific split-wrmScarlet fluorescence in the germline is undetectable when split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> is integrated using a single-copy transgene via MosSCI.</h3>
<figure>
<img src="./images/figure_S7/figure_S7.png"
alt="Figure S7" id="Figure_S7_image">
<figcaption><a href="#Figure_S7" target="_self"><strong>Figure S7</strong></a><strong>: Tissue-specific split-wrmScarlet fluorescence in the germline is undetectable when split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> is integrated using a single-copy transgene via MosSCI.</strong>
(A) Schematic of the plasmid encoding <em>Psun-1::mNeonGreen::linker::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::tbb-2 3’UTR</em> (left), which was injected into the MosSCI strain PHX1797 carrying a single, integrated copy of <em>Psun-1::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::sun-1 3’UTR</em> (right). (B) Images of animal expressing mNeonGreen::linker::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> and split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> in the germline. Despite detecting mNeonGreen fluorescence, split-wrmScarlet was undetectable in this MosSCI strain, potentially due to compromised expression, folding or maturation of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>. Scale bar, 20 µm.
</figcaption>
</figure><p></p>
<h3 id="Figure_S8">Figure S8. Generation and validation of germline-specific split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> strains.</h3>
<figure>
<img src="./images/figure_S8/figure_S8.png"
alt="Figure S8" id="Figure_S8_image">
<figcaption><a href="#Figure_S8" target="_self"><strong>Figure S8</strong></a><strong>: Generation and validation of germline-specific split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> strains.</strong>
In order to generate germline-specific split-fluorescent strains, we first tagged the C-terminus of glh-1 with T2A::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-11</sub> (A) or T2A::sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-11</sub> (B). The T2A separates GLH-1 post-translationally to disperse the fluorophore throughout germ-cell nuclei, syncytium, sperm (*) and early embryos (Upper panels). We then deleted split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> or sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> to generate the corresponding split-FP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> strains DUP237 and DUP223 respectively. As expected, these strains were non-fluorescent (Middle panels). Tagging FIB-1 with split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>, or PGL-1 with sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> confirmed that germline-specific labeling with split-wrmScarlet and split-sGFP2 can be successfully achieved using these strains (Lower panels). (C) Split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::FIB-1 is detectable in early embryos. Scale bars, 20 µm.
</figcaption>
</figure><p></p>
<h3 id="Figure_S9">Figure S9. Somatic sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> compared to full-length eGFP at the endogenous vha-13 locus.</h3>
<figure>
<img src="./images/figure_S9/figure_S9.png"
alt="Figure S9" id="Figure_S9_image">
<figcaption><a href="#Figure_S9" target="_self"><strong>Figure S9</strong></a><strong>: Somatic sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> compared to full-length eGFP at the endogenous vha-13 locus.</strong>
(A) Representative images of animals expressing sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> in somatic tissues with endogenous VHA-13 tagged with sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> (left panel), or endogenous VHA-13 tagged with eGFP in a wild-type background (right panel). Maximum intensity projections of 3D stacks shown. Scale bars, 50 µm. (B) Emission intensities from somatic sfGFP::VHA-13 and eGFP::VHA-13. Quantification was performed in the cell body, as quantifications in the excretory canal had higher variance. Mean ± s.d. Circles are individuals (n=4 for each condition). **P < 0.005.
</figcaption>
</figure><p></p>
<h3 id="Figure_S10">Figure S10. Screen for split-wrmScarlet fluorescence in mammalian cells.</h3>
<figure>
<img src="./images/figure_S10/figure_S10.png"
alt="Figure S10" id="Figure_S10_image">
<figcaption><a href="#Figure_S10" target="_self"><strong>Figure S10</strong></a><strong>: Screen for split-wrmScarlet fluorescence in mammalian cells.</strong>
(A) FACS histograms of human codon-optimized split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> expressed as a C-terminal GFP fusion. GFP expression verifies successful expression of the fusion protein in HEK293T cells by lentiviral transduction. (B) Schematic of the CRISPR-based knock-in design for screening single and double mutants of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>. Left panel shows that neither our original split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> sequence nor its mutant library enabled detectable complementation as detected by FACS. Right panel shows that the control experiment using the sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>/sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> system displays high levels of knock-in and complementation in HEK293T cells.
</figcaption>
</figure><p></p>
<h3 id="Figure_S11">Figure S11. Split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> C-terminal amino acids did not affect H2A abundance.</h3>
<figure>
<img src="./images/figure_S11/figure_S11.png"
alt="Figure S11" id="Figure_S11_image">
<figcaption><a href="#Figure_S11" target="_self"><strong>Figure S11</strong></a><strong>: Split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> C-terminal amino acids did not affect H2A abundance.</strong>
Western-blot of histone H2A (his-3) with split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>+/- MDELYK. Western blot targeting HIS-3 in wild-type animals (lane 1), somatic split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> expressing animals (lane 2), somatic split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> strain with HIS-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> ending with two glycines (lane 3), or somatic split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> + HIS-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub>. GAPDH was used as a loading control, and the HIS-3/GAPDH ratios are displayed under each lane.
</figcaption>
</figure><p></p>
<h3 id="Figure_S12">Figure S12. mScarlet ending with GG or MDELYK yields similar protein abundance in yeast.</h3>
<figure>
<img src="./images/figure_S12/figure_S12.png"
alt="Figure S12" id="Figure_S12_image">
<figcaption><a href="#Figure_S12" target="_self"><strong>Figure S12</strong></a><strong>: mScarlet ending with GG or MDELYK yields similar protein abundance in yeast.</strong>
(A) Representative images of yeast expressing mTagBFP::mScarlet fusion truncated to end with gly-gly (GG end) or MDELYK from a p416-GPD promoter plasmid. mTagBFP fluorescence is pseudocolored in green, and mScarlet in red. (B) Histogram of mTagBFP fluorescence from 2556 yeasts expressing the truncation after GG and 1777 yeasts expressing the MDELYK end.
</figcaption>
</figure><p></p>
<h2 id="Tables">Supplementary Tables</h2>
<h3><a id="TableS1">Table S1.</a> DNA Sequences of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>, split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>, sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>, sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>, sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub></h3>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 100%;">
<tr>
<th>Gene</th>
<th>DNA sequence</th>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> with 3 introns</td>
<td style="word-wrap: break-word">
ATGGTATCGAAGGGAGAAGCAGTAATCAAGGAGTTCATGCGTTTCAAGGTCCACATGGAGGGATCCATGAACGGACACGAGTTCGAGATCGAGGGAGAGGGAGAGGGACGTCCATACGAGGGAACCCAAACCGCCAAGCTCAAGGTCACCAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGAGGACCACTCCCATTCTCCTGGGACATCCTCTCCCCACAATTCATGTACGGATCCCGTGCCTTCATCAAGCACCCAGCCGACATCCCAGACTACTACAAGCAATCCTTCCCAGAGGGATTCAAGTGGGAGCGTGTCATGAACTTCGAGGACGGAGGAGCCGTCACCGTCACCCAAGACACCTCCCTCGAGGACGGAACCCTCATCTACAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTCAAGCTCCGTGGAACCAACTTCCCACCAGACGGACCAGTCATGCAAAAGAAGACCATGGGATGGGAGGCCTCCACCGAGCGTCTCTACCCAGAGGACGGAGTCCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGAGACATCACCATGGCCCTCCGTCTCAAGGACGGAGGACGTTACCTCGCCGACACCTCCACCACCTACAAGGCCAAGAAGCCAGTCCAAATGCCAGGAGCCTACCTCGTCGACCGTAAGCTCGACATCACCTCCCACAACGAGTAC</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub></td>
<td style="word-wrap: break-word">TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub></td>
<td style="word-wrap: break-word">
TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGAATGGATGAGTTATACAAG</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> with 1 intron</td>
<td style="word-wrap: break-word">
ATGTCTAAGGGAGAAGAGTTATTTACTGGAGTTGTGCCGATCCTCGTCGAGCTCGACGGAGACGTCAACGGACACAAGTTCTCCGTCCGTGGAGAGGGAGAGGGAGACGCCACCATCGGAAAGCTCACCCTCAAGTTCATCTGCACCACCGGAAAGCTCCCAGTCCCATGGCCAACCCTCGTCACCACCCTCACCTACGGAGTCCAATGCTTCTCCCGTTACCCAGACCACATGAAGCGTCACGACTTCTTCAAGTCCGCCATGCCAGAGGGATACGTCCAAGAGCGTACCATCTCCTTCAAGGACGACGGAAAGTACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagACCCGTGCCGTCGTCAAGTTCGAGGGAGACACCCTCGTCAACCGTATCGAGCTCAAGGGAACCGACTTCAAGGAGGACGGAAACATCCTCGGACACAAGCTCGAGTACAACTTCAACTCCCACAACGTCTACATCACCGCCGACAAGCAAAAGAACGGAATCAAGGCCAACTTCACCGTCCGTCACAACGTCGAGGACGGATCCGTCCAACTCGCCGACCACTACCAACAAAACACCCCAATCGGAGACGGACCAGTCCTCCTCCCAGACAACCACTACCTCTCCACCCAAACCGTCCTCTCCAAGGACCCAAACGAGAAG</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub></td>
<td style="word-wrap: break-word">CGTGACCACATGGTCCTTCATGAGTATGTAAATGCTGCTGGGATTACA</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> with 1 intron for germline expression</td>
<td style="word-wrap: break-word">
ATGGTTTCCAAGGGAGAGGCTGTTATCAAGGAATTCATGCGCTTCAAGGTTCACATGGAAGGATCTATGAACGGACACGAATTCGAAATCGAAGGAGAAGGAGAAGGACGCCCATACGAGGGAACTCAAACTGCTAAGCTTAAGGTTACTAAAGGAGGACCACTTCCATTCTCTTGGGATATCCTTTCTCCACAGTTCATGTACGGATCTCGCGCTTTCATCAAGCACCCAGCTGATATCCCAGATTACTACAAGCAGTCTTTCCCAGAAGGATTCAAATGGGAGCGCGTTATGAACTTCGAAGATGGAGGAGCTGTTACCGTTACCCAAGATACCTCCCTTGAGGATGGAACCCTTATCTACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGTTAAGCTTCGCGGAACTAATTTCCCACCAGATGGACCAGTTATGCAGAAGAAGACTATGGGATGGGAAGCTTCTACCGAGCGCCTTTACCCAGAGGATGGAGTCCTTAAGGGAGATATCACCATGGCTCTTCGTCTTAAGGATGGAGGACGTTACCTTGCTGATACCTCTACTACTTACAAGGCTAAGAAGCCAGTTCAGATGCCAGGAGCTTACCTTGTCGATCGTAAGCTTGATATCACTTCTCATAACGAATAC</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> with 2 intron for germline expression</td>
<td style="word-wrap: break-word">
ATGAGTAAAGGAGAAGAATTGTTCACTGGAGTTGTCCCAATCCTCGTCGAGCTCGACGGAGACGTCAACGGACACAAGTTCTCCGTCCGTGGAGAGGGAGAGGGAGACGCCACCATCGGAAAGCTCACCCTCAAGTTCATCTGCACCACCGGAAAGCTCCCAGTCCCATGGCCAACCCTCGTCACCACCCTCACCTACGGAGTCCAATGCTTCGCCCGTTACCCAGACCACATGAAGCGTCACGACTTCTTCAAGTCCGCCATGCCAGAGGGATACGTCCAAGAGCGTACCATCTCCTTCAAGgtaagtttaaacatatatatactaactactgattatttaaattttcagGACGACGGAAAGTACAAGACCCGTGCCGTCGTCAAGTTCGAGGGAGACACCCTCGTCAACCGTATCGAGCTCAAGGGAACCGACTTCAAGGAGGACGGAAACATCCTCGGACACAAGCTCGAGTACAACTTCAACTCCCACAACGTCTACATCACCGCCGACAAGCAAAAGAACGGAATCAAGGCCAACTTCACCgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagACCCGTCACAACGTCGAGGACGGATCCGTCCAACTCGCCGACCACTACCAACAAAACACCCCAATCGGAGACGGACCAGTCCTCCTCCCAGACAACCACTACCTCTCCACCCAAACCGTCCTCTCCAAGGACCCAAACGAGAAG</td>
</tr>
<tr>
<td style="word-wrap: break-word">Codon-optimized sequence of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> with 3 introns, engineered to avoid piRNA recognition transgene silencing. (Undetectable fluorescence in the MosSCI strain PHX1797)</td>
<td style="word-wrap: break-word">
ATGGTATCGAAGGGAGAAGCAGTCATCAAGGAGTTCATGCGTTTCAAGGTCCACATGGAGGGATCCATGAACGGACACGAGTTCGAGATCGAGGGAGAGGGAGAGGGACGTCCATACGAGGGAACCCAAACCGCCAAGCTCAAGGTCACCAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGAGGACCACTCCCATTCTCCTGGGACATCCTCTCCCCACAATTCATGTACGGATCCCGTGCCTTCATCAAGCACCCAGCCGACATCCCAGACTACTACAAGCAATCCTTCCCAGAGGGATTCAAGTGGGAGCGTGTGATGAACTTCGAGGACGGAGGAGCCGTCACCGTCACCCAAGACACCTCCCTCGAGGACGGAACCCTCATCTACAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTCAAGCTCCGTGGAACCAACTTCCCACCAGACGGACCAGTCATGCAAAAGAAGACCATGGGATGGGAGGCCTCCACCGAGCGTCTCTACCCAGAGGACGGAGTCCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGAGACATCACCATGGCCCTCCGTCTCAAGGACGGAGGACGTTACCTCGCCGACACCTCCACCACCTACAAGGCCAAGAAGCCAGTCCAAATGCCAGGAGCCTACCTCGTCGACCGTAAGCTCGACATCACCTCCCACAACGAGTAC</td>
</tr>
</table>
<h3><a id="TableS2">Table S2.</a> <em>C. elegans</em> lines expressing single-copy of split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and/or sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub></h3>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 100%;">
<tr>
<th style="font-size:100%">Strain</th>
<th style="font-size:100%">Genotype</th>
<th style="font-size:90%">Description</th>
<th style="font-size:100%">Genomic Position</th>
<th style="font-size:100%">Genetic Position</th>
<th style="font-size:100%; width:40%;">Sequence</th>
</tr>
<tr>
<td style="word-wrap: break-word">CF4582</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td style="word-wrap: break-word">Somatic split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> (eft-3 promoter)</td>
<td style="word-wrap: break-word">II: 9.83 MB</td>
<td style="word-wrap: break-word">II:1.73</td>
<td style="word-wrap: break-word"><span style="color:#0000ff">gcacctttggtcttttattgtcaacttccattggttcttccattgtttctgttaaattaatgaatttttcataaaataaagacattatacaatataaaaatgaagaatttattgaaaataaactgccagagagaaaaagtatgcaacactcccgccgagagtgtttgaaatggtgtacggtacattttcgtgctaggagttagatgtgcaggcagcaacgagagggggagagatttttttgggccttgtgaaattaacgtgagttttctggtcatctgactaatcatgttggttttttgttggtttattttgtttttatctttgtttttatccagattaggaaatttaaattttatgaatttataatgaggtcaaacattcagtcccagcgtttttcctgttctcactgtttagtcgaatttttattttaggctttcaacaaatgttctaactgtcttatttgtgacctcactttttatatttttttaatttttaaaaatattagaagtttctaggataattttttcgacttttattctctctaccgtccgcactcttcttacttttaaattaaattgtttttttttcagttgggaaacactttgctcactccgtagcagcc</span><span style="color:#ff0000">ATGGTATCGAAGGGAGAAGCAGTAATCAAGGAGTTCATGCGTTTCAAGGTCCACATGGAGGGATCCATGAACGGACACGAGTTCGAGATCGAGGGAGAGGGAGAGGGACGTCCATACGAGGGAACCCAAACCGCCAAGCTCAAGGTCACCAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGAGGACCACTCCCATTCTCCTGGGACATCCTCTCCCCACAATTCATGTACGGATCCCGTGCCTTCATCAAGCACCCAGCCGACATCCCAGACTACTACAAGCAATCCTTCCCAGAGGGATTCAAGTGGGAGCGTGTCATGAACTTCGAGGACGGAGGAGCCGTCACCGTCACCCAAGACACCTCCCTCGAGGACGGAACCCTCATCTACAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTCAAGCTCCGTGGAACCAACTTCCCACCAGACGGACCAGTCATGCAAAAGAAGACCATGGGATGGGAGGCCTCCACCGAGCGTCTCTACCCAGAGGACGGAGTCCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGAGACATCACCATGGCCCTCCGTCTCAAGGACGGAGGACGTTACCTCGCCGACACCTCCACCACCTACAAGGCCAAGAAGCCAGTCCAAATGCCAGGAGCCTACCTCGTCGACCGTAAGCTCGACATCACCTCCCACAACGAGTACTAA</span>taagtccaattactcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaacttctcttaatttctttgttttttagcttcttttaagtcacctctaacaatgaaattgtgtagattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttacttctgataaatttttttgaaacatcatagaaaaaaccgcacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttggggtttcccctattgtttgtcaagatttcgaggacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaaggtgagtagaagttgataatttgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgttt
</td>
</tr>
<tr>
<td style="word-wrap: break-word">CF4587</td>
<td style="word-wrap: break-word">muIs253[(Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td style="word-wrap: break-word">Somatic sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> (eft-3 promoter)</td>
<td style="word-wrap: break-word">II: 8.42 MB</td>
<td style="word-wrap: break-word">II:0.77</td>
<td style="word-wrap: break-word">
<span style="color:#0000ff">
gcacctttggtcttttattgtcaacttccattggttcttccattgtttctgttaaattaatgaatttttcataaaataaagacattatacaatataaaaatgaagaatttattgaaaataaactgccagagagaaaaagtatgcaacactcccgccgagagtgtttgaaatggtgtacggtacattttcgtgctaggagttagatgtgcaggcagcaacgagagggggagagatttttttgggccttgtgaaattaacgtgagttttctggtcatctgactaatcatgttggttttttgttggtttattttgtttttatctttgtttttatccagattaggaaatttaaattttatgaatttataatgaggtcaaacattcagtcccagcgtttttcctgttctcactgtttagtcgaatttttattttaggctttcaacaaatgttctaactgtcttatttgtgacctcactttttatatttttttaatttttaaaaatattagaagtttctaggataattttttcgacttttattctctctaccgtccgcactcttcttacttttaaattaaattgtttttttttcagttgggaaacactttgctc
</span>
<span style="color:#A3A3A3">
aaaa
</span>
<span style="color:#00ff00">
ATGTCTAAGGGAGAAGAGTTATTTACTGGAGTTGTGCCGATCCTCGTCGAGCTCGACGGAGACGTCAACGGACACAAGTTCTCCGTCCGTGGAGAGGGAGAGGGAGACGCCACCATCGGAAAGCTCACCCTCAAGTTCATCTGCACCACCGGAAAGCTCCCAGTCCCATGGCCAACCCTCGTCACCACCCTCACCTACGGAGTCCAATGCTTCTCCCGTTACCCAGACCACATGAAGCGTCACGACTTCTTCAAGTCCGCCATGCCAGAGGGATACGTCCAAGAGCGTACCATCTCCTTCAAGGACGACGGAAAGTACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagACCCGTGCCGTCGTCAAGTTCGAGGGAGACACCCTCGTCAACCGTATCGAGCTCAAGGGAACCGACTTCAAGGAGGACGGAAACATCCTCGGACACAAGCTCGAGTACAACTTCAACTCCCACAACGTCTACATCACCGCCGACAAGCAAAAGAACGGAATCAAGGCCAACTTCACCGTCCGTCACAACGTCGAGGACGGATCCGTCCAACTCGCCGACCACTACCAACAAAACACCCCAATCGGAGACGGACCAGTCCTCCTCCCAGACAACCACTACCTCTCCACCCAAACCGTCCTCTCCAAGGACCCAAACGAGAAGTAA
</span>
<span style="color:#A3A3A3">
atatccaccgtgctggcgggaggtcgc
</span>
catctcgcgcccgtgcctctgacttctaagtccaattactcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaacttctcttaatttctttgttttttagcttcttttaagtcacctctaacaatgaaattgtgtagattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttacttctgataaatttttttgaaacatcatagaaaaaaccgcacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttggggtttcccctattgtttgtcaagatttcgaggacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaaggtgagtagaagttgataatttgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgttt
</td>
</tr>
<tr>
<td style="word-wrap: break-word">CF4588</td>
<td style="word-wrap: break-word">muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)], muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td style="word-wrap: break-word">Somatic sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and somatic split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> (eft-3 promoter)</td>
<td style="word-wrap: break-word">II: 8.42 MB, 9.83 MB</td>
<td style="word-wrap: break-word">II:0.77, II:1.73</td>
<td style="word-wrap: break-word">
Sequences from CF4582 and CF4587 </td>
</tr>
<tr>
<td style="word-wrap: break-word">CF4610</td>
<td style="word-wrap: break-word">muIs257[Pmyo-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR] I</td>
<td style="word-wrap: break-word">Muscle split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> (myo-3 promoter)</td>
<td style="word-wrap: break-word">I: 2.85 MB</td>
<td style="word-wrap: break-word">I:-5.32</td>
<td style="word-wrap: break-word"><span style="color:#01F2FF">
agtgattatagtctctgttttcgttaattttgaattttgcttgataaggctgcaacaaagatcaggttgacatatattttcagtaatttattttaacctgtactctatcactgccggctataataagttcttgaataaaataattttcccgacaaaacatgagtatttctttcgaaaataaaagtgcaggctaattagagattattctgtaattaactgcataatttgtcacgtgccatagttttacattccactacgtcatagttcttaaaatactaatctcctgaaaatagaagtaggtgaagaaagtttaattatcagttctaaaatgacaattgatctttggaatatgttctgaaactaccgatcattgaacagatgctatttgaatgatatagaattgtatatttgcaatttctgaaacgcgttcttaaaggcacacagattaattcaaaagtaagcttctcacaccttttctctcgttatggtggccgattttgagttttgtgtgtgattgctttttcacaatcagtgttttcaggattatgtgatgaactagatcttcaagtttcgttacatttcatatgttttcggaactcacgaagtacatattgggtattgtgctcaaaaaattcagcaatcagcttcgctccgctgactttagaacccaaaaaaatagtatggccaaactgactgtgttacgatcatttcaatttttcaatacatatttaagatttctaagagtaagaaggtcaaaaactgttctggaatacatatatatttttcaggttacaattagtcaaaaagtgcactgaaatatacgttttaatttcacgaataacccaattagttcaatgtatttttggtcaaccaacgttaaagtttggcttccaaccaattatcatttctgatcaaccacaatgttttttctttatctgcaagttaattttttatttttatccagatgtttggcatatttttcaattcttcactagcgcccacttcttgcacttccggcgccctgaatctaatgcatctgttgcaagaattgaaagaccaatcaacacattgttttcttcacgagatactgaagaaaatgaataaaaacagagaaaaagagccatgtgattagtgacaactgttgctaacagataccatagcttggacttggtacgtgatggcaacgtatgggtcaacaaaaatgattgcagagggggtgcaaaacagtcaagtcgagaaaatatgaaaaacagaaaacaaagaacagaaaaatgggtttgagagtcagtataatttataaaagaaaaattgtacatagaaattaaccatttttgtagaagaagttatttttcaagcatcgttaaaaattattcaaagcaccttatttcatatttaattttaaacatggttaaatgaacaacacggtgcgcaatcaggaaaacttgaaatctgaaactgttgttgtgatcttcttcgcaactgttcagatagcactagtgtaatgttaagagtgcgcgaatataatggaatataatggatcacacctcctgccatcaggtaaacgtctctgttatcacatatttccaactattaaatttttaccttttacagttttacatttttttgaaaaaagtaactttttgtcttcaaaatccctgacgaaaatatcaaatattttaatcgagactgcagaggaaccgattgatgatttggaaaatccagctttacctgtgtaagaactgaaaagtttcataaccctagggtattcccagttacattccccactggctaacaatagcacccagtttttcatcacctttcttcaaatttctcggcgatttgttaaaaacaaaatttgtgtcccttctctgatatctctatgtctctaaacacaagttcatcggaaaacgaaggagggtaggtgttggttgggctcccgaagtgaaaatagaagagcaagaatagaatattagagagagagtgcagagagggcgggatagctcccgggattccgttttcttcttctttatcttcaacgatgatgtgtgtgcgtgttgtatagattctgttgctcccccacaactcgctccgaaggctcaatacaattcaattgatattggaggagagcctaccggagtgggaggataagaagaaacataagaagaagaagaagaagaagcatgcttctggtttttgatgctatgaaaacggcacaaaaagatgattgaggtcccttttcaataccttctctcatctttcaaatcccattgaaacctaaaacttctcaccacgctttaccattgttctccaaaaacttatagcaatgtctataacttttttatctctgaaaagcagtgttccatttttctttttcctattttatttcaattgtttctcacatttcgtttggattctttgcttgtcaaccagcttcttcttccacttttaccgtctaattttcagggcagggagccatcaaacccacgaccactagatccat
</span><span style="color:#ff0000">
ATGGTATCGAAGGGAGAAGCAGTAATCAAGGAGTTCATGCGTTTCAAGGTCCACATGGAGGGATCCATGAACGGACACGAGTTCGAGATCGAGGGAGAGGGAGAGGGACGTCCATACGAGGGAACCCAAACCGCCAAGCTCAAGGTCACCAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGAGGACCACTCCCATTCTCCTGGGACATCCTCTCCCCACAATTCATGTACGGATCCCGTGCCTTCATCAAGCACCCAGCCGACATCCCAGACTACTACAAGCAATCCTTCCCAGAGGGATTCAAGTGGGAGCGTGTCATGAACTTCGAGGACGGAGGAGCCGTCACCGTCACCCAAGACACCTCCCTCGAGGACGGAACCCTCATCTACAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTCAAGCTCCGTGGAACCAACTTCCCACCAGACGGACCAGTCATGCAAAAGAAGACCATGGGATGGGAGGCCTCCACCGAGCGTCTCTACCCAGAGGACGGAGTCCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGAGACATCACCATGGCCCTCCGTCTCAAGGACGGAGGACGTTACCTCGCCGACACCTCCACCACCTACAAGGCCAAGAAGCCAGTCCAAATGCCAGGAGCCTACCTCGTCGACCGTAAGCTCGACATCACCTCCCACAACGAGTACTAA
</span>catctcgcgcccgtgcctctgacttctaagtccaattactcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaacttctcttaatttctttgttttttagcttcttttaagtcacctctaacaatgaaattgtgtagattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttacttctgataaatttttttgaaacatcatagaaaaaaccgcacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttggggtttcccctattgtttgtcaagatttcgaggacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaag
</td>
</tr>
<tr>
<td style="word-wrap: break-word">DUP223</td>
<td style="word-wrap: break-word">glh-1(sam129[glh-1::T2A::sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>]) I</td>
<td style="word-wrap: break-word">Germline sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub></td>
<td style="word-wrap: break-word">I: 6.85 MB</td>
<td style="word-wrap: break-word">I: 1.41</td>
<td style="word-wrap: break-word">
gtttcgccacgcgccccaactacagtaacctcgacacactcatctactaaattttgggacagttcctaattctttttgctgttttcaactcaattttctggaaaaatcttaattttctgcgaaaATGTCTGATGGTTGGAGTGATAGCGAAAGTGCTGCTAAGGgtgagttttattttgaactttccaccggttttattttgattaaaaactttatttcagCCAAAACTGGATTCGGTAGTGGAGGCGGTTTCGGTGGTGGTAACAATGGAGGATCTGGGTTTGGTGGTGGTAAAAATGGAGGTACTGGATTCGGTGGAGGAAACACTGGCGGATCTGGATTCGGTGGAGGAAACACTGGCGGATCTGGATTCGGTGGAGGAAAGACTGGCGGTTCTGGATTTGGAGGTGGAAATACTTGTGGATCCGGCTTCGGTGGAGGCAGTACAGGAGGATCGCCGTATGGAGGAGCCAGTTCTGGATTCGGTGGTAGTACTGCCACATCTGGATTTGGAAGCGGTGAAAAATCAAGTGCATTTGGAGGATCAGGTGGCTTTGGAGGTAGTGCAACTGGATTCGGAAGTGGAGGAGGATCCTTTGGAGGTGGCAACTCTGGTTTTGGGGAAGGAGGACATGGCGGCGGAGAGAGAAACAATAgttcgttttttaaattgactttatataattacgtttgtttcagATTGTTTCAATTGCCAACAGCCAGGACATCGATCGAGTGACTGTCCAGAGCCGAGAAAGGAAAGAGAGCCGAGAGgttttattttgatataactttattggcagtaatttgttttattttcagTGTGCTACAATTGCCAGCAACCCGGGCACACCTCTCGTGAATGTACAGAAGAACGCAAGCCGCGTGAGGGTCGCACTGGTGGATTCGGGGGCGGAGCTGGATTTGGAAACAATGGAGGAAATGACGGTTTCGGTGGGGACGGTGGTTTTGGTGGAGGCGAAGAACGTGGTCCAATGAAATGTTTCAACTGTAAAGGCGAGGGACATCGCTCTGCTGAATGTCCGGAGCCACCCCGTGGATGTTTCAATTGTGGCGAGCAAGGTCATCGCTCGAATGAGTGCCCCAATCCAGCCAAGCCAAGGGAAGGTGTTGAAGGAGAAGGACCTAAGGCGACATACGTGCCAGTCGAAGACAACATGGAGGACGTTTTCAACATGCAGAAAATTTCGGAAGGCCTTATGTTCAACAAGTTTTTCGATGCCGAAGTTAAACTGACTTCATCCGAGAAGACTGTCGGTATCAAACCTTGCAAGACATTCGCGGAAGCTAATCTCACGGAGACCATGCAGAAAAACGTTGCTCATGCTGGATACTCCAAGACCACTCCAATTCAGCAATATGCTCTTCCACTTGTTCATCAGGGATATGATATCATGGCTTGTGCTCAAACTGGATCAGGAAAAACCGCTGCATTCCTTCTGCCTATCATGACTCGTCTCATTGACGATAATAATCTGAACACTGCCGGAGAAGGCGGTTGCTATCCCCGTTGCATCATCTTGACTCCAACTCGCGAACTCGCTGATCAAATTTACAACGAGGGAAGAAAGTTTGCTTATCAAACAATGATGGAGATCAAACCAGTTTACGGAGGATTGGCTGTCGGTTATAATAAGGGTCAGATCGAAAAGGGAGCCACGATCATTGTCGGAACTGTCGGAAGAATCAAGCACTTCTGTGAAGAGGGTACCATCAAGCTTGACAAATGCCGCTTCTTTGTTCTTGACGAGGCTGATCGTATGATCGATGCTATGGGATTCGGAACTGACATCGAAACTATTGTCAATTATGACAGTATGCCGAGGAAAGAAAATCGCCAGACACTCATGTTCAGTGCCACTTTCCCCGATTCTGTACAGGAAGCAGCTCGCGCTTTTCTCAGAGAAAACTACGTGATGATTGCAATCGACAAGATTGGAGCTGCAAACAAGTGCGTCCTACAGGAATTCGAGAGATGCGAAAGAAGCGAGAAGAAGGACAAACTTCTAGAGCTTCTGGGAATCGATATCGACAGTTACACGACCGAGAAAAgtgagtttttcgttttcttatttgatgaaataaatttcaatatttcagGTGCCGAAGTTTACACAAAGAAAACCATGGTCTTCGTTTCTCAAAGAGCAATGGCTGATACACTGGCTTCAATTTTGTCATCGGCTCAAGTTCCAGCTATCACGgttggtatatttcatttttgaccgctttttaattcaaaatgtacagATCCATGGTGCCCGTGAGCAGAGAGAGCGTTCAGAAGCTTTGAGACAATTCCGAAATGGATCGAAACCTGTTCTTATTGCTACTGCGGTCGCTGAACGTGGACTTGATATCAAAGGAGTGGATCATGTCATCAACTATGACATGCCAGACAACATTGATGACTATATCCATCGTATCGGAAGgtcagttatattttattaatgtttcaataatgcaagcattgttttcagAACTGGAAGAGTTGGAAACTCTGGAAGAGCTACAAGCTTCATCTCGGAGGATTGCAGTCTTCTGTCCGAACTTGTTGGTGTTCTCGCCGACGCACAACAGATTGTTCCAGACTGGATGCAAGGTGCTGCTGGAGGCAATTACGGAGCTAGTGGATTTGGGTCCAGTGTACCAACTCAAGTCCCGCAGGACGAGGAGGGGTGG
<span style="color:#01F2FF">
GGATCGGGA
</span><span style="color:#ff0000">
GAGGGACGTGGATCCCTTCTTACCTGCGGAGACGTCGAGGAGAACCCAGGACCA
</span><span style="color:#01F2FF">
GGAGCATCGGGAGCCTCAGGAGCATCG
</span><span style="color:#A3A3A3">
ATGAGTAAAGGAGAAGAATTGTTCACTGGAGTTGTCCCAATCCTCGTCGAGCTCGACGGAGACGTCAACGGACACAAGTTCTCCGTCCGTGGAGAGGGAGAGGGAGACGCCACCATCGGAAAGCTCACCCTCAAGTTCATCTGCACCACCGGAAAGCTCCCAGTCCCATGGCCAACCCTCGTCACCACCCTCACCTACGGAGTCCAATGCTTCGCCCGTTACCCAGACCACATGAAGCGTCACGACTTCTTCAAGTCCGCCATGCCAGAGGGATACGTCCAAGAGCGTACCATCTCCTTCAAGgtaagtttaaacatatatatactaactactgattatttaaattttcagGACGACGGAAAGTACAAGACCCGTGCCGTCGTCAAGTTCGAGGGAGACACCCTCGTCAACCGTATCGAGCTCAAGGGAACCGACTTCAAGGAGGACGGAAACATCCTCGGACACAAGCTCGAGTACAACTTCAACTCCCACAACGTCTACATCACCGCCGACAAGCAAAAGAACGGAATCAAGGCCAACTTCACCgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagACCCGTCACAACGTCGAGGACGGATCCGTCCAACTCGCCGACCACTACCAACAAAACACCCCAATCGGAGACGGACCAGTCCTCCTCCCAGACAACCACTACCTCTCCACCCAAACCGTCCTCTCCAAGGACCCAAACGAGAAGTAG</span>
aaaaccgaccaattgatagtgtttcgcatttattaatgctgtcagttcccccatattttatcctgcccttgttgatttttaattgtatttgttggtgttggttgtcgttatagtcctcgccgcataaactctgttc
</td>
</tr>
<tr>
<td style="word-wrap: break-word">DUP237</td>
<td style="word-wrap: break-word">glh-1(sam140[glh-1::T2A::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>]) I</td>
<td style="word-wrap: break-word">Germline split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub></td>
<td style="word-wrap: break-word">I: 6.85 MB</td>
<td style="word-wrap: break-word">I: 1.41</td>
<td style="word-wrap: break-word">
gtttcgccacgcgccccaactacagtaacctcgacacactcatctactaaattttgggacagttcctaattctttttgctgttttcaactcaattttctggaaaaatcttaattttctgcgaaaATGTCTGATGGTTGGAGTGATAGCGAAAGTGCTGCTAAGGgtgagttttattttgaactttccaccggttttattttgattaaaaactttatttcagCCAAAACTGGATTCGGTAGTGGAGGCGGTTTCGGTGGTGGTAACAATGGAGGATCTGGGTTTGGTGGTGGTAAAAATGGAGGTACTGGATTCGGTGGAGGAAACACTGGCGGATCTGGATTCGGTGGAGGAAACACTGGCGGATCTGGATTCGGTGGAGGAAAGACTGGCGGTTCTGGATTTGGAGGTGGAAATACTTGTGGATCCGGCTTCGGTGGAGGCAGTACAGGAGGATCGCCGTATGGAGGAGCCAGTTCTGGATTCGGTGGTAGTACTGCCACATCTGGATTTGGAAGCGGTGAAAAATCAAGTGCATTTGGAGGATCAGGTGGCTTTGGAGGTAGTGCAACTGGATTCGGAAGTGGAGGAGGATCCTTTGGAGGTGGCAACTCTGGTTTTGGGGAAGGAGGACATGGCGGCGGAGAGAGAAACAATAgttcgttttttaaattgactttatataattacgtttgtttcagATTGTTTCAATTGCCAACAGCCAGGACATCGATCGAGTGACTGTCCAGAGCCGAGAAAGGAAAGAGAGCCGAGAGgttttattttgatataactttattggcagtaatttgttttattttcagTGTGCTACAATTGCCAGCAACCCGGGCACACCTCTCGTGAATGTACAGAAGAACGCAAGCCGCGTGAGGGTCGCACTGGTGGATTCGGGGGCGGAGCTGGATTTGGAAACAATGGAGGAAATGACGGTTTCGGTGGGGACGGTGGTTTTGGTGGAGGCGAAGAACGTGGTCCAATGAAATGTTTCAACTGTAAAGGCGAGGGACATCGCTCTGCTGAATGTCCGGAGCCACCCCGTGGATGTTTCAATTGTGGCGAGCAAGGTCATCGCTCGAATGAGTGCCCCAATCCAGCCAAGCCAAGGGAAGGTGTTGAAGGAGAAGGACCTAAGGCGACATACGTGCCAGTCGAAGACAACATGGAGGACGTTTTCAACATGCAGAAAATTTCGGAAGGCCTTATGTTCAACAAGTTTTTCGATGCCGAAGTTAAACTGACTTCATCCGAGAAGACTGTCGGTATCAAACCTTGCAAGACATTCGCGGAAGCTAATCTCACGGAGACCATGCAGAAAAACGTTGCTCATGCTGGATACTCCAAGACCACTCCAATTCAGCAATATGCTCTTCCACTTGTTCATCAGGGATATGATATCATGGCTTGTGCTCAAACTGGATCAGGAAAAACCGCTGCATTCCTTCTGCCTATCATGACTCGTCTCATTGACGATAATAATCTGAACACTGCCGGAGAAGGCGGTTGCTATCCCCGTTGCATCATCTTGACTCCAACTCGCGAACTCGCTGATCAAATTTACAACGAGGGAAGAAAGTTTGCTTATCAAACAATGATGGAGATCAAACCAGTTTACGGAGGATTGGCTGTCGGTTATAATAAGGGTCAGATCGAAAAGGGAGCCACGATCATTGTCGGAACTGTCGGAAGAATCAAGCACTTCTGTGAAGAGGGTACCATCAAGCTTGACAAATGCCGCTTCTTTGTTCTTGACGAGGCTGATCGTATGATCGATGCTATGGGATTCGGAACTGACATCGAAACTATTGTCAATTATGACAGTATGCCGAGGAAAGAAAATCGCCAGACACTCATGTTCAGTGCCACTTTCCCCGATTCTGTACAGGAAGCAGCTCGCGCTTTTCTCAGAGAAAACTACGTGATGATTGCAATCGACAAGATTGGAGCTGCAAACAAGTGCGTCCTACAGGAATTCGAGAGATGCGAAAGAAGCGAGAAGAAGGACAAACTTCTAGAGCTTCTGGGAATCGATATCGACAGTTACACGACCGAGAAAAgtgagtttttcgttttcttatttgatgaaataaatttcaatatttcagGTGCCGAAGTTTACACAAAGAAAACCATGGTCTTCGTTTCTCAAAGAGCAATGGCTGATACACTGGCTTCAATTTTGTCATCGGCTCAAGTTCCAGCTATCACGgttggtatatttcatttttgaccgctttttaattcaaaatgtacagATCCATGGTGCCCGTGAGCAGAGAGAGCGTTCAGAAGCTTTGAGACAATTCCGAAATGGATCGAAACCTGTTCTTATTGCTACTGCGGTCGCTGAACGTGGACTTGATATCAAAGGAGTGGATCATGTCATCAACTATGACATGCCAGACAACATTGATGACTATATCCATCGTATCGGAAGgtcagttatattttattaatgtttcaataatgcaagcattgttttcagAACTGGAAGAGTTGGAAACTCTGGAAGAGCTACAAGCTTCATCTCGGAGGATTGCAGTCTTCTGTCCGAACTTGTTGGTGTTCTCGCCGACGCACAACAGATTGTTCCAGACTGGATGCAAGGTGCTGCTGGAGGCAATTACGGAGCTAGTGGATTTGGGTCCAGTGTACCAACTCAAGTCCCGCAGGACGAGGAGGGGTGG
<span style="color:#01F2FF">
GGATCGGGA
</span><span style="color:#ff0000">
GAGGGACGTGGATCCCTTCTTACCTGCGGAGACGTCGAGGAGAACCCAGGACCA
</span><span style="color:#01F2FF">
GGAGCATCGGGAGCCTCAGGAGCATCG
</span><span style="color:#A3A3A3">
ATGGTTTCCAAGGGAGAGGCTGTTATCAAGGAATTCATGCGCTTCAAGGTTCACATGGAAGGATCTATGAACGGACACGAATTCGAAATCGAAGGAGAAGGAGAAGGACGCCCATACGAGGGAACTCAAACTGCTAAGCTTAAGGTTACTAAAGGAGGACCACTTCCATTCTCTTGGGATATCCTTTCTCCACAGTTCATGTACGGATCTCGCGCTTTCATCAAGCACCCAGCTGATATCCCAGATTACTACAAGCAGTCTTTCCCAGAAGGATTCAAATGGGAGCGCGTTATGAACTTCGAAGATGGAGGAGCTGTTACCGTTACCCAAGATACCTCCCTTGAGGATGGAACCCTTATCTACAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGTTAAGCTTCGCGGAACTAATTTCCCACCAGATGGACCAGTTATGCAGAAGAAGACTATGGGATGGGAAGCTTCTACCGAGCGCCTTTACCCAGAGGATGGAGTCCTTAAGGGAGATATCACCATGGCTCTTCGTCTTAAGGATGGAGGACGTTACCTTGCTGATACCTCTACTACTTACAAGGCTAAGAAGCCAGTTCAGATGCCAGGAGCTTACCTTGTCGATCGTAAGCTTGATATCACTTCTCATAACGAATACTAG
</span>aaaaccgaccaattgatagtgtttcgcatttattaatgctgtcagttcccccatattttatcctgcccttgttgatttttaattgtatttgttggtgttggttgtcgttatagtcctcgccgcataaactctgttc
</td>
</tr>
</table>
<h3><a id="TableS3">Table S3.</a> <em>C. elegans</em> strains, genotypes and sources</h3>
<table style="table-layout: fixed; width: 100%;font-size:90%;line-height: 100%;">
<tr>
<th>Strain</th>
<th style="width:70%;">Genotype</th>
<th>Source</th>
</tr>
<tr>
<td>N2E</td>
<td>wild type</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4582</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4586</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; vha-13(muIs262[split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::vha-13]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4587</td>
<td style="word-wrap: break-word">muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4588</td>
<td style="word-wrap: break-word">muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)], muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4589</td>
<td style="word-wrap: break-word">muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; vha-13(muIs268[sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::vha-13]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4592</td>
<td style="word-wrap: break-word">muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; his-3(muIs255[his-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4594</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; his-3(muIs258[his-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4601</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; fib-1(muIs254[split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::fib-1]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4602</td>
<td style="word-wrap: break-word">muIs253[Peft-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)], muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; fib-1(muIs254[split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::fib-1]), his-3(muIs255[his-3::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4603</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; eat-6(muIs269[eat-6::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>]/+) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4608</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; his-3(muIs267[his-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>(x3)]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4610</td>
<td style="word-wrap: break-word">muIs257[Pmyo-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR] I</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4611</td>
<td style="word-wrap: break-word">muIs257[myo-3p::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR] I; fib-1(muIs254[split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::fib-1]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4612</td>
<td style="word-wrap: break-word">muEx690[Pmyo-3::mTagBFP2::sfCherry3<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::T2A::mNeonGreen::sfCherry3_1-10::fib-1 3'UTR]</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4613</td>
<td style="word-wrap: break-word">muEx691[Pmyo-3::mTagBFP2::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::T2A::mNeonGreen::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::fib-1 3'UTR]</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4614</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; tbb-2(muIs260[split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>::tbb-2]), unc-119(ed3) III</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4615*</td>
<td style="word-wrap: break-word">muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; tomm-20(muIs261[tomm-20::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>]) V</td>
<td>Kenyon Lab</td>
</tr>
<tr>
<td>CF4616
<td style="word-wrap: break-word"> muIs252[Peft-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III; vha-13(muIs264[split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>(x2)::vha-13]) V
<td>Kenyon Lab</td>
</tr>
<tr>
<td>COP1795</td>
<td style="word-wrap: break-word">knuSi785 [pNU1687(Plet-858::sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::unc-54 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td>Nemametrix</td>
</tr>
<tr>
<td>DUP218</td>
<td style="word-wrap: break-word">glh-1(sam124[glh-1::T2A::sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-11</sub>]) I</td>
<td>Updike Lab</td>
</tr>
<tr>
<td>DUP223</td>
<td style="word-wrap: break-word">glh-1(sam129[glh-1::T2A::sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>]) I</td>
<td>Updike Lab</td>
</tr>
<tr>
<td>DUP225</td>
<td style="word-wrap: break-word">glh-1(sam129[glh-1::T2A::sGFP2<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>]) I; pgl-1(sam126[pgl-1::GFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>]) IV</td>
<td>Updike Lab</td>
</tr>
<tr>
<td>DUP236</td>
<td style="word-wrap: break-word">glh-1(sam139[glh-1::T2A::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-11</sub>]) I</td>
<td>Updike Lab</td>
</tr>
<tr>
<td>DUP237</td>
<td style="word-wrap: break-word">glh-1(sam140[glh-1::T2A::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>]) I</td>
<td>Updike Lab</td>
</tr>
<tr>
<td>PHX731</td>
<td style="word-wrap: break-word">vha-13(syb731[wrmScarlet::vha-13]) V</td>
<td>SunyBiotech</td>
</tr>
<tr>
<td>PHX1049</td>
<td style="word-wrap: break-word">
vha-13(syb1049[gfp::vha-13]) V</td>
<td>SunyBiotech</td>
</tr>
<tr>
<td>PHX1797
<td style="word-wrap: break-word">sybSi66[Psun-1::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub>::sun-1 3'UTR, Cbr-unc-119(+)] II; unc-119(ed3) III
<td>SunyBiotech
</tr>
<tr>
<td>CA1200</td>
<td style="word-wrap: break-word">ieSi57[eft-3p::TIR1::mRuby::unc-54 3'UTR + Cbr-unc-119(+)] II; unc-119(ed3) III</td>
<td>CGC</td>
</tr>
<tr>
<td>WBM1126</td>
<td style="word-wrap: break-word">wbmIs61[myo-3p::3XFLAG::dpy-10 crRNA::unc-54 3'UTR] I</td>
<td>CGC</td>
</tr>
</table>
* Mutation present - see table S4 for the corresponding sequence
<h3><a id="TableS4">Table S4.</a> crRNAs, HDR templates and oligonucleotide sequences</h3>
<h5>S4A. Sequences of crRNA and HDR template used for split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> and sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> knock-in experiments</h5>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 100%;">
<tr>
<th style="font-size:100%">Gene name</th>
<th style="font-size:100%">Gene ID</th>
<th style="font-size:100%">Tagged term</th>
<th style="font-size:100%">gene-specific crRNA sequence</th>
<th style="font-size:100%; width:40%;">1x split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> HDR donor sequence - Ultramer ssDNA (lower case: homology arms; red: split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>; blue: linker)</th>
<th style="font-size:70%">Sequencing Primer (Forward)</th>
<th style="font-size:70%">Sequencing Primer (Reverse)</th>
</tr>
<tr>
<td>eat-6</td>
<td>B0365.3</td>
<td>C</td>
<td style="word-wrap: break-word">ACAAGCUGUUCUUUAGUAGU</td>
<td style="word-wrap: break-word">
cgacgagatccgtcgtttcttgattcgcagatatccaggaggatgggtcgagcgtgagacctactac
<span style="color:#0000ff">GGAGGAGGATCC</span>
<span style="color:#ff0000">TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA</span>
taaagaacagcttgtgaatctttgtagaattttctattttttatcttagttttttattgtttcccat
</td>
<td style="word-wrap: break-word">CCTGGTTCATGTGCTATTGCC</td>
<td style="word-wrap: break-word">CGACGACAGAAAGTAGCATCAC</td>
</tr>
<tr>
<td>fib-1</td>
<td>T01C3.7</td>
<td>N</td>
<td style="word-wrap: break-word">AUAAUCGAUUUUUGUAGUAU</td>
<td style="word-wrap: break-word">
aatacgaaaaaatcctgaaattcagtttacctccgccgccgccgccacgattgaactctggacgtcc
<span style="color:#0000ff">GGATCCTCCTCC</span>
<span style="color:#ff0000">TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
catactacaaaaatcgattatttaacaaaaacgaaaagcgaaattacgaaaaatacatacctcagcc
</td>
<td style="word-wrap: break-word">TTCTAGTCGATTCAGATCGACTGG</td>
<td style="word-wrap: break-word">CGAAACTGCCACGATCACC</td>
</tr>
<tr>
<td>his-3</td>
<td>T10C6.12</td>
<td>C</td>
<td style="word-wrap: break-word">GAAGAAAACCGGAGGAGACA</td>
<td style="word-wrap: break-word">
gattttaaatatttgtggccctaaagagggccgttgggttcggtaaaatgttttaagaaggcatcta
<span style="color:#ff0000">TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ttc<u>T</u>ttgtctcctccggt<u>C</u>ttctt<u>T</u>ggcaaaagaacagcttggatatttggaagaactcctccttgg
</td>
<td style="word-wrap: break-word">CCAAGGAGGAGTTCTTCCAAATATC</td>
<td style="word-wrap: break-word">GATTTTAAATATTTGTGGCCCTAAAG</td>
</tr>
<tr>
<td>tbb-2</td>
<td>C36E8.5</td>
<td>N</td>
<td style="word-wrap: break-word">CGCAUUGUCCGGCUUGCACG</td>
<td style="word-wrap: break-word">
tcttctctatctaaatatagtttttcaattcattacgaccttttcagcaaaaatg
<span style="color:#ff0000">TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA</span>
<span style="color:#0000ff">GGAGGAGGATCCGGAGGAGGATCCGGAGGAGGATCC</span>
agagagatcgt<u>T</u>cacgtgcaagccggacaatgcggaaaccaaatcggatccaaat
</td>
<td style="word-wrap: break-word">CAATATCGACCATGACGTGTTCTC</td>
<td style="word-wrap: break-word">CTTGAAGGTTCCGTCTGGC</td>
</tr>
<tr>
<td>tomm-20</td>
<td>F23H12.2</td>
<td>C</td>
<td style="word-wrap: break-word">ACACCGACGACUUGGAGUAA</td>
<td style="word-wrap: break-word">
gttggtgaacgaaaaatacgaagaattaaattgaaagttaataaaacttttaaatcattatccatta
<span style="color:#ff0000">TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ctccaagtcgtcggtgtcatcgataagctcttggatttgtgctggtggaggtccgtctccgaggtat
</td>
<td style="word-wrap: break-word">GAGCGAAAGCAGATGAGGC</td>
<td style="word-wrap: break-word">TCCGTGAGGAGGAAAACACC</td>
</tr>
<tr>
<td>actual tomm-20 mutation recovered</td>
<td>F23H12.2</td>
<td>C</td>
<td style="word-wrap: break-word">ACACCGACGACUUGGAGUAA</td>
<td style="word-wrap: break-word">
gttggtgaacgaaaaatacgaagaattaaattgaaagttaataaaacttttaaatcattatccatta
<span style="color:#ff0000">T<u><strong>TA</strong></u>TCC<u><strong>AT</strong></u>TGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ctccaagtcgtcggtgtcatcgataagctcttggatttgtgctggtggaggtccgtctccgaggtat
</td>
<td style="word-wrap: break-word">GAGCGAAAGCAGATGAGGC</td>
<td style="word-wrap: break-word">TCCGTGAGGAGGAAAACACC</td>
</tr>
<tr>
<td>vha-13</td>
<td>Y49A3A.2</td>
<td>N</td>
<td style="word-wrap: break-word">AUUCUGCGGCCAUCUUUUCC</td>
<td style="word-wrap: break-word">
ggtttattttgattttcttttcgatttccatatagctttctaaattcattcattccaggaaaagatg
<span style="color:#ff0000">TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA</span>
<span style="color:#0000ff">GGAGGAGGATCC</span>
gccgcagaatcttcgtacggattcgtttacggagtgtccggacctgtcgtcacagccgagaagatgg
</td>
<td style="word-wrap: break-word">GGTTTATTTTGATTTTCTTTTCGATTTCC</td>
<td style="word-wrap: break-word">CCATCTTCTCGGCTGTGAC</td>
</tr>
<!--</table>
<<h5>S4B. Sequences of crRNA and HDR template used for sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> knock-in experiments</h5>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 100%;">-->
<tr>
<th style="font-size:100%">Gene name</th>
<th style="font-size:100%">Gene ID</th>
<th style="font-size:100%">Tagged term</th>
<th style="font-size:100%">gene-specific crRNA sequence</th>
<th style="font-size:100%; width:40%;">1x sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub> HDR donor sequence - Ultramer ssDNA (lower case: homology arms; green: sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>; blue: linker)</th>
<th style="font-size:100%">Sequencing Primer (Forward)</th>
<th style="font-size:100%">Sequencing Primer (Reverse)</th>
</tr>
<tr>
<td>his-3</td>
<td>T10C6.12</td>
<td>C</td>
<td style="word-wrap: break-word">GAAGAAAACCGGAGGAGACA</td>
<td style="word-wrap: break-word">
atagaggattttaaatatttgtggccctaaagagggccgttgggttcggtaaaatgttttaagaaggcatcta
<span style="color:#00ff00">GGTGATTCCGGCGGCGTTGACGTACTCGTGGAGGACCATGTGGTCACG</span>
<span style="color:#0000ff">TCCTCCTCC</span>
ttc<u>T</u>ttgtctcctccggt<u>C</u>ttctt<u>T</u>ggcaaaagaacagcttggatatttggaagaactcctccttgggcg
</td>
<td style="word-wrap: break-word">CCAAGGAGGAGTTCTTCCAAATATC</td>
<td style="word-wrap: break-word">GATTTTAAATATTTGTGGCCCTAAAG</td>
</tr>
<tr>
<td>vha-13</td>
<td>Y49A3A.2</td>
<td>N</td>
<td style="word-wrap: break-word">AUUCUGCGGCCAUCUUUUCC</td>
<td style="word-wrap: break-word">
ggtttattttgattttcttttcgatttccatatagctttctaaattcattcattccaggaaaagatg
<span style="color:#00ff00">CGTGACCACATGGTCCTCCACGAGTACGTCAACGCCGCCGGAATCACC</span>
<span style="color:#0000ff">GGAGGAGGATCC</span>
gccgcagaatcttcgtacggattcgtttacggagtgtccggacctgtcgtcacagccgagaagatgg
</td>
<td style="word-wrap: break-word">GGTTTATTTTGATTTTCTTTTCGATTTCC</td>
<td style="word-wrap: break-word">CCATCTTCTCGGCTGTGAC</td>
</tr>
<tr>
<td>pgl-1</td>
<td>ZK381.4</td>
<td>C</td>
<td style="word-wrap: break-word">GGTGGTTACGGGGGTCGTGG</td>
<td style="word-wrap: break-word">
tacggcggagatcgtggacgtggtggttacgggggaagaggaggtagaggtggattt
<span style="color:#0000ff">GGAGCATCGGGAGCCTCAGGAGCATCG</span>
<span style="color:#00ff00">CGTGACCACATGGTCCTCCACGAGTACGTCAACGCCGCCGGAATCACC</span>
taaactccaactattgaatgtttaatttgtttttta
</td>
<td style="word-wrap: break-word">CCAAAGTTGCAAAAGGATTCGGTCAATTTG</td>
<td style="word-wrap: break-word">CATTTACCGGGAACAAGGAAAAACAGGTTG</td>
</tr>
<tr>
<th style="font-size:100%">Gene name</th>
<th style="font-size:100%">Gene ID</th>
<th style="font-size:100%">Tagged term</th>
<th style="font-size:100%">gene-specific crRNA sequence</th>
<th style="font-size:100%; width:40%;">1x split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub> HDR donor sequence - Ultramer ssDNA (lower case: homology arms; red: split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11(MDELYK)</sub>; blue: linker)</th>
<th style="font-size:100%">Sequencing Primer (Forward)</th>
<th style="font-size:100%">Sequencing Primer (Reverse)</th>
</tr>
<tr>
<td>eat-6</td>
<td>B0365.3</td>
<td>C</td>
<td style="word-wrap: break-word">ACAAGCUGUUCUUUAGUAGU</td>
<td style="word-wrap: break-word">
cgacgagatccgtcgtttcttgattcgcagatatccaggaggatgggtcgagcgtgagacctactac
<span style="color:#0000ff">GGAGGAGGATCC</span>
<span style="color:#ff0000">TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA<strong>ATGGATGAGTTATACAAG</strong></span>
taaagaacagcttgtgaatctttgtagaattttctattttttatcttagttttttattgtttcccat
</td>
<td style="word-wrap: break-word">CCTGGTTCATGTGCTATTGCC</td>
<td style="word-wrap: break-word">CGACGACAGAAAGTAGCATCAC</td>
</tr>
<tr>
<td>his-3</td>
<td>T10C6.12</td>
<td>C</td>
<td style="word-wrap: break-word">GAAGAAAACCGGAGGAGACA</td>
<td style="word-wrap: break-word">
gattttaaatatttgtggccctaaagagggccgttgggttcggtaaaatgttttaagaaggcatcta
<span style="color:#ff0000"><strong>CTTGTATAACTCATCCAT</strong>TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ttc<u>T</u>ttgtctcctccggt<u>C</u>ttctt<u>T</u>ggcaaaagaacagcttggatatttggaagaactcctccttgg
</td>
<td style="word-wrap: break-word">CCAAGGAGGAGTTCTTCCAAATATC</td>
<td style="word-wrap: break-word">GATTTTAAATATTTGTGGCCCTAAAG</td>
</tr>
<tr>
<td>tomm-20</td>
<td>F23H12.2</td>
<td>C</td>
<td style="word-wrap: break-word">ACACCGACGACUUGGAGUAA</td>
<td style="word-wrap: break-word">
gttggtgaacgaaaaatacgaagaattaaattgaaagttaataaaacttttaaatcattatccatta
<span style="color:#ff0000"><strong>CTTGTATAACTCATCCAT</strong>TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ctccaagtcgtcggtgtcatcgataagctcttggatttgtgctggtggaggtccgtctccgaggtat
</td>
<td style="word-wrap: break-word">GAGCGAAAGCAGATGAGGC</td>
<td style="word-wrap: break-word">TCCGTGAGGAGGAAAACACC</td>
</tr>
<tr>
<td>actual tomm-20 mutation recovered</td>
<td>F23H12.2</td>
<td>C</td>
<td style="word-wrap: break-word">ACACCGACGACUUGGAGUAA</td>
<td style="word-wrap: break-word">
gttggtgaacgaaaaatacgaagaattaaattgaaagttaataaaacttttaaatcattatccatta
<span style="color:#ff0000"><strong>CTTGTATAACTCATCCAT</strong>TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ctccaagtcgtcggtgtcatcgataagctcttggatttgtgctggtggaggtccgtctccgaggtat
</td>
<td style="word-wrap: break-word">GAGCGAAAGCAGATGAGGC</td>
<td style="word-wrap: break-word">TCCGTGAGGAGGAAAACACC</td>
</tr>
</table>
<h5>S4B DNA template for split-wrmScarlet tandems HDR donor sequence - plasmids</h5>
<table style="font-size:80%;line-height: 160%;">
<tr>
<th>Template name</th>
<th>Tagged term</th>
<th> DNA template for split-wrmScarlet tandems - dsDNA (lower case: homology arms; red: split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>; blue: linkers)</th>
</tr>
<tr>
<td>wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>(x2)::vha-13</td>
<td>N</td>
<td style="word-wrap: break-word">ggtttattttgattttcttttcgatttccatatagctttctaaattcattcattccaggaaaagatg
<span style="color:#ff0000">TACACCGTCGTCGAGCAATACGAGAAGTCCGTCGCCCGTCACTGCACCGGAGGA</span>
<span style="color:#0000ff">GGTGGCTCTGGAGGT</span>
<span style="color:#ff0000">TACACCGTTGTTGAGCAATACGAGAAGTCTGTTGCTCGTCACTGCACCGGAGGC</span>
<span style="color:#0000ff">GGAGGAGGATCC</span>
gccgcagaatcttcgtacggattcgtttacggagtgtccggacctgtcgtcacagccgagaagatgg
</td>
</tr>
<tr>
<td>his-3::split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">11</sub>(x3)</td>
<td>C</td>
<td style="word-wrap: break-word">gattttaaatatttgtggccctaaagagggccgttgggttcggtaaaatgttttaagaaggcatcta
<span style="color:#ff0000">TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">TCCTCCACTACCGCC</span>
<span style="color:#ff0000">TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">ACCTCCAGAGCCACC</span>
<span style="color:#ff0000">TCCTCCGGTGCAGTGACGGGCGACGGACTTCTCGTATTGCTCGACGACGGTGTA</span>
<span style="color:#0000ff">GGATCCTCCTCC</span>
ttc<u>T</u>ttgtctcctccggt<u>C</u>ttctt<u>T</u>ggcaaaagaacagcttggatatttggaagaactcctccttgg
</td>
</tr>
</table>
<h5>S4C. Primers used to PCR split-wrmScarlet tandems HDR donor sequence from plasmid</h5>
<table style="font-size:80%;line-height: 120%;">
<tr>
<th>Primer Name</th>
<th>Sequence of DNA oligo</th>
</tr>
<tr>
<td>his-3_F</td>
<td>CCAAGGAGGAGTTCTTCCAAATATC</td>
</tr>
<tr>
<td>his-3_R</td>
<td>GATTTTAAATATTTGTGGCCCTAAAG</td>
</tr>
<tr>
<td>vha-13_F</td>
<td>GGTTTATTTTGATTTTCTTTTCGATTTCC</td>
</tr>
<tr>
<td>vvha-13_R</td>
<td>CCATCTTCTCGGCTGTGAC</td>
</tr>
</table>
<h5>S4D. Sequences of crRNA and HDR template used to generate split-wrmScarlet<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> and sfGFP<sub style="font-size: 10px; line-height: 0; vertical-align: -5px;">1-10</sub> strains</h5>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 120%;">
<tr>
<th>Strain edited -> Final strain</th>
<th>crRNA#1 sequence</th>
<th>crRNA#2 sequence (if applicable)</th>
<th style="width:50%;">DNA template - Hybrid PCR amplicon (lower case: homology arms, upper case: insert)</th>
</tr>
<tr>
<td>CA1200 -> CF4582</td>
<td style="word-wrap: break-word">UACUUCUUCUGGAAACGACA</td>
<td style="word-wrap: break-word">AAGUUCGCUGGACUUGGAGG</td>
<td style="word-wrap: break-word">
tagaagtttctaggataattttttcgacttttattctctctaccgtccgcactcttcttacttttaaattaaattgtttttttttcagttgggaaacactttgctcactccgtagcagccATGGTATCGAAGGGAGAAGCAGTAATCAAGGAGTTCATGCGTTTCAAGGTCCACATGGAGGGATCCATGAACGGACACGAGTTCGAGATCGAGGGAGAGGGAGAGGGACGTCCATACGAGGGAACCCAAACCGCCAAGCTCAAGGTCACCAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGAGGACCACTCCCATTCTCCTGGGACATCCTCTCCCCACAATTCATGTACGGATCCCGTGCCTTCATCAAGCACCCAGCCGACATCCCAGACTACTACAAGCAATCCTTCCCAGAGGGATTCAAGTGGGAGCGTGTCATGAACTTCGAGGACGGAGGAGCCGTCACCGTCACCCAAGACACCTCCCTCGAGGACGGAACCCTCATCTACAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTCAAGCTCCGTGGAACCAACTTCCCACCAGACGGACCAGTCATGCAAAAGAAGACCATGGGATGGGAGGCCTCCACCGAGCGTCTCTACCCAGAGGACGGAGTCCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGAGACATCACCATGGCCCTCCGTCTCAAGGACGGAGGACGTTACCTCGCCGACACCTCCACCACCTACAAGGCCAAGAAGCCAGTCCAAATGCCAGGAGCCTACCTCGTCGACCGTAAGCTCGACATCACCTCCCACAACGAGTACTAAtaagtccaattactcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaacttctcttaatttctttgttttttagcttcttttaagtca
</td>
</tr>
<tr>
<td>COP1795 -> CF4587</td>
<td style="word-wrap: break-word">GACCAGCUGGGCGCAUAGGG</td>
<td style="word-wrap: break-word">GCCGCCCCACGAGGGCCAGG</td>
<td style="word-wrap: break-word">
aactcattttcaatttcaactgaaagattttttcattagagaatgtctagaactaggcccgggctacgtaatacgactcacttaaggcctaatttgggtctggctgcatgccaggaggtaGCACCTTTGGTCTTTTATTGTCAACTTCCATTGGTTCTTCCATTGTTTCTGTTAAATTAATGAATTTTTCATAAAATAAAGACATTATACAATATAAAAATGAAGAATTTATTGAAAATAAACTGCCAGAGAGAAAAAGTATGCAACACTCCCGCCGAGAGTGTTTGAAATGGTGTACGGTACATTTTCGTGCTAGGAGTTAGATGTGCAGGCAGCAACGAGAGGGGGAGAGATTTTTTTGGGCCTTGTGAAATTAACGTGAGTTTTCTGGTCATCTGACTAATCATGTTGGTTTTTTGTTGGTTTATTTTGTTTTTATCTTTGTTTTTATCCAGATTAGGAAATTTAAATTTTATGAATTTATAATGAGGTCAAACATTCAGTCCCAGCGTTTTTCCTGTTCTCACTGTTTAGTCGAATTTTTATTTTAGGCTTTCAACAAATGTTCTAACTGTCTTATTTGTGACCTCACTTTTTATATTTTTTTAATTTTTAAAAATATTAGAAGTTTCTAGGATAATTTTTTCGACTTTTATTCTCTCTACCGTCCGCACTCTTCTTACTTTTAAATTAAATTGTTTTTTTTTCAGTTGGGAAACACTTTGCTCaaaaatgtctaagggagaagagttatttactggagttgtgccgatcctcgtcgagctcgacggagacgtcaacggacacaagttctccgtccgtggagagggagagggagacgccaccatcg
</td>
</tr>
<tr>
<td>WBM1126 -> CF4610</td>
<td style="word-wrap: break-word">GCUACCAUAGGCACCACGAG</td>
<td></td>
<td style="word-wrap: break-word">
cacttttaccgtctaattttcagggcagggagccatcaaacccacgaccactagatccatATGGTATCGAAGGGAGAAGCAGTAATCAAGGAGTTCATGCGTTTCAAGGTCCACATGGAGGGATCCATGAACGGACACGAGTTCGAGATCGAGGGAGAGGGAGAGGGACGTCCATACGAGGGAACCCAAACCGCCAAGCTCAAGGTCACCAAGgtaagtttaaacatatatatactaactaaccctgattatttaaattttcagGGAGGACCACTCCCATTCTCCTGGGACATCCTCTCCCCACAATTCATGTACGGATCCCGTGCCTTCATCAAGCACCCAGCCGACATCCCAGACTACTACAAGCAATCCTTCCCAGAGGGATTCAAGTGGGAGCGTGTCATGAACTTCGAGGACGGAGGAGCCGTCACCGTCACCCAAGACACCTCCCTCGAGGACGGAACCCTCATCTACAAGgtaagtttaaacagttcggtactaactaaccatacatatttaaattttcagGTCAAGCTCCGTGGAACCAACTTCCCACCAGACGGACCAGTCATGCAAAAGAAGACCATGGGATGGGAGGCCTCCACCGAGCGTCTCTACCCAGAGGACGGAGTCCTCAAGgtaagtttaaacatgattttactaactaactaatctgatttaaattttcagGGAGACATCACCATGGCCCTCCGTCTCAAGGACGGAGGACGTTACCTCGCCGACACCTCCACCACCTACAAGGCCAAGAAGCCAGTCCAAATGCCAGGAGCCTACCTCGTCGACCGTAAGCTCGACATCACCTCCCACAACGAGTACTAAcatctcgcgcccgtgcctctgacttctaagtccaattactcttcaacatccctacatgct
</td>
</tr>
</table>
<h5>S4E. Primers long and short </h5>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 180%;">
<tr>
<th>Primers to mplify split-wrmScarlet for CF4582</th>
<th>Sequence</th>
</tr>
<tr>
<td>eft3p_S110(A19)_F</td>
<td style="word-wrap: break-word">tagaagtttctaggataattttttcgacttttattctctctaccgtccgcactcttcttacttttaaattaaattgtttttttttcagttgggaaacactttgctcactccgtagcagccATGGTATCGAAGGGAGAAGC
</td>
</tr>
<tr>
<td>unc54_S110(A19)_R</td>
<td style="word-wrap: break-word">tgacttaaaagaagctaaaaaacaaagaaattaagagaagttttttgataataacaaaaatagggggtgggagcacagggagaaagagcatgtagggatgttgaagagtaattggacTTATTAGTACTCGTTGTGGGAGG </td>
</tr>
<tr>
<td>S1-10_A19_F</td>
<td style="word-wrap: break-word">ATGGTATCGAAGGGAGAAGC </td>
</tr>
<tr>
<td>S1-10_A19_R</td>
<td style="word-wrap: break-word">TTAGTACTCGTTGTGGGAGGTG </td>
</tr>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 180%;">
<tr>
<th>Primers to amplify Peft-3 for CF4587</th>
<th>Sequence</th>
</tr>
<tr>
<td>E7_eft-3p_F-Long</td>
<td style="word-wrap: break-word">aactcattttcaatttcaactgaaagattttttcattagagaatgtctagaactaggcccgggctacgtaatacgactcacttaaggcctaatttgggtctggctgcatgccaggaggtaGCACCTTTGGTCTTTTATTGTCAAC
</td>
</tr>
<tr>
<td>E8_eft-3p_R-Long</td>
<td style="word-wrap: break-word">cgatggtggcgtctccctctccctctccacggacggagaacttgtgtccgttgacgtctccgtcgagctcgacgaggatcggcacaactccagtaaataactcttctcccttagacatttttGAGCAAAGTGTTTCCCAACTG
</td>
</tr>
<tr>
<td>E5_eft-3p_F</td>
<td style="word-wrap: break-word">GCACCTTTGGTCTTTTATTGTCAAC </td>
</tr>
<tr>
<td>E6_eft-3p_R</td>
<td style="word-wrap: break-word">GAGCAAAGTGTTTCCCAACTG </td>
</tr>
<table style="table-layout: fixed; width: 100%;font-size:80%;line-height: 180%;">
<tr>
<th>Primers to amplify split-wrmScarlet for CF4610</th>
<th>Sequence</th>
</tr>
<tr>
<td>PrimerS1-10_myo3F</td>
<td style="word-wrap: break-word">cacttttaccgtctaattttcagggcagggagccatcaaacccacgaccactagatccatATGGTATCGAAGGGAGAAGC </td>
</tr>