The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors  were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs ( OCT4, SOX2, KLF4, cMYC, LIN28 and NANOGHuman embryonic stem cells (hESCs) have significant therapeutic potential for various diseases, but the generation of these cells from individual patients raises ethical concerns. Recently, a technological breakthrough where somatic cells from mouse and human can be reprogrammed into hESC-like pluripotent cells, termed induced pluripotent stem cells (iPSCs), was made possible through ectopic expression of combinations of reprogramming factors including MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression post-transcriptionally . The miR-302 gene encodes a cluster of eight miRNAs on chromosome 4 (miR-302b*-b-c*-c-a*-a-d-367) that are preferentially expressed in embryonal carcinoma cells, hESCs and hiPSCs miR-223, miR-155, and miR-142-3p are enriched in differentiated cells miR-223 and miR-142-3p are found in cells of primarily hematopoietic origin miR-155 is found in hematopoietic cells as well as in many types of lymphoma and solid cancers miR-155 is also expressed 20–50 fold higher in fibroblasts than in hESCs and hiPSCs We constructed a bidirectional vector whereby the reporter gene mCherry is conjugated with perfectly complementary miRNA target sites for miR-302a, miR-302b, miR-302c, and miR-302d or miR-155 results in ablation of EGFP or mCherry expression, respectively, demonstrating sensitivity of the vector to specific miRNAs . In hematopoietic lineage cells  mCherry expression is entirely ablated whereas EGFP is maintained . After a prolonged period of culture on feeder cells, a fraction (<0.01%) of the cells reprograms and appears as hiPSC colonies The reprogramming of HFFs to hiPSCs can be achieved by the introduction of four transcription factors . Expression levels of EGFP and mCherry were monitored over the four week course of reprogramming to assess the activity of the reporter vector during generation of hiPSC  and one hiPSC clone that was not transduced with this reporter vector (hiPSC #19) for characteristics of pluripotent stem cells. These hiPSC clones transduced with the reporter vector stably maintained mCherry expression and lack of EGFP expression for over 20 generations . Althougin vitro into EBs comprising the three embryonic germ layers miR-302a/d expression as the cells differentiated. Concomitantly, mCherry expression was slightly reduced in the cells, reflecting expression of one or more of the differentiation specific miRNAs miR-223, miR-155, or miR-142-3p. However, since the EBs represent multiple lineages of differentiated cells, we were unable to conclude which cells and to what extent these miRNAs are being expressed.hESC as well as hiPSC can be differentiated SOX1, SOX3 and PAX6, markers of neuroectodermal differentiation DNMT3B, REX1 and endogenous OCT4 which are assumed to be expressed in fully-reprogrammed hiPSCs DNMT3B, REX1 and endogenous OCT4 were downregulated, whereas SOX1, SOX3 and PAX6 were upregulated in the differentiated population compared to the undifferentiated population  had reduced mCherry expression compared to two other hiPSC clones transduced with the reporter vector . AlthougThe development of this vector adds to the tools available to monitor hiPSC generation and subsequent differentiation of the hiPSC into different lineages. In addition to taking advantage of differential miRNA expression, other investigators have utilized differential promoters/enhancer expression in different lineages miR-223, miR-142-3p, and miR-155 are enriched primarily in cells of hematopoietic origin The work presented here indicates the potential great utility and flexibility of miRNA-regulatable lentiviral vectors to monitor various stages of reprogramming and the subsequent differentiation into lineage specific cells and tissues. For example, CCCGGGCGAGCAAGCTCAGTTTACACCGAATTCGGATCCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAAGTCTCACCAAAACATGGAAGCACTTAGGCCTACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGTACACACTCAAACATGGAAGCACTTAGATATCGTCGAC) and differentiated cell specific-miRNA targets (CCCGGGTCGAATTCGGTACCAGATCTGGCGCGCCGTACGTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAAGTCTGGGGTATTTGACAAACTGACAGGCCTACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAAAGTCACCCCTATCACGATTAGCATTAATTTAAATTCCATAAAGTAGGAAACACTACAGTACTCCATAAAGTAGGAAACACTACAGTACAGTTCCATAAAGTAGGAAACACTACAGTACTCCATAAAGTAGGAAACACTACAGATATCTGCATGCTTCGAAGCTAGCGGGCCC) were synthesized by Genescript . The synthesized fragment of differentiated cell specific-miRNA targets was conjugated to mCherry coding sequence amplified by PCR from pmCherry  with primers (sense: ACGCACCGGTGGATCCAAGCTTGCCACCATGGTGAGCAAGGGCGAGGA and reverse: CTGCGAATTCTCACTACTTGTACAGCTCGTCCATGCCGCCGG) and exchanged with EGFP coding sequence in FG12 lentiviral vector AGTCAGCTAGCGCCACCATGGTGAGCAAGGGCGAG and reverse: CATCGACCCGGGAATTCTCATTACTTGTACAGCTCGTCCATG), and cloned into a pAAV-MCS vector  (pAAV-EGFP). The synthesized fragment of hESC specific-miRNA targets was inserted into the pAAV-EGFP between stop codon of EGFP and hGH poly A signal. The fragment containing CMV minimal promoter, β-globin intron, EGFP, hESC specific-miRNA targets, and hGH poly A signal was amplified by PCR with primers (sense: GTACTCTCGAGCCCCATTGACGCAAATGGGCGGTAGG and reverse: CTCGTCTAGAAGGACAGGGAAGGGAGCAGTGGT) and cloned into the FG12 lentiviral vector deleted EGFP (EGFP-T/FG12) or into the mCherry-T/FG12 (EGFP-T/mCherry-T/FG12).For construction of the reporter vector, whole sequences of hESC specific-miRNA targets  were substituted with EGFP coding sequence in the FRh11. The infectious titer was determined in 293T cells by infecting with the FRh11 encoding EGFP in the presence of 8 µg/ml polybrene. Reporter gene expression was monitored by flow cytometry.For reprogramming of HFFs, we substituted the ubiqutin C promoter of FG12 lentiviral vector with the RhMLV promoter (FRh11). The RhMLV promoter is derived from the long terminal repeat (LTR) region of Moloney murine leukemia virus (MLV) in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation miR-302a, miR-302b, miR-302c, and miR302-d (PMIRH302abcdPA-2) and miR-155 (PMIRH155PA-1) from System biosciences . CopGFP sequence was eliminated from the vector.For ectopic expression of miRNAs, we purchased miRNA expressing lentiviral vectors for 2.293T hESCs (H1 clone) HFFs were isolated from the skin of 16 week-old fetus with DMEM supplemented10% FBS and 2 mM Glutamax (fibroblast medium) as reported previously CD34+ HPSCs were prepared from the liver of 16 weeks-old fetus as previously described Lentiviral vector stocks were generated using a vector plasmid, a packaging plasmid pCMV R8.2 ΔVpr, and a VSV-G envelope protein-coding plasmid by calcium phosphate-mediated transient transfection as previously described 4 cells per well of 6-well plates and infected with vectors encoding each reprogramming factor  with or without a lentiviral vector encoding EGFP-T/mCherry-T at 300 ng (around multiplicity of infection of 3–5) of p24 per each virus. The cells were cultured for 3 days in fibroblast medium and replated at 5×104 cells per 60 mm dish on irradiated mouse embryonic fibroblast (iMEF) feeder cells. On the next day, the medium was replaced with KO-DMEM (Invitrogen) supplemented with 20% Knockout Serum Replacer , 2 mM Glutamax (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol , and 50 ng/ml of recombinant human basic fibroblast growth factor (Invitrogen) (hiPSC medium). The medium was changed on a daily basis. To increase a reprogramming efficiency, the cells were treated with 0.5 mM valproic acid  and 10 µM Y27632  for first 14 days The day before lentiviral vector transduction, HFFs (passage 1–3) were seeded at 5×10Size-controlled EBs (3000 hESCs/EB) were formed using AggreWell™ 400 plates  following the manufacturer's protocol. Briefly, hESCs and hiPSCs were incubated with 10 µM Y-27632 for 24 hrs before EB formation. Cells were harvested with Accutase  as a single-cell suspension and used for EB formation. EBs were harvested into ultra low attachment plates  and maintained in Iscove's Modified Dulbecco's Medium  containing 10% FBS, 2 mM Glutamax, and 0.1 mM β-mercaptoethanol for differentiation into EBs. The medium was changed every 3 days.Induction of differentiation into neural lineages was performed as previously described with some modifications For detection of EGFP and mCherry expression, single-cell suspensions from 293T, HFF, hESC, and hiPSC were prepared using 0.25% trypsin-EDTA and collected in FACS buffer (2% FBS and 0.01% sodium azide in PBS). U937, Ramos and CEM cells were also collected in FACS buffer. For detection of hESC-specific markers, hESCs and hiPSCs were dissociated with 0.25% trypsin-EDTA into a single cell suspension. Cells were adjusted to 100,000 per sample in 100 µl of FACS buffer and then labeled with monoclonal antibodies conjugated with a fluorescent dye . For detection of iMEFs, cells were stained with an antibody specific with mouse CD29 conjugated with PE-Cy7 (eBioscience). Data were collected on a Cytomics FC500  and analyzed using FCS express .hESC and hiPSC colonies were grown on poly-L-lysine and Matrigel coated glass coverslips. Cells were fixed with 1.0% formaldehyde/PBS and permeabilized with 0.2% Triton-X 100 for 5 min on ice. Cells were then incubated with anti-human Nanog antibody  and subsequently with DyLight488 conjugated donkey anti-rabbit IgG (BioLegend) and 7-amino-actinomycin D (7-AAD) (Invitrogen) for nuclear staining. After washing, cells were visualized with a LEICA DM IRB  equipped with a SPOT camera and software .RNA extraction from hESC and hiPSC was performed using QIAGEN's RNeasy Mini kit following the manufacture's protocol . Total RNA (250 ng) was reverse-transcribed using QIAGEN's Omniscript RT-kit with a 0.5 ng/ml oligo dT primer (Invitrogen) in 20 µl reaction. PCR was performed with the HotMaster Taq DNA polymerase , using 0.5 µl of cDNA template and primers at a concentration of 3 pmol/µl. Five µl of PCR products was loaded in a 2% agarose gel containing ethidium bromide. All primer sequences were listed in Table 1.


Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of  Drosophila melanogaster centromeres, which have long remained elusive despite the high quality of this species’ genome. assembly.Long-read sequencing, CENP-A ChIP, and chromatin fiber imaging reveal the composition and organization of  Drosophila and centromeric histone H3 [CenH3] in plants), which is necessary and sufficient for kinetochore activity  × (1 pmol/330 pg) × (1/Oligopaint length in nt) = Oligopaint molarity [μM]G2/Jockey-3 oligo against the consensus of the 3′ region of G2/Jockey-3 elements found within most centromere contigs. A 5′ addition containing 5′-CAGT-3′ followed by universal forward primer binding sites separated by an XhoI cut site (5 ′-cacactccggtacgcacctgctcgagcagtgctcgttggcccacac-3′). A 3′ addition containing 5′-ACTG-3′ followed by universal reverse primer binding sites separated by a SpeI cut site (5′-agggtagtcgttgtagctcgactagtggtacgcccagaagcatccc-3′). The G2/Jockey-3 sequence was ordered as a “custom gene” (IDTDNA.com) and synthesized in the pUCIDT (AMP) vector (pUCIDT-G2). The sequence of the insert is as follows .We designed a 1,643-bp cacactccggtacgcacctgctcgagcagtgctcgttggcccacacCGGACGGCTCTTGGTGCCGCTCTGAAGCCGAAAGAGCTGAAGCGTTTGCAGATCACCTCCAGAATGCATTCACACCATTTGACAGATGCACTGGCGAAGAGCGTGCTGCAACCACCAGGTTCCTAGAGAGTCCATGTCCTCCTAGCCTGCCCATAGAGCCCGTCACCCCAGAAGAGGTTGCGCAAGAGTCGCCTCACTAAAGGCTAGCAAATCCCCAGGACTGGATCGCATCGACGCCACATCCCTTAAAATGCTGCCACCTCCCTGTTCCCAGTTGCTGGCCAACATATACAACAGATGCTTCTCACTAGGGTACTTCCCGAGATCATGGAAACGTGCAGAAGTCATTCTCATCCTCAAACCTGGAAAACCTGAAGCCAATCTTGCCTCATATAGACCGATTAGTCTGCTGGCAATCCTCTCCAAAATACTCGAAAGAGTATTTCTGCGCAGAGTGTTGCCAGTACTGGACGAGGCTGGACTGATCCCTGATCACCAGTTTGGCTTCAGGCGATCCCACGGAACACCCGAGCAATGCCACCGGCTCGTAGCACGCATCCTAGATGCATTCGAGAACAAACGATACTGTTCGGCCGTATTCCTGGATGTCAAGCAGGCGTTCGACAGAGTGTGGCATCCTGGACTCCTCTACAAACTCAAGTCCCACCTTCCCAGTTCCCACTATGCCCTACTCAAATCGTATACTGAAGGAAGAGAGTTCCAAGTGCGATGCGGTTCCTCAACCAGCACGACAAGGCCTATACGAGCCGGAGTACCTCAAGGCAGCGTCCTTGGTCCCATCCTCTACACCCTGTTTACAGCAGACCTCCCTATCATACCCTCCCGTTACCTCACAGCAGCCACCTATGCAGATGACACGGCGTTCCTTGCCACCGCAACAAACCCTCAACTAGCATCAGCCATCATCCAGAGGCAACTGGATGCATTGGATCCATGGCTGAAACGCTGGAACATCGTGATCAACGCTGATAAATCCTCCCACACCACCTTCTCTCTGCGCAGAGGAGAATGCCCCCCGGTCTCACTCGACGGCGACACAATCCCTACCTCCAGCACCCCCAAATATTTAGGGCTGACCCTGGACAGAAGGCTGACTTGGGGCCCCCACATCAACAGAAAGCGTATCCAGGCCAACATACGCCTAAAGCAACTCCACTGGCTCATCGGTAAAAAGTCCAAGCTGCGAGAGAAACTAAAGATTCTCGTCTACAAGACTATTCTCAAGCCAATCTGGACGTACGGAATTCAGCTGTGGGGCACTGCAAGCACATCACATAGAAGGAAGATCCAGCGATTTCAAAACAGATGTTTGAGAATAGTCTCCAACGCCCATCCCTACCACGAAAATTCCGCCATCCACGAGGAGCTCGGGATTCCATGGGTAGACGACGAAATCTACAGACACAGTGTGAGATATGCTAGCAGACTGGAGAACCACCACAACCACCTGGCCGTCAACCTTCTAGACCATAGCCAATCCCTAAGACGCCTGCAGAGAACGCACCCGCTTGACCTTACTCAACATACTTAATCATACTTAACCCCTACCCAAGTACACTCGATGTACTCCCCTTAAGTTAATGTTTCCCTCCAAAAAATTTAATTATTGTCCACTAGGACAGgggatgcttctgggcgtaccactagtcgagctacaacgactaccctcagt-3′5′-cagtG2 (500 ng) was digested using SpeI and XhoI restriction enzymes in 1x Cutsmart Buffer for 1 h at 37°C. The digest was run on a 1.0% SeaPlaque GTG agarose gel (Lonza), and a 1,689-bp band containing the G2 sequence was gel extracted and purified using the PureLink Quick Gel Extraction Kit (Invitrogen). DIG-labeled G2 probes were generated via PCR in 50-μl reactions consisting 0.09 ng of gel extracted G2 DNA, 0.5 μM of forward and reverse primers from the Universal_2 primer set  to provide additional support for our candidate centromeric contigs . We mappnogaster  to our anogaster  and the We calculated the significance between different categories using a Kruskal-Wallis test by ranks with Dunn’s test for post hoc analysis and the pairwise Wilcoxon rank sum test with false discovery rate (FDR) correction  of type G2/Jockey-3 sequences based on RepeatMasker annotations and custom scripts. We aligned and manually inspected G2/Jockey-3 and IGS alignments using Geneious v8.1.6 [https://doi.org/10.5061/dryad.rb1bt3j [G2/Jockey-3 and IGS using RAxML v.8.2.11 with settings “-m GTRGAMMA -T24 -d -p 12345 -# autoMRE -k -x 12345 -f a” [We extracted all IGS elements from the genome using BLAST v2.7.1  with sets v8.1.6  (see Dry.rb1bt3j ). We con45 -f a” . We used45 -f a”  to plot G2/Jockey-3 non-LTR retroelements have evidence for recent activity based on insertion polymorphism and expression. We examined RNA-seq reads from testes for evidence of G2/Jockey-3 because of the enrichment of these elements on the Y chromosome. We mapped poly-A [We investigated whether d poly-A  and totad poly-A  (S6 Tabld poly-A  and estid poly-A ).S1 FigPlot of normalized CENP-A/input for simple tandem repeats for each ChIP-seq replicate, sorted by median (red lines). Shown are only the simple tandem repeats with median CENP-A/input > 1 in all four CENP-A ChIP replicates (see details in (TIF)Click here for additional data file.S2 FigG2 and Jockey-3 sequences in D. melanogaster genome and closely related species in the simulans clade (D. simulans and D. sechellia) and D. yakuba. In D. melanogaster, G2 and Jockey-3 are interleaved across the phylogeny and thus likely correspond to the same repeat type. We therefore refer to these elements collectively as G2/Jockey-3 throughout the manuscript. (See Dryad repository files 13 and 15: https://doi.org/10.5061/dryad.rb1bt3j [A maximum-likelihood phylogenetic tree showing the relationship between .rb1bt3j ). LTR, l(TIF)Click here for additional data file.S3 FigLocations of the top 100 strongest peaks for each ChIP experiment. (A) Plot of the location of top 100 strongest peaks for each ChIP experiment on the diagonal (see details in (TIF)Click here for additional data file.S4 FigJockey elements are shown in one color even though they are distinct elements). The normalized CENP-A enrichment over input  is shown for three replicates (replicate 2 is in Organization of each CENP-A-enriched island corresponding to centromere candidates: (A) X centromere, (B) centromere 4; (C) Y centromere; (D) centromere 3; (E) centromere 2. Different repeat families are color coded Click here for additional data file.S5 FigRpL32 promoter region as a noncentromeric control. (C) Graph showing our ChIP-qPCR results using primers targeting other regions that showed CENP-A enrichment but that were not in our contigs. Again, the enrichment is calculated relative to the input and is normalized by RpL32 promoter as a noncentromeric control. We did not observe a robust CENP-A enrichment at these sites. The underlying data can be found in (A) Diagram showing putative centromere contigs showing the locations of CENP-A ChIPtigs in black and CENP-A MACS peaks in orange as in (TIF)Click here for additional data file.S6 FigPacBio reads were mapped to the genome using Minimap (v 2.11) and the setting “-ax map-pb.” Shown are (A) X centromere, (B) centromere 4, (C) Y centromere, (D) centromere 3, and (E) centromere 2. The depth of only the high-quality mapped reads (mapped Q ≥ 30) was estimated for each position and normalized by the median depth of other genomic regions (98.32× for autosomes and 49.16× for sex chromosomes) to get relative depth. The relative depths of the TE-rich islands are close to 1, whereas the depth of the flanking simple satellites is uneven, with some regions > 1 and some < 1. We therefore exclude simple repeats from any assembly-based analyses and color these regions gray in (TIF)Click here for additional data file.S7 FigProdsat (magenta) and AAGAG (blue); (C) AATAG (magenta) and AAGAG (blue) with AATAG blocks identified by white  and yellow (large block) arrows; (D) Prodsat (magenta) and dodeca (blue); (E) AATAT (magenta) and SATIII (blue); (F) AATAT (magenta) and AAGAG (blue); (G) AATAG (magenta) and Prodsat (blue) with AATAG blocks identified by white  and yellow (large block) arrows. DAPI is shown in gray. The underlying data can be found in Prodsat, Prod satellite.IF-FISH using an anti-CENP-C antibody (green) and satellite FISH probes in the following combinations: (A) AAGAT (magenta) and AAGAG (blue) with a high-contrast inset of AAGAT on the X chromosome; (B) (TIF)Click here for additional data file.S8 FigSATIII (blue); (B) dodeca (magenta) and Prodsat (blue); (C) AATAG (magenta) and Prodsat (blue) with a high-contrast inset of AATAG and Prodsat on cf(2R); (D) AAGAG (magenta) and Prodsat (blue); (E) AAGAG (magenta) and AATAG (blue) with a high-contrast inset of AATAG on chromosome 3; (F) AAGAT (magenta) and AAGAG (blue). DAPI is shown in gray. See also Prodsat, Prod satellite; S2, Schneider 2.IF-FISH using an anti-CENP-A antibody (green) and satellite FISH probes in the following combinations: (A) AATAT (magenta) and (TIF)Click here for additional data file.S9 FigG2/Jockey-3 consensus element obtained from mapping total and poly-A RNA-seq data from testes [G2/Jockey-3 copies surveyed on centromere (“Cen”) X, 4, and 3 but not Y and 2 show low levels of transcription compared to the housekeeping gene Actin. Although the primers (G2/Jockey-3 copies not included in our assembly. Error bars = SD. The underlying data for this figure can be found in (A) Shown is the plot of the normalized reads depth from uniquely mapped reads  across the m testes , 122 to  primers  are spec(TIF)Click here for additional data file.S10 FigD. melanogaster genome with related outgroups  are similar to the IGS at 3Giglio but are on small contigs, tig00022795 and id = 102159_0. Contig tig00022795 is also moderately enriched in CENP-A. CENP-A, centromere protein A; IGS, intergenic spacer of the ribosomal genes.Maximum-likelihood phylogenetic tree of all individual IGS sequences found in the .rb1bt3j ). Node s.rb1bt3j ). All ce(TIF)Click here for additional data file.S11 FigPROTOP). PROTOP are DNA transposons that have not been recently active, and their distribution is primarily in heterochromatin. TART elements are non-LTR retroelements highly enriched at telomeres and are also moderately CENP-A enriched. DM1731 is a retroelement moderately enriched for CENP-A but not enriched in the centromere islands. Doc2, G, Jockey-1, and G2/Jockey-3 are CENP-A enriched non-LTR retroelements abundant in the centromere islands  along the following chromosomes: (A) chromosome 2, (B) chromosome 3, (C) chromosome 4, (D) chromosome X, and (E) chromosome Y. Contigs from each chromosome were concatenated in order with an arbitrary insertion of 100 kb of “N.” Distances along the x-axis are approximate. The order and orientation of the Y chromosome contigs are based on gene order (see ). Each tands see  Tables. (TIF)Click here for additional data file.S12 FigMaupiti  and AAGAG (blue); (B) Lampedusa  and AAGAT (blue); (C) Lipari  and AATAT (blue); (D) Giglio  and dodeca (blue). White boxes show the separate signals at the targeted centromeres. Yellow boxes show centromeric hybridizations at other centromeres. DAPI is shown in gray. Bar 5 μm. The underlying data for this figure can be found in IF-FISH using an antibody for CENP-C (green), centromere Oligopaint FISH probes (magenta), and FISH probes for centromeric satellites (blue) in the following combinations: (A) (TIF)Click here for additional data file.S13 FigMaupiti (X), (B) Lampedusa (4), (C) Lipari (Y), (D) Giglio (3). The “Signal Adjusted” panels in (A) and (B) show high-contrast Oligopaint hybridization for visualization of weak foci. Bar 5 μm. See also IF-FISH using an antibody for CENP-A (green) and centromere Oligopaint FISH probes designed to target centromere contigs (magenta). (A) (TIF)Click here for additional data file.S14 FigP < 0.0001; *adjusted P < 0.02, pairwise Wilcoxon rank sum test with FDR correction; Kruskal-Wallis test by ranks with Dunn’s test for post hoc analysis. The underlying data for this figure can be found in Plots showing intra- and interchromosomal interactions between regions in Hi-C data from: (A) stage 16 embryos (end of embryogenesis) and (B) embryonic cycles 1–8 . The di(TIF)Click here for additional data file.S15 FigRsp locus ; (B) 100-kb Oligopaint for a heterochromatic region on chromosome 3L ; (C) 100-kb Oligopaint for a euchromatic region approximately 600 kb from the telomere of chromosome 3L . Arrows show the region of the fiber that was measured. Bar 5 μm. (D) Scatterplot showing the quantification of fiber lengths. Mean lengths were used to estimate the size in kb (approximately 10 kb/1 μm). Error bars show the standard deviation. P = 0.085 (n.s.) for each pair of measurements compared (two-tailed t test). The underlying data can be found in Rsp, Responder.Stretched chromatin fibers from female third instar larval brain cells using the following probes: (A) (TIF)Click here for additional data file.S16 FigMaupiti (magenta), and AAGAG probe (cyan) on female third instar larval brain cells. DAPI is shown in gray. CENP-A occupies Maupiti and the AAGAG satellite. We observed some variation in FISH signals and Maupiti and CENP-A domain lengths, likely because of the efficiency of Oligopaint binding and variable stretching in this region. Arrows show the region of the fiber that was measured. (H) Scatterplot showing the quantification of the length of Maupiti FISH and CENP-A IF signals. Error bars show the standard deviation. N = 24 fibers. Bar 5 μm. The underlying data for this figure can be found in (A-G) Examples of fibers visualized with IF with anti-CENP-A antibody (green), FISH with Oligopaints for (TIF)Click here for additional data file.S17 FigLampedusa (magenta), and AAGAT probe (cyan). DAPI is shown in gray. CENP-A occupies predominantly the island Lampedusa. Arrows show the region of the fiber that was measured. (H) Scatterplot showing the quantification of the length of Lampedusa FISH and CENP-A IF signals. Error bars show the standard deviation. N = 25 fibers. Bar 5 μm. The underlying data for this figure can be found in (A-G) Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH Oligopaint FISH for (TIF)Click here for additional data file.S18 FigLipari (magenta). DAPI is shown in gray. We did not include satellite FISH because no centromeric satellites are known for the Y. Note that the Oligopaints only target part of Lipari  Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH with Oligopaints for pari see . CENP-A (TIF)Click here for additional data file.S19 FigGiglio (magenta), and a probe for the centromere 3–specific dodeca satellite (cyan). DAPI is shown in gray. CENP-A occupies primarily Giglio and a small stretch of dodeca satellite. Note that the binding of the dodeca (an LNA probe) is quite variable between fibers and results in several gaps that could be a result of the higher stringency conditions needed for Giglio Oligopaint FISH. Arrows show the region of the fiber that was measured. (F) Scatterplot showing the quantification of the length of Giglio FISH and CENP-A IF signals. Error bars show the standard deviation. N = 30 fibers. Bar 5 μm. The underlying data can be found in (A-E) Examples of fibers visualized by IF with anti-CENP-A antibody (green), FISH with Oligopaints for (TIF)Click here for additional data file.S20 Figdodeca from the experiment in Giglio (magenta), and FISH with dodeca probe (cyan). DAPI is shown in gray. Note the presence of Giglio signal on the dodeca CENP-A region. Multiple, overlapping panels were often acquired to follow an individual fiber. Panels were then cropped and juxtaposed in the figure, with white lines showing the separate images. White boxes show the CENP-A domain on Giglio, and yellow boxes show the smaller domain on dodeca. N = 5 (these are rare fibers to find in our preparations because of their length). Bar 5 μm. CENP-A, centromere protein A; FISH, fluorescence in situ hybridization; IF, immunofluorescence.(A-D) Examples of longer fibers tracked along (TIF)Click here for additional data file.S21 FigProdsat (magenta) and AATAG (cyan). (C-D) Examples of fibers with AAGAG (cyan) and Prodsat (magenta). (E) Example of fiber with AAGAG (magenta) and AATAG (cyan). We propose that Capri is located between flanking blocks of AAGAG and AATAG satellites that reside very close to where the Prodsat begins. Arrows show the region that was measured for each fiber. (F) Scatterplot of CENP-A IF signal lengths. (G) Model for the organization of centromere 2 showing a possible location of Capri. Error bars show the standard deviation. N = 18 fibers. Bar 5 μm. The underlying data can be found in Prodsat, Prod satellite.(A-D) Examples of fibers visualized with IF with anti-CENP-A antibody (green) and FISH with satellites. DAPI is shown in gray. (A-B) Examples of fibers showing colocalization of CENP-A (green) with (TIF)Click here for additional data file.S1 TableWe used kseek  to estim(XLSX)Click here for additional data file.S2 TableRows correspond to complex repeat families (TEs and complex satellites), with the counts per family in the ChIP and input reads from every dataset. We calculated enrichment for each repeat type by normalizing by total mapped reads for each dataset and taking the ratio of normalized values for each ChIP and its corresponding input. ChIP, chromatin immunoprecipitation; TE, transposable element.(XLSX)Click here for additional data file.S3 TableWe mapped all ChIP-seq data to the de novo assembled ChIPtigs and called peaks using MACS with high-quality reads . We also mapped ChIPtigs to the genome to determine its genomic location and assigned repeat IDs based on BLAST results.(XLSX)Click here for additional data file.S4 TableWe mapped the ChIP and input reads to our genome assembly and used the high-quality reads  to call ChIP peaks with MACS. We show the peak locations for each dataset. ChIP, chromatin immunoprecipitation.(XLSX)Click here for additional data file.S5 TableWe used IDR to compare MACS peaks from different ChIP-seq replicates. We show the statistics for shared peaks from each comparison. ChIP-seq, chromatin immunoprecipitation sequencing; IDR, irreproducible discovery rate; OreR, Oregon-R.(XLSX)Click here for additional data file.S6 TableWe list reads and mapping summaries of all Illumina and long-read datasets generated in this paper or downloaded from NCBI’s SRA.(XLSX)Click here for additional data file.S7 TableList of primers used for qPCR in this study. The centromere contig that each target is associated with  is designated in the “Centromere” column. Note that in silico PCR for the 3_G2 primers predicted three specific products from centromere 3 as well as two products on contig tig00022795 and additional nonspecific products from the X chromosome when three or more mismatches are allowed all of the same 145-bp size. qPCR, quantitative PCR.(XLSX)Click here for additional data file.S8 TableG2/Jockey-3 outside of the centromeric islands. ChIP, chromatin immunoprecipitation; IDR, irreproducible discovery rate; OreR, Oregon-R.We listed peaks outside canonical centromeres with any agreement between replicate ChIP experiments (IDR ≤ 0.05). We also report any genes or repeat annotations that overlap the MACS peaks. Note that there is no general enrichment in (XLSX)Click here for additional data file.S9 TableP values. G2/Jockey-3, G, Doc2, and Jockey-1 are significantly enriched in centromeres relative to other heterochromatic regions and to the whole genome. Asterisk signs show that TART and ProtoP are significantly underrepresented in centromeres relative to other heterochromatic regions. FDR, false discovery rate; FISH, fluorescence in situ hybridization; TART, Telomere-associated retrotransposon; TE, transposable element.We show the copy numbers of TEs in different genomic regions. The sums of base pairs in the assembly size in centromeres , pericentromeric heterochromatin , and other regions  were used to compute the distribution statistics of TEs. We created a 2-by-2 contingency table for each TE comparing observed to expected (based on the sum of bp) for each comparison: centromere to heterochromatin (“cen-het”) regions or centromeres to whole genome (“cen-genome”). We computed a Fisher’s exact test with FDR correction to get adjusted (XLSX)Click here for additional data file.S10 TableHybridization conditions used for FISH with specific Oligopaints. FISH, fluorescence in situ hybridization.(XLSX)Click here for additional data file.S11 TableInformation on the fluors used and sequences of satellite FISH probes used in this report. * = “+N” designates the incorporation of an LNA. FISH, fluorescence in situ hybridization; LNA, locked nucleic acid.(XLSX)Click here for additional data file.S12 TableInformation on the 5′ secondary oligo adapter site and sequence of satellite probes used in this report.(XLSX)Click here for additional data file.S13 TableSequence and fluors of secondary oligo probes used for fluorescence detection of Oligopaints and unlabeled satellite probes.(XLSX)Click here for additional data file.S14 TableList of primer sets used for library amplification and G2 probe synthesis.(XLSX)Click here for additional data file.S15 TableList of primer sets used for sublibrary amplification and Oligopaint synthesis.(XLSX)Click here for additional data file.S16 TableWe assigned contigs from the assembly to a chromosome and a chromatin status Click here for additional data file.S17 TableG2/Jockey-3. CENP-A, centromere protein A; IDR, irreproducible discovery rate; S2, Schneider 2.We compared the MACS peaks shared between “normal” S2 (this study) and S2 with CENP-A overexpression using the IDR test. Some noncentromeric regions should have more CENP-A enrichment after CENP-A overexpression; however, only four peaks have IDR ≤ 0.05. None of these peaks have (XLSX)Click here for additional data file.S18 TablePercentage of probe signals that overlap with different cytological locations  in S2 cells. The underlying data can be found in (XLSX)Click here for additional data file.S19 Tables, using an anti-CENP-A antibody to mark the centromere. Locations were designated as centromeric (“Cen”), pericentric (“Peri”), or heterochromatic (“Het”). See also s, small chromosome 4; CENP-A, centromere protein A; cf(2L), centric fragment of chromosome 2L; cf(2R), centric fragment of chromosome 2R; S2, Schneider 2; X;4, Robertsonian translocation between chromosomes X and 4.Summary of the locations of satellite repeats determined by IF-FISH on S2 cell chromosomes X, X;4, 2, cf(2R), cf(2L), 3, 4, and 4(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 DataThe columns indicate the centromere contig ID, start and end coordinates of sequence, followed by the oligo sequence, and the melting temperature . Included are also the same Oligopaint sequences with 5' and 3' extensions containing the universal primer followed by library-specific barcodes (oligos.with.adaptors).(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Concerns were raised that some of the sequences reported in this article  may not In Table 1, the sequences are written as 5’ to 3’, and for shRNA sequences, both rows in the first white band comprise a single sequence per column. The shRNA sequences were designed according to the polyclonal loci of pGenesil-1 vector, with the shRNA structure: BamHI+ Sense + Loop (TTCAAGACG) + Antisense+ Termination signal+ Sal I+ Hind III.As noted in Table 1, the COMMD7 shRNA Sense sequence is as follows:CTCTGGGTCTTAGTGAGGATTCAAGACGTCCTCACTAAGACCCAGAGTTTTTTGTCGACA-3’5’-GATCCGFor clarity, the targeting  sequences are in bold. The targeting sequence aligns to nucleotides 910–928 of COMMD7 transcript variant 1 mRNA sequence (NM_053041.2).There is an error in Table 1 in that the COMMD7 shRNA Antisense sequence is reported in 3’ to 5’ orientation. The COMMD7 shRNA antisense sequence used in the study with targeting sequences in bold is as follows:GAGACCCAGAATCACTCCTAAGTTCTGCAGGAGTGATTCTGGGTCTCAAAAAACAGCTGTTCGA-5’3’-GCIn Table 1, the Sense and Antisense sequences for COMMD7 (for PCR) are switched. The 5’ to 3’ sequence listed in Table 1 for COMMD7 Antisense (for PCR) is actually the Sense sequence, corresponding to nucleotides 758–779 of COMMD7 transcript sequence NM_053041.2. The sequence listed for COMMD7 Sense is actually the Antisense sequence, corresponding to nucleotides 938–915 of COMMD7 transcript sequence NM_053041.2.A 2009 report , as a ce3 are reported. As noted in the Methods, tumor sizes were estimated using the equation, V = L x W2 . This resulted in reported tumor volume estimates twice as large as would be calculated using the standard technique for this type of experiment in which tumor volumes are instead estimated as V = (L x W2)/2. [In Fig 5, tumor sizes up to 5000 mmx W2)/2. .The authors also provide here additional information about the controls used for the reported experiments:“Control” indicates HepG2 cells without plasmid transfection.“Scramble” group was transfected with an shRNA construct generated using the following oligonucleotides:GACTTCATAAGGCGCATGCTTCAAGACGGCATGCGCCTTATGAAGTCTTTTTTGTCGACA-3’(Sense) 5’-GATCCCTGAAGTATTCCGCGTACGAAGTTCTGCCGTACGCGGAATACTTCAGAAAAAACAGCTGTTCGA-5’(Antisense) 3’-GThe targeting sequence (bold) for the scramble construct includes a 14 nucleotide stretch that is 100% identical to the human ODF2 transcript (variant X34).For electrophoretic mobility shift assays, the core κB binding sequence  in the rThe data underlying results in this article are no longer available.


This article presents data on initial experiments that were carried out to investigate new thermostable transketolase (TK) activities with l-arabinose. Transaminase (TAm) sequences from an in-house library of thermophilic strains were analyzed to compare homologies to characterized TAms with desired activity. DNA and amino acid sequences are presented for all the enzymes investigated. Calibration curves for products of the TK and TAm reactions are also presented along with chromatographic analysis of the various one-pot reactions.The dataset presented in this article is related to the research article entitled “One-pot, two-step transaminase and transketolase synthesis of  Specifications TableValue of the data•The data presented in this article gives new insight into the activities of thermostable enzymes not published before.•The data represents a rationale behind why TKs and TAms were selected for the one-pot reaction.•l-gluco-heptulose, is a pharmaceutically-relevant compound.Product of one-pot reaction, 1l-Arabinose is a major monosaccharide of sugar beet pulp (SBP), a by-product of sucrose extraction which is currently produced and sold as a low value animal feed l-gluco-heptulose, a high value, pharmaceutically relevant compound from l-arabinose using a two-step thermostable enzyme cascade. A thermostable TK catalyzed the synthesis of l-gluco-heptulose from l-arabinose and β-hydroxypyruvate (HPA) in which the latter was produced in situ from l-serine and α-ketoglutaric acid using a thermostable TAm.l-arabinose via the Seliwanoff assay l-gluco-heptulose.22.1E.coli BL21 DE3. Cell lysates were used to determine activity towards l-arabinose using the colorimetric assay, Seliwanoff assay. The Seliwanoff assay distinguishes between ketoses and aldoses using 6 M HCl and resorcinol (Seliwanoff׳s reagent) l-arabinose, Seliwanoff reagent was added to the reaction and heated at 100 °C. Colour formation due to the presence of the ketose, l-gluco-heptulose, was observed within 15 min  equipped with a Dionex AminopacTM PA1 anion exchange column 4×250 mm2 fitted with a Dionex AminopacTM PA1 guard column 4×50 mm2, an electrochemical detector system, and an eluent generator with a KOH 500 cartridge. The elution times of each compound can be observed in Dgeo. Standard calibration curves of l-gluco-heptulose and HPA were used for quantification purposes (Quantitative analysis of purposes , Fig. 5.2.4>TKDgeoDNA sequences were retrieved from the NCBI database >TKDgeoATGAGTCCCGAACAGCAGGCCGTGCGTCAGGATGTCGATCAGCTGAGCATCAACACCATCAGGACGCTTGCCATCGATGCGGTGCAGCGGGCCAACAGTGGCCACCCCGGCGCGCCGCTCGGCATGGCCCCGATGGGCTACGTGCTGTGGCAGCGCTTCCTGCGCCACAATCCGAAACATCCCGAGTGGCCGGGCCGCGACCGCTTCGTGCTGTCGGCAGGGCACGCCAGCATGCTGATCTACTCGCTGCTGCACCTCACCGGCTACGACCTGCCGCTGGAGGACATCAAGAACTTCCGCCAGTGGGGCAGCAAGACGCCTGGGCATCCCGAGTTCTTCCACACCCCAGGCCTAGACGCCACCACCGGCCCGCTCGGTCAGGGTGCGGCGATGACGGTGGGCATGGCGATGGCCGAAGCGCACCTCGCCGCACGCTACAACCGCCCCGGCTTCAAGGTCTTTGACAACTACACCTACGCGATCTTGGGGGACGGCGACCTGCAAGAAGGCGTCAACCACGAGGCCGCGTCGCTGGCAGGGCACCTCAAGCTGGGCAAGCTGATCTGGCTGCACGACGACAACCAGGTGCAGCTGGACACCGCCACGTTCAAGGCGGCCAACGAGGATACTGCGGAGCGTTACCGCGCCTACGGCTGGGAAGTTCTGCGTGTGCAAGACGGCAACAATCTCACGGAGATCGAGAACGCGATCCGCCAGGCACGGATGAACACCGAGCAGCCCACCCTGATCCAGGTTCGCACGGTGATCGGCTTCGGCAGTCCCCGTGCGGGCACCAGCAAGGCGCACGGCGAGCCGCTGGGCGAGGAAGGCGTGCAGGAGACCAAGGCGGCCCTGGGCTGGGACTACCCGCCCTTCACGGTGCCCGACGAGGTCAAGGCGCATATGGACGCGACTGAGCGTGGCGCGGAGTGGGAGGCCGACTGGAACGCGCTGATGGAGCGCTACCGTGCCGAGTACCCCGATCTCGCGGCGGAGGTTGACGCGCTGCTGGCGCGCGAACTGCCCGCCAATCTCGCCGAAGTGCTCCCCTCCTACGAAGTGGGCAGCAAGGCCATCGCCACCCGCAACGCGAGCGGTGAAGTCATCAATGCGCTGGCGCAGGTGGTGCCGGGCCTGATGGGGGGCAGTGCGGATCTCTCCGGCAGCACCAAAACCACCATCAAGGACGGCGGCGAGTTTCTGCCAGGAAACTACGGGGGCCGCAACGTCTACTTTGGCGTCCGCGAGTTTGGGATGGCCGCAGCGGGCAATGGCCTTTCGCTCTACGGAGGTGTTCGGCCCCTGGTGGGGACCTTCCTGGTGTTTGCGGACTACCTCAAGCCCGCCTTCCGCCTCTCCGCCCTTCAGTTCCAGCCGGTTACCTATGTCCTGACCCATGACTCCATTGGCCTGGGCGAAGACGGCCCAACCCACCAGCCTATTGAGCAGCTCGCCATGCTGCGCGCCGTGCCGGGTGCCCACGTGATTCGCCCCGCCGACGCCAACGAGACGGCGGCGGCCTGGCAGATGGCGCTGGAGTACGACAAGGGACCAACCGCTCTGGCCCTCTCCCGCCAGGATCTCCCAGTGCTGCCCCGCAACCACGCGGGCGTGAAGAAGGGCGCCTACGTGGTTCGCGACGCCGAAGGGGGGCCGGCACAGATCATCTTGATCGCCACCGGCTCGGAGGTGAGCCTGGCGCTGGATGCTGCCCAAGCGCTGGCGGAGGAAGGCATCCAGGCTCGGGTCGTCTCAATGCCCTGCATGGAAGTCTTCCGCCAGCAGGACGCCAGTTATCGGGACAGCGTGCTCACCCCCGGCGTGAAACGCGTGGCCATCGAGGCTGCCAGCCCGCTCCCCTGGTATGAGTGGGTGGGCTTTGACGGCGCGGTGATCGGAATGACCACCTTTGGCGCCTCGGCCCCAGCCAAAGTCCTCTTTGAGAAATTCGGCTTCAACGTGCCGAACGTCGTGCAGGTCGTCAAGGGCGTTTTGCAGAGGTGA>TKDradMSPEQQAVRQDVDQLSINTIRTLAIDAVQRANSGHPGAPLGMAPMGYVLWQRFLRHNPKHPEWPGRDRFVLSAGHASMLIYSLLHLTGYDLPLEDIKNFRQWGSKTPGHPEFFHTPGLDATTGPLGQGAAMTVGMAMAEAHLAARYNRPGFKVFDNYTYAILGDGDLQEGVNHEAASLAGHLKLGKLIWLHDDNQVQLDTATFKAANEDTAERYRAYGWEVLRVQDGNNLTEIENAIRQARMNTEQPTLIQVRTVIGFGSPRAGTSKAHGEPLGEEGVQETKAALGWDYPPFTVPDEVKAHMDATERGAEWEADWNALMERYRAEYPDLAAEVDALLARELPANLAEVLPSYEVGSKAIATRNASGEVINALAQVVPGLMGGSADLSGSTKTTIKDGGEFLPGNYGGRNVYFGVREFGMAAAGNGLSLYGGVRPLVGTFLVFADYLKPAFRLSALQFQPVTYVLTHDSIGLGEDGPTHQPIEQLAMLRAVPGAHVIRPADANETAAAWQMALEYDKGPTALALSRQDLPVLPRNHAGVKKGAYVVRDAEGGPAQIILIATGSEVSLALDAAQALAEEGIQARVVSMPCMEVFRQQDASYRDSVLTPGVKRVAIEAASPLPWYEWVGFDGAVIGMTTFGASAPAKVLFEKFGFNVPNVVQVVKGVLQRATGACAGACCAGAGCGTTTCCCAAAACGTGGCGCGGCTGAGTGTGAACACCATTCGCACGCTCGCCATTGACGCGGTGCAGGCCGCCAACTCGGGCCACCCCGGTGCGCCGCTGGGCATGGCCCCGATGGGCTACGTGCTGTGGCACAAGTTCCTGCGCCACAACCCCGCGCACCCTGAGTGGCCGGGCCGCGACCGCTTCGTGCTGTCGGCGGGGCACGCCTCCATGCTGATCTACAGCCTGCTGCACCTGACCGGCTACCAGGAAATGACGCTCGACGACCTGCGCCACTTCCGGCAGTGGGGCTACCACACCCCCGGCCACCCCGAGTTTTTCCACACCAAGGGTCTGGACGCGACCACCGGCCCGCTTGGGCAGGGCGCGGCGATGACGGTGGGCATGGCGATGGCCGAAGCACACCTCGCCGCCCGCTACAACCGCGAAGGCTTTCCGATTTTCGACAACCGCACCTACGCCATCATGGGCGACGGCGATCTGCAAGAAGGCATCAACCACGAAGCCGCCGCGCTCGCCGGGCACCTGAAACTCGGCAAGCTGATCTGGCTGCACGACGACAACCACATCCAGCTCGACACGCCCACGAACAAGGCCGAGTCCGAGGACACCGCCGCCCGCTTCCGCGCCTATGGCTGGAACGTGCTGAAGGTGGAAGACGGCGACAATCTGGACGAAATTGAAAAGGCGATTGCCGAGGCCCGCAGCCAAAGCGAGCGGCCCACGCTGATTCAGGTGCGCACCATCATCGGCTTCGGCAGCCCGCGCGCCGGCACGAGCAAGGCGCACGGCGAGCCGCTCGGCGAAGAGGGCGTGGCCGAGACGAAGAAAGCGTTGGGCTGGGAGTACCCCGCTTTTACCGTGCCCGACGAAGTGGCTGCGCACATGAACGCTCGCGCTAAGGGTGCTCAACTCGAAGCCGACTGGGAAAAACTGATGGCCGACTACCGCACCGCGCACCCCGACCTCGGCAAGGAAGTGGACGCGCTGCTCGCCCGTGAACTGCCCGCCAACCTCGCCGACCTGCTGCCCAAGTACGAAGTCGGCGGCAAGGCGGCGGCCACCCGCAACGCGAGCGGCGAAGTCATCAACGCGCTGGCGAAGGTGCTTCCCGGTTTGATGGGCGGCAGCGCGGACCTCTCGGGCTCGACCAAGACCACCATCAAGGACGGCGGCGAGATGGAAGCGGGCACCATGGGCGGGCGCAACGTGCTGTTCGGCGTGCGCGAGTTCGGCATGAGCGCCGCGGGCAATGGCCTGAGCCTCTACGGCGGCCTGCACCCGATGGTAGGCACCTTCCTGGTATTCGCCGACTACCTCAAGCCGGCTTTCCGCCTCTCGGCGCTGCAAATGCAGCCGGTGACTTACGTGCTGACCCACGACTCCATCGGTCTGGGCGAAGACGGGCCGACCCACCAGCCGGTGGACCAGCTCGCCATGCTGCGAGCGGTGCCGGGCGCCCACGTCATTCGCCCCGCCGACGCCAACGAAACCGCCGCCGCGTGGCTGATGGCGCTGGAATACGACAAGGGCCCCACCGCGCTCGCCCTCTCGCGCCAGGATCTGCCGATTCTGCCCGCCAACATCGAAGGCGTGAAGAAGGGCGCGTATGTCCTCCGAGATGTGGACGGTGCCGATGGTCAGGGGGCTCAAGTCATCCTGATC>TKDradGCCAGCGGCTCGGAAGTCGCCCTGGCCCTGAGCAGCGCCGAGCGGCTGGCCGAAGAGGGCGTGCAGGCCCGCGTGGTGTCCATGCCGTGCATGGAGGTCTTTCGCCAGCAGGAGCAGAGCTACCGCGACAGCGTGCTGACCCCCGGCGTGAAGCGCGTCGCCATCGAGGCCGCCAGCCCGCAGCCCTGGTACGAGTGGACGCTCGGCGGCCCAGTCATCGGCATGACGACCTTCGGTGCGTCGGCCCCGGCCAAGGTGCTGTTTGAGAAGTTCGGCTTCAGCGTGGAAAACGTGGTGAAGGTGGTCCACTCCGTGCTGTAA>pQR1743MTDQSVSQNVARLSVNTIRTLAIDAVQAANSGHPGAPLGMAPMGYVLWHKFLRHNPAHPEWPGRDRFVLSAGHASMLIYSLLHLTGYQEMTLDDLRHFRQWGYHTPGHPEFFHTKGLDATTGPLGQGAAMTVGMAMAEAHLAARYNREGFPIFDNRTYAIMGDGDLQEGINHEAAALAGHLKLGKLIWLHDDNHIQLDTPTNKAESEDTAARFRAYGWNVLKVEDGDNLDEIEKAIAEARSQSERPTLIQVRTIIGFGSPRAGTSKAHGEPLGEEGVAETKKALGWEYPAFTVPDEVAAHMNARAKGAQLEADWEKLMADYRTAHPDLGKEVDALLARELPANLADLLPKYEVGGKAAATRNASGEVINALAKVLPGLMGGSADLSGSTKTTIKDGGEMEAGTMGGRNVLFGVREFGMSAAGNGLSLYGGLHPMVGTFLVFADYLKPAFRLSALQMQPVTYVLTHDSIGLGEDGPTHQPVDQLAMLRAVPGAHVIRPADANETAAAWLMALEYDKGPTALALSRQDLPILPANIEGVKKGAYVLRDVDGADGQGAQVILIASGSEVALALSSAERLAEEGVQARVVSMPCMEVFRQQEQSYRDSVLTPGVKRVAIEAASPQPWYEWTLGGPVIGMTTFGASAPAKVLFEKFGFSVENVVKVVHSVL> pQR1743ATGGCGCATTCGATCGAAGAATTAGCGATTACCACCATTCGAACGCTGTCGATTGACGCGATCGAAAAAGCGAAATCCGGGCACCCGGGCATGCCGATGGGCGCGGCCCCGATGGCGTATACGCTCTGGACGAAATTTATGAACCATAATCCAGCGAATCCCAACTGGTTTAACCGCGACCGGTTTGTTTTGTCCGCTGGGCACGGGTCGATGCTGCTTTACAGCCTGCTTCATCTAAGCGGCTACGATGTCACGATGGACGACTTGAAACAGTTCCGCCAATGGGGAAGCAAAACGCCGGGCCATCCGGAATACGGCCATACGCCAGGGGTGGAGGCAACGACCGGCCCGCTCGGCCAAGGGATTGCGATGGCGGTCGGCATGGCGATGGCGGAACGGCATTTGGCGGCTGCATACAATCGCGATGGATTTGACATTATCAACCACTACACGTATGCGATTTGCGGCGACGGCGATTTGATGGAAGGAGTGGCGAGCGAAGCGGCGTCACTCGCCGGCCACTTGAAGCTCGGCCGTCTGATCGTCCTGTATGACTCGAACGACATTTCGCTGGACGGCGAGCTCAACTTGTCGTTTTCGGAAAACGTCGCCCAACGTTTCCAAGCGTACGGCTGGCAATATTTGCGCGTTGAGGACGGCAACAATATTGAAGAAATCGCCAAAGCGCTCGAGGAGGCGCGGACGGACCTCAGCCGGCCGACGCTCATTGAAGTAAAAACGACGATTGGCTACGGCGCGCCAAATAAAGCGGGCACGTCCGGCGTCCACGGCGCTCCGCTCGGCGCCCAAGAGGCGAAGTTGACGAAAGAAGCGTACCGCTGGACGTTTTCCGAAGATTTCTACGTGCCGGATGAAGTGTACGCTCATTTCCGGGAAACGGTGCAAGAAGCCGGAGCGAGAAAAGAAGCGGAGTGGAATGAGCGCTTCGTTGCTTACGAGCGGGCGCATCCGGAATTGGCCGCCGAGCTGAAGCAGGCGATTGAAGGGAAGCTTCCGGATGGCTGGGAAACATCGCTGCCGGTGTATGAAGCGGGCAAAAGCTTGGCGACCCGCTCATCGTCCGGGGAAGTGATCAACGCCATCGCCAAAGCGGTGCCGCATTGTTTGGCGGTTCGGCGGACTTGGCAAGCTCGAATAAAACGCTTATCAAAGGCGGCGGCAACTTCTTGCCGGACAGCTACGAAGGGCGCAACATTTGGTTTGGCGTGCGCGAGTTTGCCATGGGCGCGGCGTTAAACGGCATGGCGCTTCACGGCGGGTTGAAAGTGTTCGGCGGCACGTTCTTCGTGTTCTCCGACTACTTGCGCCCGGCGATTCGGCTGGCGGCGCTCATGGGCTTGCCGGTGACGTACGTGCTGACGCACGACAGCATCGCCGTCGGGGAAGACGGCCCGACGCATGAGCCGGTCGAGCATCTCGCTTCACTTCGGGCGATGCCGAACTTGTCAGTCATCCGGCCGGCTGACGCAAACGAAACGGCGGCCGCCTGGCGGCTGGCGCTCGAGTCGACGAACAAGCCGACTGCGCTCGTCTTGACGCGTCAAGATGTGCCGACATTGCCGACAACCGCTCAGTTGGCGTATGAAGGCGTGAAAAAAGGCGCGTACGTCGTTTCACCGGCGAAAAACGGCGCTCCGGAGGCGCTGTTGTTGGCGACTGGCTCGGAAGTCGGTCTGGCCGTCAAAGCGCAAGAAGCGCTCGCCGCTGAGGGCATCCATGTCTCCGTCATCAGCATGCCATCGTGGGACCGCTTCGAAGCGCAGCCAAAATCGTACCGCGATGAAGTGCTGCCGCCGGCCGTGACGAAGCGGCTCGCCATTGAAATGGGCGCCTCGCTCGGTTGGGAGCGCTACGTCGGCGCCGAGGGCGACATTTTGGCCATCGACCGATTCGGTGCTTCCGCTCCGGGAGAGAAAATCATGGCCGAGTATGGCTTTACGGTTGACAACGTCGTCCGCCGCACAAAAGCGCTGCTCGGCAAGTAA>pQR1744MAHSIEELAITTIRTLSIDAIEKAKSGHPGMPMGAAPMAYTLWTKFMNHNPANPNWFNRDRFVLSAGHGSMLLYSLLHLSGYDVTMDDLKQFRQWGSKTPGHPEYGHTPGVEATTGPLGQGIAMAVGMAMAERHLAAAYNRDGFDIINHYTYAICGDGDLMEGVASEAASLAGHLKLGRLIVLYDSNDISLDGELNLSFSENVAQRFQAYGWQYLRVEDGNNIEEIAKALEEARTDLSRPTLIEVKTTIGYGAPNKAGTSGVHGAPLGAQEAKLTKEAYRWTFSEDFYVPDEVYAHFRETVQEAGARKEAEWNERFVAYERAHPELAAELKQAIEGKLPDGWETSLPVYEAGKSLATRSSSGEVINAIAKAVPQLFGGSADLASSNKTLIKGGGNFLPDSYEGRNIWFGVREFAMGAALNGMALHGGLKVFGGTFFVFSDYLRPAIRLAALMGLPVTYVLTHDSIAVGEDGPTHEPVEHLASLRAMPNLSVIRPADANETAAAWRLALESTNKPTALVLTRQDVPTLPTTAQLAYEGVKKGAYVVSPAKNGAPEALLLATGSEVGLAVKAQEALAAEGIHVSVISMPSWDRFEAQPKSYRDEVLPPAVTKRLAIEMGASLGWERYVGAEGDILAIDRFGASAPGEKIMAEYGFTVDNVVRRTKALLGK>pQR1744ATGAACACCGGCACCCCAAAGACCCTGGACTGGTCTGATCTCGATAGACGTACCGTAGACGTGGTTCGTGCCCTGGCGATGGACGCGGTCGAAGAAGCGGGATCCGGGCACCCTGGAACCGCGATGAGTCTGGCGCCTGTGGCCTACCTGCTCTTCCAGAAGGTGATGCGGCACGATCCGACAGATCCGAAGTGGATCGGCCGCGACCGCTTCGTCCTGTCCTGCGGGCACTCCAGCCTCACGCTCTACATCCAGCTCTACCTGGCTGGCTACGGGCTGAGCCTGAACGACATCAAGCGGCTGCGCCAGTGGGGCAGCCTCACCCCGGGCCACCCCGAATACGGGCACACCGCCGGGGTGGAAACCACCACCGGCCCCTTGGGGCAGGGCATCGGCAACGCGGTCGGCATGGCCATGGCCGCCCGCCGGGAGCGGGGCCTGTTCGACCCGGACACCCCGATCGGGGAAAGCCCGTTCGACCACTACATCTACGTCCTGTGCTCTGACGGCGACGTCCAGGAGGGCATCAGCCACGAAGTAAGTGCCCTCGCCGGCACGCAGAAGCTCGGCAACCTCATCGTCATCTGGGACGACAACCGCATCTCCATCGAAGACGACACCCAGATCGCATTCACCGAAGACGTCGTCGCCCGCTACGCCGCCTACGGCTGGCACGTCCAAGAGGTCGAGTGGGTCGGCGAGGACGGCTCCTACCACGAAGACGTGGCGGCGCTGTACGACGCGATCCGGGCCGCCCAGGCGGAGACGGAACGTCCCTCTTTCATCCGGCTGCGCACCATCATCGGCTGGCCGTCCCCGAACAAGCAGAACACGGGGGCGATCCACGGCGCCGCGCTGGGGGCTGAAGAGGTCGCCGCCACCAAGCGGGTGCTGGGCCTCAACCCTGAGGCGCAGTTCGACGTGCCCAACGAGCTGCTGGAGCACGCCCGGGGCGTGGTGGCGCGGGGCCGCGCCGCCCGCCAGGAATGGGAGGCCTTGTTCGCCAAGTGGCGGGCCAACGCGGGCGAGCGTGCCGAACTGTTCGACCGGCTGATGGCAGGCTCGCTCCCGGACGGTTGGGAGAAGGCGATCCCGACCTTCGAGCCCAGCGCTAAGGGCATGGCCACCCGGAAAGCGTCCGGTGAGGTGCTGAGCGCGATCGCCCCGGTGCTGCCGGAGCTGTGGGGCGGCTCGGCGGACTTGGCCGGATCCAACAACACCACGCCTAAGGGCGAGCCGTCGTTCATCCCCGAGGAGCGGTCCACGAAGGCGTTCTCCGGCCACCGCTACGGCCGGGTGCTGCACTTCGGGATCCGTGAACACGGCATGGGGGCGATCCTCAACGGGATCGCGCTGCACGGCCCCACCCGCCCCTACGGTGGCACCTTCCTCGTGTTCAGCGACTACATGCGGCCGTCGGTGCGGCTGGCTGCCCTGATGAAGCTGCCGGTCACGTACGTGTGGACCCACGACTCGATCGGTCTGGGCGAAGACGGACCCACCCACCAGCCGGTGGAGCACCTGTGGTCGCTGCGCGCCATCCCCGGCCTGGCGGTGGTGCGTCCCGCCGACGCCAACGAGACGGCAGTGGCCTGGCGCACCATCCTGGAACGCAATGACGGCCCGGTGGCGCTCGCGCTGACCCGGCAGTCGGTTCCGGTTCTGGACCGCTCCGAGCTCGCCTCTGCGGAGCTGGTCTCCCGCGGCGGGTACATCCTGGCCGAAGCCAGCAACGGCCGTCCGGAGGCGATCATCATCGCCACCGGAAGTGAGGTGCAGATCGCGTTGGAGGCGCGTTCCCGCCTGGAGGAGTCGGGTACTCCTACCCGTGTGGTGTCGATGCCGTGCCTGGAGTGGTTCAACGAGCAGGACGACGCCTACCGCCAGCAGGTGCTTCCACCGTCGGTCCGGGTCCGGGTCTCCGTGGAAGCCGGGGTCGCCTTGGGCTGGCGCGAGCTGGTGGGCGAGTATGGCGAGTCGGTGAGTCTGGAACACTTCGGCGCTTCGGCTCCGTACGCGACTCTCTACGAGCAGTTCGGGCTCACCGCCGACCGGGTAGTGGCAGCCGTACACTCCAGCGCTGCCAAGCTCGGCGGTGACCGTGGATCAACGACCGGCAACTGA>pQR1745MNTGTPKTLDWSDLDRRTVDVVRALAMDAVEEAGSGHPGTAMSLAPVAYLLFQKVMRHDPTDPKWIGRDRFVLSCGHSSLTLYIQLYLAGYGLSLNDIKRLRQWGSLTPGHPEYGHTAGVETTTGPLGQGIGNAVGMAMAARRERGLFDPDTPIGESPFDHYIYVLCSDGDVQEGISHEVSALAGTQKLGNLIVIWDDNRISIEDDTQIAFTEDVVARYAAYGWHVQEVEWVGEDGSYHEDVAALYDAIRAAQAETERPSFIRLRTIIGWPSPNKQNTGAIHGAALGAEEVAATKRVLGLNPEAQFDVPNELLEHARGVVARGRAARQEWEALFAKWRANAGERAELFDRLMAGSLPDGWEKAIPTFEPSAKGMATRKASGEVLSAIAPVLPELWGGSADLAGSNNTTPKGEPSFIPEERSTKAFSGHRYGRVLHFGIREHGMGAILNGIALHGPTRPYGGTFLVFSDYMRPSVRLAALMKLPVTYVWTHDSIGLGEDGPTHQPVEHLWSLRAIPGLAVVRPADANETAVAWRTILERNDGPVALALTRQSVPVLDRSELASAELVSRGGYILAEASNGRPEAIIIATGSEVQIALEARSRLEESGTPTRVVSMPCLEWFNEQDDAYRQQVLPPSVRVRVSVEAGVALGWRELVGEYGESVSLEHFGASAPYATLYEQFGLTADRVVAAVHSSAAKLGGDRGSTTGNATGGAAAGGTTTCCCTATGAAAAACTTCCAGAAAGCGAACTCAAAGAGTTGAAAGAACTCGGAAGGCTCTGCCGTGGCGACATACTGAAAATGACCTACATAGCTAACTCAGGCCATCCTGGAGGATCCATGTCTTCGATCGATCTTTATCTTACCGTCTTCAAGTACGCAAAACTCAGACCCGTCGATGATCCTGCAAGAGACAGAATCGTGATCAGCCATGGACACACTTCTCCGGGTGTCTACGCAGCTATGGCTCGTTTGGGGTTTGTCGATCTCGATGAAGTCCTCGCAGGATTCAGACACCCCGCTTCCGTTTTTGAAGGACACGTGACCCGAGGTGTTGGGATCATCGACTGGACAACCGGAAACCTCGGTCAGGGTCTTTCAGCCGGACTCGGTTTTGCCCTCGCATCCAGGTTCACAGGAAAAGATTACCACGTCTTTGTTCTCATGAGTGACGCAGAACAGGCAAAAGGACAGGTGGCGGAGGCAAGAAGAGTGGCGAAAAAGTACGGTGTCACGAATCTCACAGTGATCATCGACTACAACGACGCCCAGATCAGTGGCCGTGCCAGAGACGTCATGCCCGTGAACATAAAGGAAAACTACTTAGCGGACGGCTGGAGGGTCATCGAGATCGATGGGCACGACTACGAACAGATCTATCTCGCACTGAAAGAAGCGGTAGAAGACGAAC> pQR1745TGAATCCCGTTGCCATAATCGCCAAAACGGTCATGGGAAAAGGCGTATCTTTCATGGAAAACGAGGTGAAATACCACGGAAAGCCTTTGAACAGAGAAGAACTCGAAAAAGCCCTCGCGGAACTCGGAATTGAAAACGATGTTGATGTGTACATCGAAAAAAGAAAACAACTTCCAGTGGAAAAACACAAGAAAGTCTACAAAACTTACCCGATCAAGATCGACACGGGAGAGCCCATCACCTACACCTCACCCACTGACAACAGAAGCGCATTCGGAAAAGCTATTCTGGATCTGGTGAAGAAGAACGTAAACAATCCAGAAACCACACCCATCGTCGCTGTGGACTGCGACCTGAAGGGATCGGTCAAACTCGACCTGCTCGACAAAGAGTTCCCTGAGAGACTCCTGGAAGTGGGCGTTCAGGAACACAACGCTGCCGCTATGGCGGGGGCACTCTCCGCAGAGGGTGTGATCACGTTCTTCGCTGATTTTGGTGTTTTTGGAATTTCTGAAACCTACAACCAGCACAGGCTGAACGCCATCAATGGAACGAACCTCAAAGTCGTTGTCACACACTGCGGACTCAACGTGGGAGAGGACGGAAAAACTCATCACGGACTCGACTACGTTTCCGGGCCGATGAACTGGTACGGTTTCAAAGTGATCGTCCCTGGTGATCCCAACCAGACGGATAGAGTTGTCAGATACGCCGCGAAGGAATACGGGAACTTCGTAATCGCCATGGGAAGATCTAAGCTTCCCATCATCCTCGATGAAAACGGGAAACCTTTCTTCGGAGAGGGTTACACCTTCGAATATGGGAAGATCGATGTCGTTAGAAAAGGTGACGACGCGGTGATCATAACTTACGGTTCTACACTCTGTGAAGCCGTAAATGCCGCAGACGAACTCAAGAAAGAAGGAGTAAACGTAGCCGTTCTGAATGTCTCCTGTCCGGTGGATCTCGATATAGAGACCTTGAAGATGGTCGATGGAAAACCCGTTCTCGTTGTGGAGGATCACAACGTTTTCACAGGACTTGGAAGCTTCCTTGGAACCACCCTTCTTGAAAACGGCATCATCCCGAAGAAATACGTGAGAGTAGGTGTTCCAGAATTCGCCGTGTCCGGCAGTTACACGATGCTCTACAAACTCTACGGCCTGGATAAAGATGGAATAATTTCCAGACTCAGAGAGATGCTCTAAMERFPYEKLPESELKELKELGRLCRGDILKMTYIANSGHPGGSMSSIDLYLTVFKYAKLRPVDDPARDRIVISHGHTSPGVYAAMARLGFVDLDEVLAGFRHPASVFEGHVTRGVGIIDWTTGNLGQGLSAGLGFALASRFTGKDYHVFVLMSDAEQAKGQVAEARRVAKKYGVTNLTVIIDYNDAQISGRARDVMPVNIKENYLADGWRVIEIDGHDYEQIYLALKEAVEDELNPVAIIAKTVMGKGVSFMENEVKYHGKPLNREELEKALAELGIENDVDVYIEKRKQLPVEKHKKVYKTYPIKIDTGEPITYTSPTDNRSAFGKAILDLVKKNVNNPETTPIVAVDCDLKGSVKLDLDKEFPERLLEVGVQEHNAAAMAGALSAEGVITFFADFGVFGISETYNQHRLNAINGTNLKVVVTHCGLNVGEDGKTHHGLDYVSGPMNWYGFKVIVPGDPNQTDRVVRYAAKEYGNFVIAMGRSKLPIILDENGKPFFGEGYTFEYGKIDVVRKGDDAVIITYGSTLCEAVNAADELKKEGVNVAVLNVSCPVDLDIETLKMVDGKPVLVVEDHNVFTGLGSFLGTTLLENGIIPKKYVRVGVPEFAVSGSYTMLYKLYGLDKDGIISRLREML2.5>TAmGsteDNA sequences were retrieved from the NCBI database >TAmGsteATGAAATTGGCAAAACGGGTGGCGTCGCTGACGCCATCGGCGACTTTGGCCATTACGGAGAAAGCAAAAGAACTAAAAGCGGCCGGGCATGACGTGATTGGTCTCGGAGCTGGCGAACCGGATTTCAACACGCCACAGCACATTCTTGATGCCGCCATCAAGGCAATGAACGAAGGACATACGAAATATACACCATCGGGCGGTTTGCCGGCGTTAAAGGAGGAAATTATAAAAAAATTCGCCCGCGACCAAGGCTTGGATTATGAGCCGGCTGAAGTGATTGTATGCGTCGGAGCGAAGCACGCCCTTTACACGCTGTTCCAAGTATTGCTCGATGAAGGCGACGAAGTGATCATTCCGACGCCATACTGGGTGAGCTATCCGGAACAAGTGAAACTGGCGGGCGGTGTTCCGGTTTACGTCGAAGGGCTTGAACAAAATCATTTTAAAATTACGCCGGAGCAGCTGAAACAGGCAATCACGCCGCGGACGAAAGCGGTTATCATCAACTCGCCGAGCAACCCGACTGGCATGATTTATACAGCCGAAGAGTTGAAGGCGCTTGGTGAGGTGTGCCTAGCGCATGGTGTATTGATCGTGTCAGATGAAATTTACGAAAAATTGACTTACGGCGGGGCGAAGCATGTGTCCATCGCTGAGTTGTCGCCGGAGCTGAAGGCGCAGACAGTCATCATTAACGGCGTGTCAAAGTCGCATTCGATGACGGGCTGGCGCATTGGTTATGCGGCGGGGCCGAAAGATATTATTAAGGCAATGACAGATTTGGCGAGCCACAGCACGTCCAACCCGACGTCAATCGCCCAATACGCGGCCATCGCTGCTTACAGCGGGCCGCAGGAGCCGGTCGAACAAATGCGCCAAGCGTTTGAACAACGGCTCAATATCATTTACGACAAGCTCGTGCAAATTCCAGGATTCACGTGCGTTAAGCCACAAGGGGCGTTTTATTTGTTCCCGAACGCCCGCGAAGCGGCTGCAATGGCCGGCTGCCGCACGGTCGACGAGTTCGTCGCTGCCTTGTTGGAGGAAGCGAAAGTCGCGCTTGTGCCCGGCTCTGGGTTTGGAGCGCCGGATAACGTTCGCTTGTCATACGCGACATCGCTCGATGCACTGGAAACCGCCGTGGAACGCATCCACCGGTTTATGGAAGCGCGCGCTTAA>TAmDGeoMKLAKRVASLTPSATLAITEKAKELKAAGHDVIGLGAGEPDFNTPQHILDAAIKAMNEGHTKYTPSGGLPALKEEIIKKFARDQGLDYEPAEVIVCVGAKHALYTLFQVLLDEGDEVIIPTPYWVSYPEQVKLAGGVPVYVEGLEQNHFKITPEQLKQAITPRTKAVIINSPSNPTGMITAEELKALGEVCLAHGVLIVSDEIYEKLTYGGAKHVSIAELSPELKAQTVIINGVSKSHSMTGWRIGYAAGPKDIIKAMTDLASHSTSNPTSIAQYAAIAAYSGPQEPVEQMRQAFEQRLNIIYDKLVQIPGFTCVKPQGAFYLFPNAREAAAMAGCRTVDEFVAALLEEAKVALVPGSGFGAPDNVRLSYATSLDALETAVERIHRFMEARA>TAmDGeoATGTTCGAGGACACGCCCGCACCCTTTCCACCGCACATTCTGCTGACGCCCGGTCCGACACCGATTCACCCCCGGGCCCAGCGGGCGCTGCTGCGCGAGATGCTGGGGCACATGGACCCTGAGGTGTTCGCCCTGAACCGCGAGATCCAGGCGGACTTGCGGGTGATGTACGGGACGGGGCCCCAGACCTTTACGGCGCTGCTGGCGGGCACCGGGAGCCTGGGCATGGAGGCGGGCTTCGCCAACTTGGTGGAGAGGGGAGACGACGTGCTGATCTGCGTCAATGGTGCCTTTGGTCAGCGCATGGCCGAGATGGCGGCGCGCTACGGTGCGAATGTACGGCGGGTGACCGCGCCGCTGGGCGAGCCGATCGACCCGGCCGACGTGGCTGCGCGGTTGAGCGGCGCGCGGCTGGTGGCGGTGGTGCATGGGGAGACGAGCACGGGTGTGCTCAATCCGCTTCCGGAGATTGCCGAGGCCGTGCGCGGGAGCGGGGCATTGCTGGCCGTGGACGCCGTGACGACCGCCGGGATGGAACCCTTCCATATGGCGGACTGGGGCGTGGACTACGCCTATACCGGCGCGCAGAAGTGCCTCTCGGCACCGCCCGGCCTGGCCCCGGTGGCGATCAGCGACCGTGCTCTCGCTCGCCACGCGGCCCGCCGCACGCCCACGCCGCTGTGGTACTGCGATTTTGAGGGCCTGCGCGACTACTGGGACCGGCACAGCTACCACCACACGGTCCCGGTGAATCTGCACTACGCCTTCCACGCCGCCCTGCGCGCCGCACTCGAAGAAGGCCTCCAAGCCCGGCAGGCCCGCGTGCGCGACCTTGGCCAGGCGGTGCTGGCGGCCCTGACGCCGCTGGGCTTCACGCCGTATGTGGCCGATCCCGCCGCGCGGCTGCCCACCGTCTTGGCCCTGCGTCTTCCTCCCGGCTTCGACGACGCGGGCGTTCGCCAGGCCCTACGGGAACGCGGGATCAGCGTGACCGGCGGCCTGGGACCGACGGCAGGGCTGATCTGGCGTCTGGGCCTGATGGGGGAAGCGGCTCGCCCCGCGCCCTACCGCGCGCTGATGCTCGCCCTGGAAGACCTGCTGGGCGAGCGGGGCTTGGTGGCGCGCTTCGAGGAGGCGCTGGGCGTCGCGGCCTGA>pQR1746MFEDTPAPFPPHILLTPGPTPIHPRAQRALLREMLGHMDPEVFALNREIQADLRVMYGTGPQTFTALLAGTGSLGMEAGFANLVERGDDVLICVNGAFGQRMAEMAARYGANVRRVTAPLGEPIDPADVAARLSGARLVAVVHGETSTGVLNPLPEIAEAVRGSGALLAVDAVTTAGMEPFHMADWGVDYAYTGAQKCLSAPPGLAPVAISDRALARHAARRTPTPLWYCDFEGLRDYWDRHSYHHTVPVNLHYAFHAALRAALEEGLQARQARVRDLGQAVLAALTPLGFTPYVADPAARLPTVLALRLPPGFDDAGVRQALRERGISVTGGLGPTAGLIWRLGLMGEAARPAPYRALMLALEDLLGERGLVARFEEALGVAA>pQR1746ATGACCTCTCCTTTCCGCCTCTCCGCCCGCGCCCAGAGCCTCAAGCCGTCTGCGACAGTGGCGGTCACGTCCCGCGCCCTGGAACTCCAGCGTCAGGGCCTGGACGTGATTTCCATGAGCGTGGGCGAGCCGGATTTCGACACGCCGCCACATGTCAAGGCCGCCGGCATCGCCGCCATCGAGGAAGGCAAGACCAAATACACCCCGGTCAGCGGCATTCCCGAACTGCGCGAGGCCATCAGCGCCAAGTTTCGGCGCGAAAACGGCCTGGACTACGCGCCGAACGCCGTGACGGTAACGAGCGGCGGTAAACAGGCGCTGTTCAACGCCTTTTTCGCGTTGCTGAACCCCGGCGACGAGGTGCTGATTCCCGCGCCCCACTGGGTCAGCTACCCCGAAATGGTCGCGCTGACCGGCGCGGTGCCGGTAACCGTACCCACTACGCCGCAGCAGGGCTTTCAACTCGACCCGGACGCCCTCGCCGCCGCCATCACGCCGCGCACCCGCATGGTGATTCTCAACAGCCCCGGCAACCCGACGGGCGCGGTGTTTCCGCCGGAAACCTTGCGGGCGGTGGCCGACCTCGCCACGCAGCACGGCTTGATGATCGTCACCGACGAAATCTACGAGCACCTCGTCTACGACGCCGAGCAGGTCAGCATCGGCACCTACGCGCCGGAGCACACCCTGACCATCAATGGCGCGAGCAAAGCGTATGCCATGACCGGCTGGCGCATCGGCTACGCGGGCGGGCCGCGCGAGGTGATTGCCGCCATGAACGCGCTGCAATCGCAAAGCACCAGCAACGCCAGCAGCGTCAGCCAGTACGCCGCCCTCGCCGCGCTGGAACAGCACGAGGAAACCATGCGCTTCATCGACAGGGCCCGCACCGCCTACCGCGAACGGCGCGACCGCATCGTGGCGGGCCTCAACGCGCTGGGGTTGCCCACGCCCACGCCGCAAGGGGCCTTTTACGTGATGGCCGACACCCGCGCCATTCACACCGACGAACTCGAAGCCGCCCGCATCATTCTGGATGAGGCGCAGGTCGCCGTCGTGCCCGGCACCGATTTCGCCGCGCCGGGACAGGTGCGCCTGAGCTACGCGACCAGCATGGACAACATCGAGGAAGTGCTGCGGCGGCTGGAAGGGGTCGTGCGGCGCTAA>pQR1748MTSPFRLSARAQSLKPSATVAVTSRALELQRQGLDVISMSVGEPDFDTPPHVKAAGIAAIEEGKTKYTPVSGIPELREAISAKFRRENGLDYAPNAVTVTSGGKQALFNAFFALLNPGDEVLIPAPHWVSYPEMVALTGAVPVTVPTTPQQGFQLDPDALAAAITPRTRMVILNSPGNPTGAVFPPETLRAVADLATQHGLMIVTDEIYEHLVYDAEQVSIGTYAPEHTLTINGASKAYAMTGWRIGYAGGPREVIAAMNALQSQSTSNASSVSQYAALAALEQHEETMRFIDRARTAYRERRDRIVAGLNALGLPTPTPQGAFYVMADTRAIHTDELEAARIILDEAQVAVVPGTDFAAPGQVRLSYATSMDNIEEVLRRLEGVVRR>pQR1748ATGACTGACCGACCTCGTATCTCCGCACGCATCGGCGGTATCTCCGAGTCAGCGACCCTGGCGGTGGACGCCAAGGCCAAGGCCCTGAAGGCCGCTGGGCATCCCGTGATCGGCTTCGGCGCCGGGGAGCCTGACTTCCCCACGCCCGACTACATCGTGGAGGCAGCGGTCGCCGCCTGCCGCGACTCGCGCTTCCACCGCTACACCCCGGCGGGAGGCCTCCCCGAACTCAAGGAAGCCATCGCGGCTAAGACGCTGCGCGACTCCGGCTACCGGGTGGAGCCGAACCAAGTCCTGGTCACCAACGGCGGCAAGCAAGCGATCTACGAGGCGTTCGCCACGCTGCTCGATCCGGGCGACGAGGTCATCGTGATCGCGCCCTACTGGACCACCTACCCTGAATCGATCCGGCTGGCCGGAGGAACCCCGGTCTACGTGGTCACCGACGAGTCCACTGGCTACCTGGCCACGGTCGAGCAGCTGGAGGCGGCCCGCACCGACCGCACCAAGGTGCTGCTGTTCGTCTCCCCCTCGAACCCGACCGGCGCGGTGTACTCGCCCGAGCAGGTCCGCGAGATCGGCCGGTGGGCCCTCGAACACAACCTGTGGGTGCTCACCGACGAGATCTACGAGCACCTCGTCTACGGGGACGCCCGGTTCTCCTCGATGCCGGTGGAAGTTCCGGAACTGGCCGACCGCACCGTGGTGGTCAACGGGGTGGCCAAGACCTACGCCATGACCGGGTGGCGGGTCGGCTGGCTCATCGGCCCCGTGGACGTGGTCAAGGCTGCGACCAACCTGCAGTCGCACGCCACCTCCAATGTGGCCAACGTCTCGCAGGCCGCGGCTCTGGCAGCGGTCTCCGGCGACCTGTCGGCCGTGGAGGAGATGAAGCAGGCCTTCGACCGGCGGCGGCAGACCATTGTGCGGATGCTCAACGAGATCCCCGGTGTGGTGTGCCCCGAGCCCCAGGGCGCGTTCTACGCCTACCCGTCGGTCAAGGAGATCCTCGGCAAGGAGATCCGCGGTCAGCGTCCGCAGACCTCCAGCGAGCTGGCGTCGCTGATCCTGGAGCACGCCAAGGTCGCGGTGGTCCCGGGCGAGGCGTTCGGCACTCCGGGCTACCTGCGGTTGTCCTACGCGTTGAGCGACGCCGATCTGGTCGAAGGGGTCAGCCGGATCGCCAAGCTGCTGAGCGAAGCCCACTGA>pQR1749MTDRPRISARIGGISESATLAVDAKAKALKAAGHPVIGFGAGEPDFPTPDYIVEAAVAACRDSRFHRYTPAGGLPELKEAIAAKTLRDSGYRVEPNQVLVTNGGKQAIYEAFATLLDPGDEVIVIAPYWTTYPESIRLAGGTPVYVVTDESTGYLATVEQLEAARTDRTKVLLFVSPSNPTGAVYSPEQVREIGRWALEHNLWVLTDEIYEHLVYGDARFSSMPVEVPELADRTVVVNGVAKTYAMTGWRVGWLIGPVDVVKAATNLQSHATSNVANVSQAAALAAVSGDLSAVEEMKQAFDRRRQTIVRMLNEIPGVVCPEPQGAFYAYPSVKEILGKEIRGQRPQTSSELASLILEHAKVAVVPGEAFGTPGYLRLSYALSDADLVEGVSRIAKLLSEAH>pQR1749ATGGTATCCAGGAGAATATCAGAGATTCCCATATCGAAAACCATGGAACTCGACGCGAAGGCCAAAGCCCTCATAAAAAAGGGAGAAGACGTGATCAATCTAACGGCTGGTGAGCCGGATTTTCCCACACCGGAACCCGTCGTGGAAGAAGCGGTGAGATTTCTCCAGAAAGGAGAAGTGAAATACACAGATCCTCGTGGTATCTACGAACTCAGAGAGGGTATAGCGAAAAGGATAGGCGAGAGATACAAAAAAGATATCTCACCGGATCAGGTCGTGGTGACGAATGGAGCGAAACAGGCTCTGTTCAATGCTTTCATGGCCCTTCTCGATCCCGGTGACGAAGTGATCGTGTTTTCTCCCGTCTGGGTCAGCTACATTCCTCAGATCATCCTTGCTGGTGGCACGGTGAACGTGGTTGAGACGTTCATGAGTAAAAATTTCCAGCCCAGTCTGGAAGAGGTGGAAGGGCTTCTTGTTGGGAAAACGAAAGCCGTTCTTATCAACTCGCCGAACAATCCCACTGGTGTGGTGTACAGAAGAGAGTTCCTTGAAGGACTTGTGAGACTTGCCAAGAAGAGGAATTTCTACATAATCAGCGACGAAGTCTACGATTCCCTTGTTTACACGGATGAATTCACATCGATACTCGATGTTTCTGAAGGATTCGACCGGATAGTTTACATAAACGGCTTCTCGAAGTCTCACTCCATGACCGGCTGGAGGGTGGGTTACCTGATATCGAGCGAAAAAGTAGCGACCGCTGTGTCGAAGATCCAGTCTCACACCACCTCCTGTATCAACACGGTAGCACAGTACGCCGCCTTGAAGGCTCTGGAAGTGGACAACTCTTACATGGTTCAGACCTTTAAAGAAAGAAAAAATTTCGTGGTGGAAAGATTGAAAAAGATGGGTGTTAAGTTCGTGGAACCAGAAGGTGCGTTCTACCTCTTTTTCAAAGTCCGGGGTGACGATGTGAAATTCTGTGAAAGGCTCCTCGAAGAAAAGAAGGTTGCACTCGTTCCAGGATCCGCTTTTCTGAAGCCTGGATTTGTGAGGCTTTCTTTTGCCACATCTATAGAAAGACTTACGGAGGCGCTGGATAGAATTGAAGACTTCCTCAATTCTCGTTGA>pQR1751MVSRRISEIPISKTMELDAKAKALIKKGEDVINLTAGEPDFPTPEPVVEEAVRFLQKGEVKYTDPRGIYELREGIAKRIGERYKKDISPDQVVVTNGAKQALFNAFMALLDPGDEVIVFSPVWVSYIPQIILAGGTVNVVETFMSKNFQPSLEEVEGLLVGKTKAVLINSPNNPTGVVYRREFLEGLVRLAKKRNFYIISDEVYDSLVYTDEFTSILDVSEGFDRIVYINGFSKSHSMTGWRVGYLISSEKVATAVSKIQSHTTSCINTVAQYAALKALEVDNSYMVQTFKERKNFVVERLKKMGVKFVEPEGAFYLFFKVRGDDVKFCERLLEEKKVALVPGSAFLKPGFVRLSFATSIERLTEALDRIEDFLNSR>pQR1751ATGGCACCTGACCTGCGCCACCTGCACACCTTCGGCGAACTGGATCCGCCGCAACGCCTGTTGATGGGCCCCGGCCCGGTCAATGCGCATCCACGCGTGCTGCGTGCGATGGCGGCCGACCTGCTTGGCCAGTTCGACCCGGAAATGACCACCTACATGAACGAGGTGATGGCGCTGTACCGCCCCTTGTTCGGCACCCAGAACCGCTGGACCTTTCTGGTCGATGGCACGGCGCGCGCCGGCATCGAAGCCGCGCTGGTGTCGCTGGTGCAGCCGGGCGACCGTGTGCTGGTGATCAACTTCGGCCGCTTCGGTTTGTTGCTGACCGAAATCCTTGGCCGGCTCGGCGCCGACGTCCACACCGTGGATGCGCCGTGGGGCGAGGTGGTGCCGCTGGCGGCGATTGCCGAGGCGATCGCAAGCGTGGCACCCAAGCTGGTGGCCACCGTGCACGGCGACACCTCCACCACCATGGCGCAGCCGCTCGATGGCCTAGGCGCGCTATGCCGGGCGGCCGGCGCGCTGAGTTACGTAGACGCCACAGCCACCATCGGCGGCATGGACATCGCCAGCGACCGCTGGGAGGTGGACGTGGTCACCGCGGGGCTGCAGAAATGCCTGGGCGGGCCGTCCGGCTCGGCGCCGATCACTGTCTCTGCCGCGGCAGCGGAGGCGATCTTTGCGCGGCGGCATGTCGAACGCGGCATCGTGCGCGAGGACATCGCCAACGGCAGCGGCCCACGCATCGCCTCGAATTATTTCGACCTGGCGATGATCATGGATTACTGGTCCGACAAGCGCCTCAATCACCACACCGAAGCCACCACCATGCTGTACGGCGCGCGCGAATGCGCACGCGTGGCCTTGCAGGAAGGCCTGGAGGCGCGCTACGCCCGGCATGCGGCTGCCGGCCGCGCGGTCAGCGCCGGCGTGCGCGCACTGGGGCTGGAGGTGTTCGGCGACGATGCGCACCGCATGAGCAATGTCACCGGCGTGGTGATCCCGCACGGCGTCGACAGTGAAGCAGTGCGGCGGCGCATGCGCGAGGATTTCGAAATCGAGATCGGCACCGCGTTCGGCCCGCTGCAAGGCAGGATCTGGCGCATCGGTGCGATGGGCTACAACGCGATGAAGCACAAGGTGCTGCTCACCCTGGCCGCACTGGAAGCGGTGCTGCGCGCCGAGGGCTACGCGTGCACCCAAGGCCTGGCGGTCGAAGCCGCACGCGCCGCCTGGCATGCGGAGCCGGCTGCATGA>pQR1752MAPDLRHLHTFGELDPPQRLLMGPGPVNAHPRVLRAMAADLLGQFDPEMTTYMNEVMALYRPLFGTQNRWTFLVDGTARAGIEAALVSLVQPGDRVLVINFGRFGLLLTEILGRLGADVHTVDAPWGEVVPLAAIAEAIASVAPKLVATVHGDTSTTMAQPLDGLGALCRAAGALSYVDATATIGGMDIASDRWEVDVVTAGLQKCLGGPSGSAPITVSAAAAEAIFARRHVERGIVREDIANGSGPRIASNYFDLAMIMDYWSDKRLNHHTEATTMLYGARECARVALQEGLEARYARHAAAGRAVSAGVRALGLEVFGDDAHRMSNVTGVVIPHGVDSEAVRRRMREDFEIEIGTAFGPLQGRIWRIGAMGYNAMKHKVLLTLAALEAVLRAEGYACTQGLAVEAARAAWHAEPAA>pQR1752ATGGGAAAGTTTCTTAAGAAACACTACATAATGGCACCTGGACCAACACCAGTCCCAAACGATATTTTAACAGAAGGAGCGAAGGAAACAATACACCACAGAACACCTCAGTTTGTTTCCATAATGGAAGAGACCCTCGAAAGTGCAAAGTACATCTTTCAGACAAAACACAACGTGTACGCCTTTGCTTCCACAGGAACTGGCGCTATGGAAGCGGCGGTGGCGAATCTTGTGAGCCCTGGAGACAAAGTGATCGTGGTTGTGGCTGGAAAGTTCGGTGAAAGATGGAGAGAGCTCTGTCAGGCTTACGGTGCTGATATCGTAGAAATCGCCCTCGAATGGGGAGACGCGGTCACACCTGAACAGATCGAAGAGGCTCTCAACAAAAACCCCGATGCGAAGGTCGTCTTCACCACCTACAGTGAAACATCGACGGGTACAGTCATAGACCTCGAAGGAATTGCCAGAGTCACGAAGGAAAAAGACGTTGTTCTTGTGACAGACGCTGTCAGCGCTCTTGGAGCAGAACCACTGAAGATGGATGAATGGGGTGTGGATCTCGTTGTCACAGGTTCACAGAAGGGTTTGATGTTACCTCCAGGACTGGCGCTCATCTCTCTCAACGACAAAGCGTGGGGGCTCGTGGAAAAATCCAGATCTCCAAGGTACTACTTCGATCTGAGGGCCTACAGGAAATCTTACCCCGACAATCCTTACACCCCCGCAGTAAACATGATATACATGTTGAGAAAGGCTCTTCAGATGATAAAAGAGGAAGGCATAGAAAACGTATGGGAAAGGCACAGAATACTGGGAGACGCAACAAGAGCAGCGGTGAAAGCACTTGGACTGGAACTCCTCTCGAAAAGACCGGGAAACGTTGTAACAGCCGTGAAAGTGCCTGAGGGCATCGATGGAAAACAGATTCCCAAGATCATGAGAGACAAGTACGGTGTGACCATCGCCGGTGGACAGGCTAAACTCAAGGGAAAAATATTCAGGATAGCACACCTCGGATACATGTCACCTTTCGACACCATAACTGCCATTTCCGCTCTTGAATTAACCTTGAAGGAACTCGGTTATGAGTTCGAACTCGGAGTCGGTGTTAAGGCAGCCGAAGCTGTCTTCGCTAAAGAATTCATTGGGGAGTGA>pQR1755MGKFLKKHYIMAPGPTPVPNDILTEGAKETIHHRTPQFVSIMEETLESAKYIFQTKHNVYAFASTGTGAMEAAVANLVSPGDKVIVVVAGKFGERWRELCQAYGADIVEIALEWGDAVTPEQIEEALNKNPDAKVVFTTYSETSTGTVIDLEGIARVTKEKDVVLVTDAVSALGAEPLKMDEWGVDLVVTGSQKGLMLPPGLALISLNDKAWGLVEKSRSPRYYFDLRAYRKSYPDNPYTPAVNMIYMLRKALQMIKEEGIENVWERHRILGDATRAAVKALGLELLSKRPGNVVTAVKVPEGIDGKQIPKIMRDKYGVTIAGGQAKLKGKIFRIAHLGYMSPFDTITAISALELTLKELGYEFELGVGVKAAEAVFAKEFIGE>pQR1755ATGACTCAGATTTTTAATTTTAGCGCCGGTCCAGCAATGCTGCCGGTTGAAGTACTGCGTCGTGCTGAACAGGAATTGTGTAATTGGAATGGCCTGGGCACATCGGTTATGGAAATCAGCCACCGCAGTAAAGAGTTTATGCAGGTTGCCGCTGAATCCGAACAGGATCTGCGTGATTTGCTGAAAATCCCCTCCAACTACAAAGTGCTCTTTTGCCACGGCGGTGCTCGTGCGCAATTCGCCGCAGTGCCGTTAAATCTTCTGGGCGAACGCTCAACGGCCGACTACATCGACGGCGGGTATTGGGCGCACAGCGCAATCAATGAAGCAGAAAAATACTGCACGCCTAACGTGATTGACGTGAAAATGCGCGTGGGCGAACTGCGTGGCATTAAGCCGATGCGTGAATGGAAATTGTCTGATGACGCGGCGTTTGTGCATTACTGCCCGAATGAAACCATCGACGGTATTGCGATCGAAGAAGAGCCGGACTTTGGCGATAAAATTGTGGTCGCCGACTATTCTTCCAGCATCCTGTCTCGTCGTATTGATGTCAGCCGTTACGGCGTGATCTATGCCGGTGCGCAGAAAAATATCGGCCCTGCCGGCCTGACGCTGGTTATCGTACGTGAAGATTTGCTGGGCAAGGCGCGCCGTGAGCTGCCATCGATTCTGGATTACCAGGTTCTGGCGGACAATGACTCCATGTTTAACACGCCACCGACCTTTGCCTGGTACCTGTCCGGTATGGTCTTCAAATGGCTGAAAGAGTACGGCGGTCTGGCTGAAATGGAAAAACGTAACCAGGAGAAGGCTGACCTGCTGTATAGCGCGATTGACGGTAACGATTTCTATCGTAATGACGTTGCGGTAGCGAACCGTTCTCGCATGAATGTGCCATTCCTGTTGGCGGATTCTGCGCTGGATAAAGTCTTCCTGGAAGAATCAGTCGCTGCAGGTCTGCACGCGCTGAAAGGCCATCGCGTAGTAGGCGGCATGCGTGCCTCTATCTACAATGCGATGCCGTTGGAAGGCGTGAAAGTGCTGACGGAATTTATGGCTGACTTCGCTCGTCGCCACGGTTGAMTQIFNFSAGPAMLPVEVLRRAEQELCNWNGLGTSVMEISHRSKEFMQVAAESEQDLRDLLKIPSNYKVLFCHGGARAQFAAVPLNLLGERSTADYIDGGYWAHSAINEAEKYCTPNVIDVKMRVGELRGIKPMREWKLSDDAAFVHYCPNETIDGIAIEEEPDFGDKIVVADYSSSILSRRIDVSRYGVIYAGAQKNIGPAGLTLVIVREDLLGKARRELPSILDYQVLADNDSMFNTPPTFAWYLSGMVFKWLKEYGGLAEMEKRNQEKADLLYSAIDGNDFYRNDVAVANRSRMNVPFLLADSALDKVFLEESVAAGLHALKGHRVVGGMRASIYNAMPLEGVKVLTEFMADFARRHG


To produce synthetic nucleotides of notifiable dengue virus (1–4 types), Japanese encephalitis, yellow fever and Zika flaviviruses. These notifiable flaviviruses, particularly dengue and Zika, are problematic mosquito-borne infections in the Philippines, as well as in those countries with tropical and subtropical climates.An algorithmic design formulation of overlap extension – polymerase chain reaction (OE-PCR) was performed to propagate 50–60 oligomer lengths of select notifiable flaviviral RNAs to DNA nucleotides via the two-step process of OE-PCR.Algorithmic OE-PCR design formulation efficiently produced 253–256 bp of notifiable flaviviruses. Comparing the newly designed algorithmic OE-PCR with existing executable programs demonstrated it to be efficient and useful in generating accurate sequences of synthetic flaviviral nucleotides.The efficiently and accurately produced novel synthetic nucleotides of notifiable dengue virus 1–4, Japanese encephalitis, yellow fever and Zika flaviviruses using OE-PCR is useful in understanding the dynamics of flaviviral species and holds potential for the development of synthetic nucleotide-based immunogens. Dengue virus (1–4), Japanese encephalitis and Zika fevers are notable mosquito-borne infections that continue to be problematic in the Philippines and its southeast Asian neighbors. Flaviviral yellow fever affects other tropical countries. This study aimed to effectively and efficiently synthesize flaviviral nucleotides through a predictively accurate algorithmic overlap extension-polymerase chain reaction design. The cost-saving arithmetic formulation has enabled the advanced production of 253–256 bp flaviviral nucleotide lengths. The newly synthesized and sequenced products are now ready for further experimentation and open the door for immunological exploration.  Dengue and Zika fevers, including Japanese encephalitis (JEV), are particularly burdensome mosquito-borne infections in the Philippines and are also selectively reported in Japan . The chaThe OE-PCR in this experiment was performed via two processes: the first one is using the reference or original OE-PCR protocol ,12 and t1.Preparation of the first-stage primer mixed solution(A) Both ends primer  2.5 μl(B) Central primer (2–5) 0.5 μl(U) Deionized water (DW) 18 μl2.Preparation of reaction solution for the first-step PCR(A) First-stage primer mixed solution × 6 μl(A) GoTaq 12.5 μl(U) DW 6.5 μl3.First-stage PCR reaction(A) 95°C 2 min21(C) 72°C - 2 min4.Preparation of the second-step PCR solution(A) First-step PCR reaction solution 1 μl(B) Both ends primer (stock solution) 1 μl × 2(C) GoTaq 12.5 μl(D) 9.5 μl of DW5.Second-step PCR reaction(A) 95°C 2 min(A) (95°C - 1 min → 72°C - 2 min) × 35-times (C) 72°C - 2 min1. Preparation of the first-stage primer-mixed solution(A) Both ends primer  2.5 μl(B) Central primer (2–5) 0.5 μl(U) DW 193 μl(A) GoTaq 12.5 μl(U) DW 6.5 μl3. First-stage PCR reaction(A) 95°C 2 min(A) (95°C - 1 min → 48°C - 1 min → 72°C - 1 min) × 20(C) 72°C - 2 min4. Preparation of the second-step PCR solution(A) First-step PCR reaction solution 1 μl(B) Both ends primer (stock solution) 1 μl × 2(C) GoTaq 12.5 μl(D) 9.5 μl of DW5. Second-step PCR reaction(A) 95°C 2 min(B) (95°C. -1 min → 72°C. -2 min) × 25(C) 72°C. -2 minDepicted in In the second PCR, the most outside oligomers work for PCR primers and amplified the whole size of target DNA.An electrophoresis of OE-PCR products was done for PCR product-size prediction after amplification of both OE-PCR and pathogen-specific PCR. Single product was confirmed on 1.5% agarose gel electrophoresis with 1 × Tris/Borate/EDTA (TBE) buffer and UV-visualized with 0.7 micro-g/ml-ethidium bromide . The PCRThe following notable flaviviruses, namely dengue (4 types), YF, JEV and Zika, were specifically processed for production of artificial DNAs. The algorithmic formulated OE-PCR was subsequently carried out in two-step cycles, with each cycle's DNA products depicted via UV-illuminated bands following gel electrophoresis . In the DNA sequencing was carried out in similar steps as previously described ,7 Eurof.Approaches to determine efficiency and cost–effectiveness of the current algorithmic OE-PCR tool versus the existing programs are presented in It must be noted that the price of oligomers varies greatly depending on the length in base and according to the viral species involved. Owing to this, an algorithm was designed to achieve the maximum length within the lowest cost range in base size. Unfortunately, this is difficult to realize using the currently available reported design algorithms. In the stated design algorithms, the difficulty lies in how to control the length of the oligomer. This is because the setting of overlapping region of the oligomer has been designed first. With the current study, the basic length has been decided to begin with; coupled with a lowered priority on the thermodynamic stability of the overlapping region. This has resulted in fewer problems in the conduct of OE-PCR.The ongoing dilemma of largely problematic and imperfectly developed flaviviral immunogens prompted us to responsibly contribute on the need to develop an efficient algorithmic OE-PCR program design for synthetic nucleotide production of select notifiable dengue (1–4), JEV, YF and Zika flaviviruses. To this end, we developed an OE-PCR program that can be utilized as reference tool in the furtherance of flaviviral research. It can be deduced that the program formulation was found to be demonstrably reproducible, cost-effective and efficient in yielding substantial nucleotides of select notifiable flaviviral DNAs, which can be sourced out at the databases of DDBJ/NCBI with the respective accession numbers of LC227563, LC227561, LC227562, and LC227568 (dengue 1–4); LC227560 (JEV); LC227567 (YF); and LC227589 (Zika). The OE-PCR is a reliable method that can be utilized when designed and accurately predicted using algorithmic manipulation. Henceforth, we finally conclude that the DNA primer design using algorithmic OE-PCR is a valuable tool in producing synthetic nucleotide products of pathogenic species of flaviviruses.in vivo would result in pathological or immunological response.The novel nucleotide sequences generated from this study can be utilized for studies looking to further understand the genomic diversity of a variety of flaviviral strains, including dengue (1–4), YF, JEV and Zika. In addition, clinical trials involving initial utilization of whole nucleotide sequence followed by booster immuno-stimulation of antigenic protein may serve as a starting point for generating long-term immune responses for flaviviral infections such as dengue and Zika, where trials for DNA-based vaccines are ongoing, yet faced with challenging dose-dependency concerns. Further experiments should be conducted to determine whether administration of synthetic nucleotides Box 1. flavi |2|1: ATGTATGCCGATGACACCGCAGGATGGGATACAAGAATCACACTAGAAGACCTAAAAAAT: 60flavi |2|2: TTGTGTTCTCCTTCCATGTGGTTTGTTACCATTTCTTCATTTTTTAGGTCTTCTAGTGT: 59flavi |2|3: CATGGAAGGAGAACACAAGAAACTAGCCGAGGCCATTTTCAAACTAACGTACCAAAACA: 59flavi |2|4: TGTGCCTCTTGGTGTTGGTCTTTGCACACGCACCACCTTGTTTTGGTACGTTAGTTTG: 58flavi |2|5: CAACACCAAGAGGCACAGTAATGGACATCATATCGAGAAGAGACCAAAGAGGTAGTGGA: 59flavi |2|6: TGGTGAAAGTATTGAGTCCATAGGTGCCAACTTGTCCACTACCTCTTTGGTCTC: 54flavi |3|1: ATGTATGCTGATGACACAGCAGGCTGGGACACAAGAATCACTGAGGATGACCTTCAAA: 58flavi |3|2: TGGTGGGGAGCCATCTGTTCCGTGATCAGTTCCTCATTTTGAAGGTCATCCTCAGT: 56flavi |3|3: TGGCTCCCCACCACAAGATCCTAGCCAAAGCCATTTTCAAACTAACCTATCAAAACAA: 58flavi |3|4: TCCCCGCGGTGTGGGTCTGAGGACTTTCACCACTTTGTTTTGATAGGTTAGTTTG: 55flavi |3|5: CACCGCGGGGAGCGGTGATGGATATCATATCCAGGAAAGACCAAAGAGGTAGTGGA: 56flavi |3|6: TGGTGAATGTGTTCAAACCATATGTTCCAACTTGTCCACTACCTCTTTGGTCTT: 54flavi |5|1: ATGTATGCAGATGACACAGCCGGATGGGACACAAGAATAACAGAGGATGATCTTCAGAA: 59flavi |5|2: CATGTTCAGGTTCCATGATGTCAGTGATTTTGGCCTCATTCTGAAGATCATCCTCTGT: 58flavi |5|3: CATGGAACCTGAACATGCCCTATTGGCCACGTCAATCTTTAAGCTAACCTACCAAAACA: 59flavi |5|4: GTTCCATTTTTCGCTGGTCTCTGCACCCTTACTACCTTGTTTTGGTAGGTTAGCTTA: 57flavi |5|5: AGCGAAAAATGGAACCGTGATGGATGTCATATCCAGACGTGACCAGAGAGGAAGTGG: 57flavi |5|6: TGGTGAAGGTGTTTAAGCCATAGGTTCCAACCTGTCCACTTCCTCTCTGGTCA: 53flavi |8|1: ATGTATGCTGATGACACAGCTGGTTGGGACACAAGAATAACAGAAGATGACCTGCACA: 58flavi |8|2: TGTGTTCAGGGTCCATTTGCTGTATGATCTTTTCCTCATTGTGCAGGTCATCTTCTG: 57flavi |8|3: AATGGACCCTGAACACAGGCAGTTAGCGAACGCTATATTCAAGCTCACATACCAAAACA: 59flavi |8|4: TGCCCGTTGGAGTCGGTCGTTGAACTTTGACCACTTTGTTTTGGTATGTGAGCTTG: 56flavi |8|5: ACTCCAACGGGCACGGTAATGGATATTATATCTAGGAAAGACCAAAGGGGCAGT: 54flavi |8|6: TGGTGAATGTATTCAGGCCATAAGTTCCCAGTTGTCCACTGCCCCTTTGGTC: 52flavi |15|1: ATGTACGCAGATGACACTGCTGGCTGGGACACCCGCATTAGTAAGTTTGATCTGGAGAA: 59flavi |15|2: GTGCCCTTCCTCCATTTGGTTGGTAATCAGAGCTTCATTCTCCAGATCAAACTTACT: 57flavi |15|3: TGGAGGAAGGGCACAGAACTCTGGCGTTGGCCGTGATTAAATACACATACCAAAACAA: 58flavi |15|4: TTTTTCCTCCTTCAGCTGGTCTGAGAACCTTCACCACTTTGTTTTGGTATGTGTATTTA: 59flavi |15|5: CAGCTGAAGGAGGAAAAACAGTTATGGACATCATTTCAAGACAAGACCAGAGAGGGAG: 58flavi |15|6: TGGTGAATGTGTTGAGAGCATAAGTGACAACTTGTCCACTCCCTCTCTGGTCTTGTC: 57flavi |25|1: TTCTACGCGGATGACACCGCTGGATGGGACACGCGCATCACAGAGGCAGACCTTGAT: 57flavi |25|2: TGTGATGTGGGCTCATGTAGTTCAAGATCTCCTGTTCATCATCAAGGTCTGCCTCTG: 57flavi |25|3: ATGAGCCCACATCACAAAAAACTGGCACAAGCAGTGATGGAAATGACATACAAGAACA: 58flavi |25|4: TTCCCTCCTGGGGCTGGTCTCAACACTTTCACCACTTTGTTCTTGTATGTCATTTCC: 57flavi |25|5: GCCCCAGGAGGGAAAGCCTACATGGATGTCATAAGTCGACGAGACCAGAGAGGAT: 55flavi |25|6: TGGTGATGGTGTTCAGAGCATAAGTCACTACCTGCCCGGATCCTCTCTGGTCTCGT: 56flavi |27|1: ATGTACGCTGATGACACCGCCGGATGGGACACTAGAATTACCAGAACTGATTTAGAAAA: 59flavi |27|2: GGTGTTCACCGTCTAGGAGCTCCAGCACCTTGGCTTCATTTTCTAAATCAGTTCTGGT: 58flavi |27|3: CCTAGACGGTGAACACCGCATGCTCGCTCGAGCCATAATTGAACTGACTTACAGGCACA: 59flavi |27|4: CTTTCCTTCTGCTGCAGGTCTCATGACCTTGACCACTTTGTGCCTGTAAGTCAGTTCA: 58flavi |27|5: TGCAGCAGAAGGAAAGACCGTAATGGACGTGATATCAAGAGAAGATCAAAGGGGGAG: 57flavi |27|6: TCGTGAAAGTGTTAAGGGCATAAGTGACCACCTGTCCACTCCCCCTTTGATCTTCT: 56This project aimed to develop synthetic nucleotides using overlap extension-polymerase chain reaction (OE-PCR), particularly of flaviviral organisms that are notorious for their ability to cause both animal and human diseases.A method is provided for generating template designs that could help to address epidemics caused by select flaviviral dengue, Zika and related species.Expansion of OE-PCR as a tool in developing templates for viral studies is presented, addressing concern regarding the lack of reported commercial production of artificial nucleotides of these flaviviruses.This research has delivered a reproducibly effective and efficient algorithmic OE-PCR design that is suitable for yielding substantial nucleotides of these flaviviruses.These synthetic nucleotides could pave the way for future collaborative research exploring the possibility of generating synthetic flaviviral nucleotide-based immunogens.