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Terminator Regions, Genetic

Terminator Regions, Genetic: Specific DNA sequences that inhibit the expression or transcrption of adjacent genes.
These regions can be used to prevent the unintended spread of genetically modified organisms by limiting the ability of the organism to reproduce.
They play a key role in ensuring the safety and containment of genetic modifications, and are an important consideration in the development of biotechnological products and protocols.
Terminator Regions help enhance the reproducibility and reliability of genetic research by providing a robust mechanism for controlling genetic expression.

Most cited protocols related to «Terminator Regions, Genetic»

Genomic data for the 343 organisms was downloaded on 6 June 2006 from [25 ]. Annotations were taken from the .ptt file accompanying the genomic sequence. Accession and version numbers for the sequences used are available as Additional data file 3. TransTermHP was run using the default search parameters and the -p scoring parameter to produce predictions for the best terminator following each gene in these organisms (available on the web [18 ], --bag option to TransTermHP) as well as the best terminators in each robust tail-to-tail region (Additional data file 2, --t2t-perf option to TransTermHP). The former were used when comparing performance to [3 (link)], while the latter was used to assess the importance of Rho-independent termination within an organism. Experiments were run on a 3.2 GHz Intel Xeon processor running Linux. High-confidence terminators are those with scores ≥ 76.
The C++ source code for TransTermHP is available (Additional data file 1) under an open source license.
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Publication 2007
Genes Genome Tail Terminator Regions, Genetic
In most cases the respective inserts were PCR amplified to create the GreenGate entry modules. The nucleotides 5′-AACA-GGTCTC-A-NNNN (nn)-3′ were added to the forward primer in front of the gene specific sequence. GGTCTC is the BsaI recognition site, AACA was added because the enzyme does not cut if the restriction site is at the extreme ends of PCR products. NNNN represents the module specific overhang and 2 nucleotides (nn) are needed in case of the coding sequence and C-tag modules to bring the modules into frame (NNN represents an in-frame coding triplet in the overhangs). The sequence 5′-AACA-GGTCTC-A-NNNN-3′ was added to the reverse primers, followed by the reverse complement of the sequence of interest. NNNN stands for the reverse complement of the module specific overhang, the coding triplet being underlined.
After amplification, the PCR reactions were separated on agarose gels, the product bands excised, purified with innuPREP DOUBLEpure Kit (Analytik Jena AG, Jena, Germany) and digested with BsaI. The respective empty entry modules (∼ 100 ng) were also cut with this enzyme, usually in the same tube (1 h, 37°C). The digestion was purified with the above mentioned kit and ligated with T4 DNA ligase (1 h room temperature, overnight 4°C). After heat-inactivation (10 min, 70°C) the reaction was transformed via heat shock into ccdB sensitive E. coli strains (Mach1™-T1R, DH5α, XL1-Blue MR). Transformants were checked by colony PCR, plasmid DNA was isolated from positive clones and checked by sequencing and test digestion.
If internal BsaI recognition sites were present in the module sequence, they were removed by nucleotide substitution. For protein-coding sequences, silent mutations were chosen. In promoter and terminator sequences, the nucleotides to be changed were selected at random, but for later constructs we switched to always replace the first guanine by a cytosine. For simplicity, we used scar-free BsaI-cloning to create the substitutions. Primers were designed on both sides of the internal BsaI recognition sites introducing the mismatch and flanked on their 5′-ends by external BsaI recognition sites. The overhangs generated by the external BsaI cut were designed to be part of the gene-specific sequence and being different from the module specific overhangs.
Shorter modules were assembled as oligonucleotide duplices created from overlapping primers with unpaired 5′-overhangs complementary to the module specific overhangs. The oligonucleotides (10 µM or 100 µM) were mixed in equimolar ratios with each other, soused with boiling water, allowed to cool slowly down to room temperature and then ligated into BsaI digested and purified entry vector.
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Publication 2013
Cicatrix Cloning Vectors Cytosine Digestion Enzymes Escherichia coli Gels Genes Guanine Heat-Shock Response Nucleotides Oligonucleotide Primers Oligonucleotides Open Reading Frames Plasmids Reading Frames Sepharose Silent Mutation Strains T4 DNA Ligase Terminator Regions, Genetic Triplets

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Publication 2013
Cells Cloning Vectors Codon Deoxyribonuclease EcoRI Doxycycline Fluorescence Genes Genome Inverse PCR Leucine Ligation lithium acetate Mammals MXI1-0 protein, human Nuclear Localization Signals Plasmids Proteins Repression, Psychology Simian virus 40 Strains Terminator Regions, Genetic Uracil

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Publication 2009
Alkaline Phosphatase alkaline phosphatase, placental Alleles BP 42 Codon, Terminator FLP recombinase Germ Line Homologous Recombination Homo sapiens Mouse Embryonic Stem Cells Mus Simian virus 40 Terminator Regions, Genetic Transcription, Genetic
DNA encoding the actin binding domain of Drosophila moesin and the SV40 terminator sequence were cloned by PCR and inserted into NotI-BglII and BglII-EcoRI sites of pCASPER2 respectively. 1160bp of Drosophila genomic DNA immediately upstream of the patched coding region was fused to DNA encoding eGFP by PCR and the resulting fragment was cloned into pCR4TOPO (Invitrogen) according to the manufacturer's instructions. Genomic DNA from 11200 to 1160bp upstream of ptc was then inserted upstream of this as a BamHI-NdeI fragment. Finally, the complete patched-GFP fragment was inserted into pCASPER2 upstream of moesin as a BamHI-NotI fragment.
Publication 2008
Actins Deoxyribonuclease EcoRI Drosophila Genome moesin, Drosophila MSN protein, human Simian virus 40 Terminator Regions, Genetic

Most recents protocols related to «Terminator Regions, Genetic»

The DNA fragments MMV FLt (-193 to +63 bp), MMV Sgt (-306 to -125 bp), and FMV Sgt (-270 to -63 bp) were chemically synthesized (Gene Universal, Inc., Newark, USA) (Supplemental Table 1). The FM′M promoter was first synthesized as a single DNA fragment. Subsequently, each domain was amplified by PCR using the specific primers MMFg-F, MMSg-F, FsMf-F, FsMf-R, M′FM-R, M′FM-F, and FM′M-R (Supplemental Table 2). To insert the UAS×4 motif and/or the single or double zinc finger binding motifs into the FM′M promoter, the primers FM′M-U-F and FM′M-R or FM′M-US-F and FM′M-US-R or FM′M-UD-F and FM′M-UD-R were used for overlapping PCR (Supplemental Table 2). All promoter fragments were digested with PstI and XbaI, and ligated into pCAMBIA1300, digested with the same restriction endonucleases. The BiP : GFP:HDEL recombinant construct (Islam et al., 2020 (link)) was ligated to each of the hybrid promoters following digestion with the restriction endonucleases XbaI and XhoI. The reporter gene encoding BiP : RBD:SD1:6×His : HDEL (Bangaru et al., 2020 (link)) was digested with the restriction endonucleases XbaI and XhoI, and ligated to the FM′M-UD or CaMV 35S promoters that had previously been digested with the same restriction endonucleases. The recombinant construct, BiP : MP:CBM3:bdSUMO:hIL6:HDEL (Islam et al., 2019 (link)), was ligated to the FM′M-UD or CaMV 35S promoters following digestion with XbaI and XhoI restriction endonucleases. All these constructs contained the RD29B terminator from Arabidopsis thaliana RD29B (D.13044.1) or the recombinant 3PR terminator (Supplemental Table 1). The 3PR terminator sequence was chemically synthesized (Gene Universal, Inc., Newark, United States). These terminators were ligated into the constructs after digestion with restriction endonucleases XhoI and EcoRI. The DNA fragments encoding the transcription factors GAL4:VP16, GAL4:TAC3d2, and ZinC7:TAC3d2 were chemically synthesized (Gene Universal, Inc., Newark, United States), and contained XbaI and XhoI restriction endonuclease sites. The MacT promoter and RD29B terminator were used for the expression of these transcription factors. All the primer sequences used in this study are listed in Supplemental Table 2.
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Publication 2023
Arabidopsis thalianas Deoxyribonuclease EcoRI Digestion DNA Restriction Enzymes endodeoxyribonuclease XhoI Genes Genes, Reporter Hybrids Oligonucleotide Primers Terminator Regions, Genetic Transcription Factor VP-16 Zinc Fingers
In order to investigate the activity of PrWRI1 visually, the coding sequence of PrWRI1 was cloned into the SacII and BamHI restriction sites of pK34 entry vector, and then the recombinant pK34 vector with double CaMV 35S promoters and a terminator sequence was digested with AscI for entry into plant expression vector, pB110. The vector was then transiently expressed in Nicotiana benthamiana leaves by Agrobacterium-mediated transformation.
The ORF of PrWRI1 were inserted into the vector pCAMBIA1300 under the control of Arabidopsis seed-specific promoter 2S2 by the digestion of KpnI and BamHI. The generated constructs were transformed into Agrobacterium tumefaciens GV3101 using the freeze–thaw method, and then they were used for the transformation of wild-type Arabidopsis by the floral dip method.
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Publication 2023
Agrobacterium Agrobacterium tumefaciens Arabidopsis Cloning Vectors Digestion Freezing Nicotiana Open Reading Frames Plants Terminator Regions, Genetic
Four PhaC amino acid sequences were chosen based on the BLASTP search results. These phaC genes were chemically synthesized with optimized codon usage in E. coli by Eurofins Genomics Co. Ltd. (Tokyo, Japan) for plasmid construction and evaluation. E. coli LSBJ, a fadB fadJ double-deletion strain of E. coli LS5218 [fadR601, atoC (Con)] (Tappel et al., 2012a (link)), was used as the host strain for PHA biosynthesis. This strain is an ideal host for non-native PHA production because of its ability to take up a wide variety of substrates to be incorporated into PHA homo- and copolymers, and bench-level scale-up methodologies available for overall production (Tappel et al., 2012b (link); Levine et al., 2016 (link); Pinto et al., 2016 (link); Fadzil et al., 2018 (link); Furutate et al., 2021 (link); Scheel et al., 2021 (link)). A broad-host-range plasmid pBBR1MCS-2 (Kovach et al., 1995 (link)) harboring the genes encoding the PhaCs to be evaluated, the lac promoter region, the (R)-specific enoyl-CoA hydratase gene from A. caviae (phaJAc), the 3-ketothiolase gene (phaA) from Ralstonia eutropha H16, and the acetoacetyl-CoA reductase gene (phaB) from R. eutropha H16, termed pBBR1-phaCsABReJAc, was used for the expression of PhaCs (Supplementary Figure S1). For phaAB expression, the R. eutropha pha promoter and terminator regions were located upstream and downstream of their genes, respectively. To enhance the supply of 3HHx, 3H4MV, and 3H2MB monomers, the plasmid pTTQ-PCT (Furutate et al., 2017 (link)) containing the propionyl-CoA transferase (PCT) gene from Megasphaera elsdenii (pct) (Taguchi et al., 2008 (link)) was introduced into the E. coli LSBJ strain (Supplementary Figure S1).
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Publication 2023
acetoacetyl-CoA reductase Acetyl-CoA C-Acyltransferase Amino Acid Sequence Anabolism Codon Usage Coenzyme A-Transferases Cupriavidus necator Deletion Mutation Escherichia coli Genes Homo Host Range Megasphaera elsdenii Plasmids propionyl-coenzyme A R-specific trans-2,3-enoylacyl-CoA hydratase Strains Terminator Regions, Genetic Transferase
The split marker method was used to construct the CcPtc1 gene deletion (ΔCcPtc1) mutants as previously described (Catlett et al., 2003 (link); Goswami, 2012 (link)). According to this method, the upstream (∼1.3 kb) and downstream (∼1.2 kb) flanking sequences of CcPtc1 were amplified by primer pairs CcPtc1-5Ffor/CcPtc1-5Frev and CcPtc1-3Ffor/CcPtc1-3Frev, respectively (Supplementary Table 1). The hygromycin B resistance cassette (HPH) was amplified by specific primer pairs hygromycinfor and hygromycinrev, which included approximately 20 bp overlapped 5′ and 3′flanking sequences, respectively. Then, the resulting upstream and downstream fragments were fused with two-thirds of the hygromycin B resistance cassette by overlap PCR using primer pairs CcPtc1-5Ffor/HY-R and YG-F/CcPtc1-3Frev, respectively. The two overlapping fragments were directly transformed into protoplasts of the WT strain by using the PEG-mediated transformation, and the transformants were selected on TB3 agar medium supplemented with 20 μg/ml hygromycin B. All transformants were identified by PCR assays with the primer pairs External-CcPtc1for/External-CcPtc1rev and Internal-CcPtc1for/Internal-CcPtc1rev to screening the successful replacement transformants. In addition, to analyze homologous recombination events in the transformants, southern blotting analysis was performed with the DIG High Prime DNA Labeling and Detection Starter Kit I, following the manufacturer’s protocol (Roche, Germany). BglI was used to digest the genomic DNA extracted from the WT strain and the transformants. The probes were amplified by the primers ProbeHPHfor and ProbeHPHrev from HPH and the primers ProbeCcPtc1for and ProbeCcPtc1rev for CcPtc1.
To generation the CcPtc1 gene complementation construct, a fragment containing the entire length of the CcPtc1 coding region along with native promoter sequence and terminator sequence was cloned from gDNA using the primer pair CcPtc1-Compfor/CcPtc1-Comprev. The resulting PCR products were co-transformed into protoplasts of the ΔCcPtc1-11 strains with a geneticin-resistant cassette. After that, we selected the transformants in TB3 medium supplemented with 40 μg/ml geneticin. Successful complementation was confirmed by PCR with the primer pair Internal-CcPtc1for/Internal-CcPtc1rev. The complementation strain was named ΔCcPtc1/PTC1 in this study. All primers used in gene deletion and complementation were listed in Supplementary Table 1.
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Publication 2023
3' Flanking Region Agar Biological Assay Gene Deletion Genes Geneticin Genome Homologous Recombination Hygromycin B Oligonucleotide Primers Protoplasts PTCH1 protein, human Southern Blotting Strains Terminator Regions, Genetic
Plasmids used for testing and comparing the different 2A peptides were assembled using the GoldenBraid cloning system, which has been described previously [12 (link)]. Each 2A peptide used was codon optimized for expression in D. discoideum using the IDT Codon Optimization Tool (Integrated DNA Technologies), synthesized by GenScript, and then cloned into a GoldenBraid pUPD2 vector with the appropriate overhangs to act as a C-terminal linker part. Each transcriptional unit was assembled with an actin15 promoter and an actin8 terminator, and finally assembled into a backbone with a G418-resistance cassette and a D. discoideum origin of replication. We used mNeonGreen as a fluorescent protein in all constructs for comparison, mCherry as a red fluorescent protein for all western blotting experiments due to the availability of an antibody with high affinity and specificity, and mScarlet-I as a red fluorescent protein for flow cytometry because it is brighter than mCherry and thus easier to assay [33 (link)].
Plasmids for comparing dual cassette and P2A-linked antibiotic resistance gene expression were based on pDM1203 [15 (link)]. Note that as a result, all the plasmids for this set of experiments use different actin15 promoter and actin8 terminator sequences derived from this parent plasmid. To make the dual cassette plasmid pDMDC a15-mNG coaA-HygroR, a codon-optimized mNeonGreen was first cloned into the BglII/SpeI cloning site of pDM1203 and the G418-resistance cassette was swapped for a hygromycin-resistance gene by restriction digest with NheI/NotI and Gibson assembly. The hygromygin resistance fragment was obtained using pDM1501 [15 (link)] as a PCR template. In contrast to all other plasmids in this study, the resulting dual cassette plasmid is bidirectional, with the antibiotic resistance gene encoded in one direction on the plasmid and the mNeonGreen encoded in the other direction. To make the P2A-linked plasmid pDMP2A a15-mNG-P2A-HygroR, the resistance cassette was removed from pDM1203 by XhoI/BamHI digest followed by blunt-ending and religating the resultant plasmid. A codon-optimized mNeonGreen was then inserted into the BglII/SpeI site along with P2A-HygroR by Gibson assembly. The expression cassettes of the generated plasmids were verified by Sanger sequencing with GENEWIZ (Azenta Life Sciences).
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Publication 2023
antibiotic G 418 Antibiotic Resistance, Microbial Biological Assay Cloning Vectors Codon Flow Cytometry Gene Expression Genes hygromycin A Immunoglobulins mCherry fluorescent protein Parent Peptides Plasmids Replication Origin Staphylococcal Protein A Synapsin I Terminator Regions, Genetic Transcription, Genetic Vertebral Column

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More about "Terminator Regions, Genetic"

Terminator sequences, genetic containment, genetic safeguards, expression control, biological confinement, transcriptional regulation, genetic modifications, biotechnology protocols, research reproducibility, genetic experimentation, gene expression, DNA sequences, genetic engineering, molecular biology, In-Fusion cloning, Phusion polymerase, PCR purification, Sanger sequencing, DNA ligase, hot start master mix, cloning vectors, antibiotic resistance, site-directed mutagenesis.
Terminator Regions are specific DNA sequences that inhibit the expression or transcription of adjacent genes.
These genetic safeguards play a crucial role in ensuring the safety and containment of genetically modified organisms by limiting their ability to reproduce.
They help prevent the unintended spread of GMOs and enhance the reliability and reproducibility of genetic research.
Terminator Regions provide a robust mechanism for controlling gene expression, which is an important consideration in the development of biotechnological products and protocols.
PubCompare.ai's innovative tools leverage Terminator Regions and genetic comparisons to help researchers identify the best protocols and products for their studies, simplifying workflows and improving the accuracy of their findings.
By incorporating techniques like In-Fusion cloning, Phusion polymerase, PCR purification, Sanger sequencing, DNA ligase, hot start master mix, cloning vectors, and site-directed mutagenesis, researchers can further enhance the reproducibility and reliability of their genetic experiments.