11 results about "Transcription initiation" patented technology

The invention discloses a tool for realizing efficient specific gene inhibition in a plant by utilizing a SunTag system of CRISPR-dCas9 (clustered regularly interspaced short palindromic repeats-das9). According to the method, gRNA near a transcription start site of a targeted target gene is designed, dCas9 and scFv-GFP are accurately recruited to a target site by using a SunTag system, the transcription start process of RNA polymerase II is interfered, and the expression of the target gene is remarkably inhibited. In view of relatively high activity of the system in a plant rdr6 mutant, an artificial miR-RDR6 fragment is further introduced into a vector and is used for inhibiting expression of RDR6 in a wild plant, so that the applicability and functionality of the system are expanded. Compared with a traditional method depending on a plant endogenous effector, the method has the advantages that dependence on endogenous regulatory factors is avoided, the specificity and safety of gene inhibition are remarkably improved, and the method can be used for plant genetic function research and character improvement.

Provided herein are recombinant microorganisms that express a subject polypeptide. Microorganisms can comprise an expression construct comprising a flagellin promoter operatively linked with a heterologous nucleotide sequence encoding the subject polypeptide. The flagellin promoter sequence can comprise a genetic modification that reduces CsrA inhibition of translation. Microorganisms also can comprise a genetic modification that reduces FlgM inhibition of SigD initiation of transcription. The target polypeptide can be an aldehyde dehydrogenase. Such microorganisms are useful in the treatment of alcohol hangover.

The application discloses a kind of gene markers in esophagus, stomach, intestine multiple cancer early screening application, early screening model construction method and detection device, belong to the early non-invasive detection technical field of digestive tract tumor.It establishes a new type of multiple cancer screening system by analyzing the whole genome characteristics of circulating free DNA in peripheral blood.Based on low-depth whole genome sequencing data, three dimensions of molecular markers are detected: genome copy number variation pattern, DNA fragment distribution characteristics of specific length and epigenetic signals of transcription initiation region.Advanced converter neural network architecture is used, and the model can efficiently capture the complex feature correlation in the whole genome range through its unique self-attention mechanism.The model design specially considers the particularity of genomic data, and introduces an adaptive position coding system to accurately reflect the spatial distribution relationship of DNA fragments on the chromosome.The system can still maintain excellent detection performance at very low sequencing depth.

The present disclosure provides inducible constructs comprising transcription initiation and / or transcriptional regulatory elements. These constructs can be used to drive gene expression in response to specific cellular conditions (e.g., in the context of antigenic stimulation). In some embodiments, the constructs are incorporated as part of regulatory circuits useful for controlling the expression and regulation of cells expressing the engineered receptor.

The present disclosure provides inducible constructs comprising a transcription initiator and / or a transcription regulator. These constructs can be used to drive gene expression in response to a particular cellular state, such as in the context of antigen stimulation. In some embodiments, these constructs are integrated as part of a regulatory circuit, useful for controlling expression and regulation of cells expressing engineered receptors.

Disclosed are a combination and a method for synthesizing Cap2 structure 5'-capped RNA. The combination comprises a tetranucleotide cap analog and a DNA template. The transcription initiation combination can realize efficient preparation of 5'-capped RNA at a lower concentration of the tetranucleotide analog.

The invention discloses a recombinant conditionally replicated HIV (Human Immunodeficiency Virus) strain plasmid and application thereof. The plasmid is a lentivirus plasmid containing a coding gene of fluorescent protein, a coding gene of herpes stomatitis virus G protein, a Teton (R) 3G protein coding gene and a tetracycline control system; the transcription starting element on the plasmid is subjected to function inactivation; the truncated TRE3GS promoter on the plasmid is located in a truncated U3 region in the 3 'LTR and a truncated U3 region in the 5' LTR. The recombinant expression vector with a specific structure designed by the invention can be used for preparing conditional replicated viruses and used as a positive reference substance for replicated virus detection, the detection efficiency, sensitivity and accuracy are remarkably improved, and the recombinant expression vector is suitable for various detection methods, reliable in result and high in safety.

The invention discloses a method for improving plant pilot editing efficiency, which comprises the following steps: replacing AtU6 with an NtU26 promoter through a restriction enzyme site directional cloning technology, and respectively connecting the NtU26 promoter with two serially connected PegRNA sites. The method comprises the following steps: constructing a '35S promoter-Cmylc enhancer-NtU6-26' expression unit to replace a '35S promoter-Cmylc enhancer-AtU6', and ensuring that the transcription initiation of pegRNA is regulated and controlled by a 35S promoter-Cmylc enhancer-NtU6-26 promoter; the unique transcriptional regulation ability of the NtU6-26 promoter in tobacco cells is utilized, sufficient PegRNA substrates are provided for assembly of an editing compound (nCas9-RT / pegRNA) by improving the pegRNA transcriptional level and increasing the accumulation amount of the PegRNA, the order of magnitude improvement of tobacco pilot editing efficiency is achieved, precise replacement of AtU6 by the NtU26 promoter is achieved by utilizing restriction enzyme sites, uncertainty caused by random integration is avoided, and the method has the advantages that the method is simple and convenient to operate and high in practicability. The stability and repeatability of the gene expression unit are ensured; the NtU6-26 is designed for tobacco cells, and the unique transcriptional regulation ability of the NtU6-26 can significantly improve the transcriptional level of pegRNA and increase the accumulation amount of pegRNA in the cells.

The invention discloses a plant pilot editing method which comprises the following steps: replacing AtU6 with an NtU26 promoter through a restriction enzyme site directional cloning technology, and respectively connecting the NtU26 promoter with two PegRNA sites which are connected in series. The method comprises the following steps: constructing a '35S promoter-Cmylc enhancer-NtU6-15 'expression unit to replace a '35S promoter-Cmylc enhancer-AtU6', and ensuring that the transcription initiation of pegRNA is regulated and controlled by a 35S promoter-Cmylc enhancer-NtU6-15 promoter; the unique transcriptional regulation ability of the NtU6-15 promoter in tobacco cells is utilized, sufficient PegRNA substrates are provided for assembly of an editing compound (nCas9-RT / pegRNA) by improving the pegRNA transcriptional level and increasing the accumulation amount of the PegRNA, the order of magnitude improvement of tobacco pilot editing efficiency is achieved, precise replacement of AtU6 by the NtU26 promoter is achieved by utilizing restriction enzyme sites, uncertainty caused by random integration is avoided, and the method has the advantages that the method is simple and convenient to operate and high in practicability. The stability and repeatability of the gene expression unit are ensured; the NtU6-15 is designed for tobacco cells, and the unique transcriptional regulation ability of the NtU6-15 can significantly improve the transcriptional level of pegRNA and increase the accumulation amount of pegRNA in the cells.

The invention discloses a method convenient for plant pilot editing, which comprises the following steps: respectively carrying out cis-connection on a Cmylc promoter and two core editing elements through a restriction enzyme site directional cloning technology; a Cmylc promoter-nCas9-RT expression unit is constructed on the upstream of a gene (the sequence comprises a nuclear localization signal peptide coding region, an nCas9 nicking enzyme structural domain coding region and a reverse transcriptase functional domain coding region) for coding an nCas9-RT fusion protein, and it is ensured that transcription initiation of the fusion protein is regulated and controlled by a Cmylc promoter; the editing efficiency is enhanced from two dimensions by utilizing the unique transcriptional regulation ability of the Cmylc promoter in tobacco cells, on one hand, by improving the transcriptional level of nCas9-RT fusion protein and increasing the accumulation amount of functional fusion protein in cell kernels, a sufficient substrate is provided for assembly of an editing compound (nCas9-RT / pegRNA), and on the other hand, the editing efficiency is improved from two dimensions; finally, through cooperative high expression of'protein-nucleic acid 'double elements, the core limitation of'insufficient element expression quantity' in a traditional editing system is broken through, and the order of magnitude improvement of the tobacco leading editing efficiency is achieved.

Provided herein are recombinant microorganisms that express a subject polypeptide. Microorganisms can comprise an expression construct comprising a flagellin promoter operatively linked with a heterologous nucleotide sequence encoding the subject polypeptide. The flagellin promoter sequence can comprise a genetic modification that reduces CsrA inhibition of translation. Microorganisms also can comprise a genetic modification that reduces FlgM inhibition of SigD initiation of transcription. The target polypeptide can be an aldehyde dehydrogenase. Such microorganisms are useful in the treatment of alcohol hangover.