14 results about "Replication Initiation" patented technology

The invention discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for targeted knockout of Pten and Trp53 genes, and the recombinant plasmid comprises an EPI vector system. The recombinant plasmid takes an ori element as a replication start site, and sequentially comprises an sgRNA sequence of a targeted Trp53 gene and Pten controlled by double U6 promoters, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, an orip element and an EBNA1 protein expression element. The invention also discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and the established ovarian cancer cell model. By adopting the method to construct the ovarian cancer cell model, the period from cell editing to animal tumor formation is shortened, the stability and immune integrity of the genetic background of the model are ensured, and large-scale drug screening and high-throughput experiments are facilitated.

The invention provides a DNA (deoxyribonucleic acid) construct. The construct comprises: 1) a replication start site; 2) screening elements; 3) a target gene; 4) auxiliary elements; and 5) a telomerase recognition site. The constructs can be used to prepare linear endless DNA. The invention also provides a method for producing the endless linear DNA, and an application of the method.

The present invention addresses the problem of providing a new replication initiation region capable of increasing the yield of plasmid DNA.　Provided is a double-strand circular DNA vector having a replication initiation region in which at least the base corresponding to position 22 in the base sequence represented by SEQ ID NO: 1 is an adenine base.

The present invention addresses the problem of providing a novel vector using a replication initiation point derived from Thermus thermophilus. The problem is solved by providing a shuttle vector that includes a specific first replication initiation point derived from Thermus thermophilus and a second replication initiation point derived from a biological species other than the Thermus thermophilus.

Provided is a precursor plasmid used for preparing a screening-tag-free plasmid, the precursor plasmid comprising: (1) a conditioned replication initiation site having a plasmid replication initiation capacity that is dependent on regulatory proteins, wherein the conditioned replication initiation site has a first replication initiation state in the presence of a first regulatory protein and a second replication initiation site in the presence of a second regulatory protein, and has a stronger ability to initiate plasmid replication in the second replication initiation state than in the first replication initiation state; 2) a first regulatory protein expression cassette expressing the first regulatory protein; 3) a sequence encoding a repressor protein; 4) a screened tag gene; 5) a target gene, or a cloning site for inserting the target gene; and 6) paired recombination sites. Also provided are a host cell suitable for the recombination of the precursor plasmid and the production of a screening-tag-free plasmid, and a method for large-scale production of the screening-tag-free plasmid.

The application belongs to the technical field of synthetic biology, and discloses a high-purity and high-yield industrial preparation method of a high-molecular-weight polynucleotide (PN). The method uses a plasmid containing a PN base sequence as a template, generates long-chain DNA by using a rolling circle amplification (Rolling Circle Amplification) technology, and then realizes efficient separation of the PN segment by restriction enzyme digestion. The template plasmid is designed, the replication initiation site and the enzyme cutting site are embedded in the corresponding ends of the PN sequence, a single continuous segment seamlessly fused with the PN sequence is formed, 100% theoretical recovery of the PN segment is realized, and the subsequent purification process is significantly simplified. Through the fusion design of the plasmid backbone structure and the PN sequence, the application realizes low-cost and easy-to-scale production of the PN prepared by the rolling circle amplification, and improves the yield and purity of the target product. The process realizes industrial preparation of the high-molecular-weight polynucleotide (PN), and provides a new technical path of safe and reliable, efficient and convenient, and scalable amplification for industrial production of the high-molecular-weight PN.

The invention discloses a yarrowia lipolytica gene editing system based on CRISPR / AsCas12f1 and application of the yarrowia lipolytica gene editing system, and belongs to the field of molecular biology. A gene editing helper plasmid containing an AsCas12f1 expression cassette, a corresponding AssgRNA expression cassette, a related selection marker and a replication starting element is constructed through a gene cloning technology, and the helper plasmid and a proper donor DNA fragment are co-transformed into yarrowia lipolytica, so that efficient traceless knockout of a target gene can be realized. The gene editing kit of the yarrowia lipolytica is enriched, and the gene editing kit has very important application value in the field of targeted genetic modification of the yarrowia lipolytica.

The invention discloses a novel agrobacterium carrier system and application thereof. The invention provides an agrobacterium vector system, and the agrobacterium vector system comprises an expression vector and an auxiliary vector, the expression vector comprises elements such as a pVS1 oriV nucleotide sequence of a replication start site from a pVS1 replication system and the like; the auxiliary vector comprises a nucleotide sequence from a replication start site oriV of a pRK2 replication system and other elements. The agrobacterium vector system provided by the invention can keep high copy in escherichia coli and agrobacterium respectively, has very high plant transformation efficiency and very small skeleton, is very easy and flexible in construction, and is very suitable for construction of libraries. Moreover, as the framework is small and the construction efficiency is high, the step of transforming escherichia coli can be omitted, the agrobacterium can be directly transformed, and the positive rate is high, so that the whole experimental period is shortened, and the workload is reduced.

The invention discloses construction of recombinant yarrowia lipolytica capable of efficiently synthesizing fructo-oligosaccharide, and belongs to the technical field of biological engineering. According to the recombinant yarrowia lipolytica disclosed by the invention, a fructosyl transferase mutant is over-expressed by using a high-copy free plasmid p-Sce-mtORI5-HygB constructed on the basis of a saccharomyces cerevisiae mitochondrial replication starting sequence Sc5 (the 66th valine residue of the fructosyl transferase derived from aspergillus oryzae is mutated into an asparagine residue). The invention provides the recombinant yarrowia lipolytica FT06 capable of efficiently synthesizing the fructo-oligosaccharide, the engineering strain is converted for 48 hours in a 5L fermentation tank, the titer of the fructo-oligosaccharide can reach 335g / L, and the yield is as high as 67%.

The invention belongs to the technical field of synthetic biology, and particularly relates to an industrial preparation method of high molecular weight polynucleotide (PN). According to the method, a plasmid containing a PN base sequence is used as a template, long-chain DNA is generated by using a rolling circle amplification technology, and then efficient separation of PN fragments is realized through restriction enzyme digestion. The plasmid is subjected to optimization design, a restriction enzyme cutting site is introduced into a replication start site and a selection marker gene, after enzyme cutting treatment, a plasmid skeleton is degraded into small fragments smaller than 250 bp, and efficient separation of a target PN fragment is achieved by means of molecular weight difference. Through optimization design and large-scale preparation of a plasmid template, low-cost and easy-to-amplify production of PN prepared by rolling circle amplification is realized, and meanwhile, the length and sequence of a final PN product can be precisely designed, regulated and controlled. Particularly, a specific enzyme cutting site is introduced into a plasmid template, so that the subsequent precise removal of a plasmid skeleton sequence is facilitated, and the purity of a target product is remarkably improved. According to the process, the industrial preparation of the high molecular weight polynucleotide (PN) greater than 1500 bp is successfully realized, and a safe, efficient, convenient and large-scale brand new path is provided for the production of the high molecular weight PN.

Provided are an agrobacterium vector system and use thereof. The agrobacterium vector system comprises an expression vector and a helper vector. The expression vector comprises elements such as a nucleotide sequence of a replication origin pVS1oriV from a pVS1 replication system. The helper vector comprises elements such as a nucleotide sequence of a replication origin oriV from a pRK2 replication system. The provided agrobacterium vector system can maintain a high copy number in both E. coli and A. tumefaciens, exhibits very high plant transformation efficiency, features a very small backbone, and is easily and flexibly constructed, making it very suitable for library construction. Moreover, due to its small backbone and high construction efficiency, this agrobacterium vector system can be directly transformed into A. tumefaciens without the need for an E. coli transformation first, and the positive rate is high, thereby shortening the entire experimental cycle and reducing the workload.

The present application relates to a kind of orthogonal type DNA self-replicating vector and its use in mammal to express gene of interest.The vector includes the expression cassette of the replication essential gene of type I herpes simplex virus and replication initiation sequence, wherein the replication essential gene of type I herpes simplex virus is UL9, UL29, UL30, UL42, UL5, UL8 and UL52, and the replication initiation sequence is OriS and / or OriL.

Provided in the present application is a DNA construct which comprises: 1) a replication origin; 2) a screening element; 3) a gene of interest; 4) an auxiliary element; and 5) a telomerase recognition site. The construct can be used for preparing a linear end-free DNA. Further provided in the present application are a method for producing an end-free linear DNA, and the use of the method.

The application discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and particularly discloses a recombinant plasmid for knocking out Pten and Trp53 genes, which comprises an EPI vector system; the recombinant plasmid takes an ori element as a replication start site, and sequentially comprises sgRNA sequences for targeting Trp53 genes and Pten controlled by a double U6 promoter, a CAG promoter, a SauriCas9 nuclease expression unit, a fluorescent protein expression element, a resistance gene, and an orip element and an EBNA1 protein expression element. The application further discloses a method for rapidly establishing an ovarian cancer model based on SauriCas9, and an ovarian cancer cell model obtained by construction. The ovarian cancer cell model is constructed by adopting the method, the period from cell editing to animal tumorigenesis is shortened, the stability and immune integrity of the genetic background of the model are ensured, and large-scale drug screening and high-throughput experiments are facilitated.