24 results about "Replication Initiation" patented technology

The invention discloses a mammalian cell expression vector, an expression plasmid and an expression system. The expression vector sequentially comprises the following elements: (a) a promoter; (b) a Kozak sequence; (c) a polyadenine sequence, the polyadenine sequence sequentially comprises a bGH PolyA sequence and a 5 'UTR PolyA sequence, the nucleotide sequence of the bGH PolyA sequence is as shown in SEQ ID NO.5, and the nucleotide sequence of the 5' UTR PolyA sequence is as shown in SEQ ID NO.6; (d) copying the start site; and (e) screening marker genes. According to the present invention, the expression vector is optimized by the expression element, such that the exogenous gene can be conveniently, efficiently and flexibly inserted and edited by the expression vector so as to achieve the stable extracellular or intracellular expression of the exogenous protein, and the expression efficiency of the exogenous protein can be improved by carrying out the expression plasmid construction based on the expression vector; and more efficient expression and purification of the foreign protein are realized.

Provided is a method with which it is possible to simply and exponentially amplify circular DNA, particularly long-chain circular DNA, in a cell-free system. Specifically, provided is a method for amplifying circular DNA, which comprises an isothermal reaction of a reaction mixture produced by mixing circular DNA having a replication initiation sequence (oriC) with a reaction solution containing the following enzyme groups: (1) a first enzyme group that catalyzes replication of the circular DNA, (2) a second enzyme group that catalyzes replication of the circular DNA, (3) a third enzyme group that catalyzes replication of the circular DNA, (4) a fourth enzyme group that catalyzes replication of the circular DNA, and (5) a third enzyme group that catalyzes replication of the circular DNA. (2) a second enzyme group which catalyzes a linking reaction of an okazaki fragment to synthesize two sister circular DNAs forming a catenary body; and (3) a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs.

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 discloses an escherichia coli-bacillus subtilis shuttle plasmid and application, and belongs to the field of gene engineering. The shuttle plasmid comprises the following elements: a promoter sequence, a coding sequence of bacillus subtilis signal peptide, a replication initiation sequence of bacillus subtilis, a replication initiation sequence of escherichia coli and an antibiotic resistance gene. According to the plasmid design, a modular structure is adopted, and quick replacement of key elements such as a promoter, a signal peptide and RBS is supported through optimized enzyme cutting site layout; a 6 * His tag introduced at the tail end of a multiple cloning site (MCS) can be adapted to nickel column affinity chromatography, and the purification process of the recombinant protein is simplified. The plasmid has the characteristics of small molecular weight and convenience in operation, can be efficiently cloned and constructed in escherichia coli and then directly converted into bacillus subtilis for protein expression, is particularly suitable for development of a gram-positive bacterium secretory expression system, and provides an efficient tool for large-scale production of industrial enzyme preparations.

The invention discloses a micro-ring plasmid synthesis method, and belongs to the technical field of gene editing. Two pairs of primers are designed, eukaryotic expression plasmids containing selection markers and target fragments are used as templates for PCR amplification to obtain two target fragments, vector plasmids loxp-R6K with specific sequences and the target fragments form recombinant plasmids, the recombinant plasmids contain sequences such as the target fragments, restriction enzyme cutting sites and R6K replication start sites, and the recombinant plasmids are used for preparing the recombinant plasmids. The recombinant plasmid is transformed in Escherichia coli GT115 competent cells, the recombinant plasmid is verified to have the recombination efficiency of 81% after bacterial colony PCR amplification, a correctly cloned seed solution is screened and subjected to induced expression to form a bacterial solution containing a screening-label-free micro-plasmid, the micro-ring plasmid is separated and purified, and the screening-label-free micro-ring plasmid is obtained. The sequence of the microring plasmid only contains a target fragment and an R6K replication start site, the accuracy of the size sequence of the microring plasmid in a bacterial colony cultured and cloned by the microring plasmid is as high as 90%, and the microring plasmid can be efficiently and stably obtained.

The present invention provides an efficient method for producing circular DNA. This method for producing circular DNA includes: (1) preparing circular DNA that will serve as a template, said circular DNA including (i) a replication initiation sequence capable of binding to an enzyme that has a DnaA activity, and (ii) a gyrase binding sequence, and said circular DNA not including a DnaA box cluster at a different position from the replication initiation sequence; (2) forming a reaction mixture of the circular DNA prepared in (1) and a reaction liquid including a first enzyme group that catalyzes replication of the circular DNA, a second enzyme group that synthesizes two sister circular DNAs forming a catenane, and a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) at a temperature of 80°C or lower, thereby obtaining a reaction product that includes the circular DNA, which has been amplified.

The invention belongs to the technical field of biology, and particularly relates to a novel recombinant adenovirus vector and a preparation method thereof. The technical problem to be solved by the invention is to provide a novel recombinant adenovirus vector to overcome the defects of long time consumption, complex screening process and the like of the existing adenovirus vector system. The technical scheme adopted by the invention is to provide a recombinant adenovirus vector. The recombinant adenovirus vector comprises an adenovirus genome, a resistance gene and a replication start site element ori. The recombinant adenovirus vector system is a single plasmid system, contains adenovirus gene information and restriction enzyme sites, is simpler and more convenient compared with a two-plasmid system of AdMax and AdEasy which are widely used at present, and has a very good application prospect in the field of preparation of replication-defective adenoviruses and oncolytic adenoviruses.

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 belongs to the technical field of microbial fermentation, and particularly relates to an escherichia coli recombinant strain with high plasmid yield as well as a construction method and application of the escherichia coli recombinant strain. The Escherichia coli recombinant strain is obtained by taking Escherichia coli DH5 alpha as an original strain and overexpressing RNAII (Ribonucleic Acid II) on a genome of the Escherichia coli DH5 alpha. The escherichia coli DH5alpha is subjected to engineering modification, RNAII is overexpressed on the genome of the escherichia coli DH5alpha, plasmid replication initiation is enhanced, the plasmid yield is increased by optimizing the strength of a promoter and balancing plasmid production and strain growth, the plasmid yield is further increased by overexpressing nrdAB genes on the genome and enhancing dNTP synthesis, and the plasmid yield on a 2L fermentation tank reaches 2.20 g / L.

The invention discloses an RNAi recombinant vector resistant to both bemisia tabaci and tomato yellow leaf curl virus as well as a construction method and application of the RNAi recombinant vector resistant to both bemisia tabaci and tomato yellow leaf curl virus, a DNA beta promoter accompanied by geminivirus expressed in phloem is constructed to an RNAi vector pCambia 1391 vector, and a 1391:: DNA beta pro vector skeleton is obtained; an RT-PCR (reverse transcription-polymerase chain reaction) method is used for amplifying part of fragments of key genes BtACTB of bemisia tabaci, key segments (including a gene spacer region with virus replication initiation, part of coat protein and replication-related protein) of TYLCV and inverted repeat sequences of the key segments to construct a gene which is driven by a phloem promoter and is capable of expressing the bemisia tabaci gene BtACTB. The recombinant vector is of a reverse hairpin structure and simultaneously targets key sequences of bemisia tabaci and tomato yellow leaf curl virus. The constructed recombinant vector is introduced into tomatoes through an agrobacterium transformation method, and tomato plants capable of reducing the survival rate of bemisia tabaci and inhibiting TYLCV infection are obtained through screening.

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.

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 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.

Provided is a precursor plasmid that can be used to prepare a plasmid without a screening tag, which includes: 1) a conditional replication origin site, whose plasmid replication initiation ability depends on a regulatory protein, having a first replication initiation state in the presence of a first regulatory protein and a second replication initiation state in the presence of a second regulatory protein, and having a stronger ability to initiate plasmid replication when the conditional replication origin site is in the second replication initiation state than when it is in the first replication initiation state; 2) a first regulatory protein expression cassette for expressing the first regulatory protein; 3) a coding sequence of a repressor protein; 4) a screening tag gene; 5) a target gene, or a cloning site for inserting a target gene; and 6) a pair of recombination sites. Also provided are host cells suitable for recombination of the precursor plasmid therein to produce a plasmid without a screening tag, and a method for large-scale production of a plasmid without a screening tag.

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.