70 results about "Replication Initiation" patented technology

The invention discloses construction and application of a CRISPR / Cas9 gene editing vector for microorganisms. The constructed CRISPR / Cas9 gene vector consists of a replication start site, a selection marker gene, a Cas9 protein gene, gRNA coding DNA, a homologous recombinant element and an operon. The CRISPR / Cas9 gene vector constructed by the invention is capable of editing (including performing such operations as knocking out, replacing, interpolating and the like on gene or DNA sequence) escherichia coli or corynebacterium glutamicum genome; and the gene vector has the advantages of being short in test cycle, time-saving and cost-saving, high in efficiency and the like.

The invention discloses a CRISPR / Cas9-mediated large DNA fragment assembling method. Specifically, the invention provides a nucleic acid construct capable of carrying out constitutive expression of Cas9 in yeast; the nucleic acid construct contains a yeast Tef1 promoter operationally connected with a Cas9 gene, a replication origin from pBR322 and a screening mark thereof, and a replication region from CEN6ARS4 and a screening mark thereof; and the nucleic acid construct is in the form of single-copy replication in the yeast and in the form of high-copy replication in Escherichia coli. The invention also provides a nucleic acid construct capable of carrying out constitutive expression of sgRNA in yeast, a nucleic acid construct used as a receptor vector and a DNA assembling method. According to the invention, two or more to-be-assembled large DNA fragments are successfully assembled in microzyme to form a large plasmid in one shot; so low-efficiency in-vitro digestion recovery, genetic transformation and the like of large DNA fragments are avoided, and the method provided by the invention is more convenient and efficient compared with traditional methods.

The present invention relates to nucleic acid molecules and in particular to vectors, comprising at least one gene of interest, at least one scaffold / matrix attached region (S / MAR), at least one origin of replication, and at least one replication initiation factor, cells comprising these, processes for their propagation, and their use, in particular for the high level expression of proteins which can be used as medicaments.

The present invention relates to methods for producing a recombinant polypeptide of interest, the method comprising the steps of: a) providing a polynucleotide library encoding one or more polypeptides of interest, wherein the library was prepared in an expression cloning vector comprising at least the following elements: i) a polynucleotide encoding a selectable marker; ii) a fungal replication initiation sequence, preferably an autonomously replicating sequence (ARS); and iii) a polynucleotide comprising in sequential order: a promoter derived from a fungal cell, a cloning-site into which the library is cloned, and a transcription terminator; b) transforming a mutant of a parent filamentous fungal host cell with the library, wherein the frequency of non-homologous recombination in the mutant has been decreased compared to the parent; c) culturing the transformed host cell obtained in (b) under conditions suitable for expression of the polynucleotide library; and d) selecting a transformed host cell which produces the polypeptide of interest.

The invention discloses a single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof. The bacillus subtilis shuttle expression vector provided by the invention comprises the following elements: a bacillus subtilis formed strong promoter P43, a sequence which encodes signal peptide, multiple cloning sites, an antibiotics resistance gene (kalamycin or chloramphenicol), a bifunctional promoter sequence which can start resistance gene expression of kalamycin or chloramphenicol in escherichia coli and bacillus subtilis, and copy homing sequences of bacillus subtilis and escherichia coli. The expression vector disclosed by the invention not only can be stably copied in escherichia coli, but also can be stably copied in bacillus subtilis, and expresses homologous and heterologous proteins. All vectors just contain one resistance gene, the plasmid of which is less than 400bp, so that gene operation and plasmid conversion are easy to carry out, and the conversion ratio is high. Besides the expression vector of an exogenous gene, the vector can be further used for screening promoters and constructing a gene knock-out vector.

The invention discloses a method for constructing a recombinant expression vector simultaneously expressing a plurality of genes. An MISSA system consists of a strain and a vector, wherein the strain comprises a donor strain and an acceptor strain; the vector comprises a donor vector and an acceptor vector; replication initiation protein carried by a chromosome of the donor strain is responsible for replicating and amplifying a suicidal donor vector, and the carried conjugal transfer protein Tra is responsible for conjugal transfer of the donor vector; the acceptor strain can induce and express two sets of locus specific recombinant proteins, namely Cre recombinant enzyme and lambda phage locus specific recombinant proteins; when the donor strain is mixed with the acceptor strain, the donor strain and the acceptor strain are conjugated, and the donor strain is transferred into the acceptor vector because the donor vector in the donor strain carries an oric element; and the donor vector and the acceptor vector undergo recombination reaction in the acceptor strain under the action of the two sets of locus specific recombinant proteins, so that a target gene carried by the donor vector is assembled onto the acceptor vector. The process is repeated, so a plurality of target genes can be assembled onto the acceptor vector according to the preset direction and order.

The invention discloses an Escherichia coli-Bacillus subtilis shuttle expression vector and application thereof. The Escherichia coli-Bacillus subtilis shuttle expression vector comprises the following elements: a coding sequence of secretory signal peptide, a coding sequence of replication initiation protein of Bacillus subtilis, a promoter sequence of the Bacillus subtilis, and a replication initiation sequence of Escherichia coli. The vector can perform steady replication in the Escherichia coli, and can also replicate and express in the Bacillus subtilis. The vector can be applied to establishing a protein mutant gene library, mutant genes are connected to the vector first to ensure that various mutant genes are replicated in the Escherichia coli and then are transferred into the Bacillus subtilis to be expressed and screened, the efficiency of constructing the mutant library is equivalent to that of an Escherichia coli operation system, and the vector can simply and flexibly control the capacity of the library and can also fully utilize the external secretion expression ability of host cells of the Bacillus subtilis, thereby being convenient for the detection and screening of the performance of the mutant library.

The invention provides a text replication method and device, and an intelligent terminal. The method comprises the following steps: receiving a text of characters corresponding to a replication starting point drawn on a screen of the intelligent terminal to locate the replication starting point; receiving a text of characters corresponding to a replication ending point drawn on the screen of the intelligent terminal to locate the replication ending point; and determining the contents between the replication starting point and the replication ending point as target contents. The invention further provides a text replication device and an intelligent terminal. According to the method provided by the invention, the texts of the characters corresponding to the replication starting point and the replication ending point are drawn on the screen of the intelligent terminal, the characters having the same text in the screen are displayed in high brightness to determine the replication starting point and the replication ending point so as to determine the replication target contents, thereby avoiding the wrong touch phenomenon in a use process of a user, the replication position and contents to be selected by the user can be accurately located, multiple times of selection can be repeated, and no tedious finger dragging operation is needed, so that the user experience is improved.

The invention discloses an oligonucleotide-linker-mediated DNA assembly method and an application thereof. The assembly method comprises steps of synthesis of a long sequence, preparation and assembly of a module carrier, remolding and assembly of a skeleton carrier, preparation of a short sequence, and enzyme-cut and link-up integration of the module carrier, the remolded skeleton carrier and the short sequence. According to the method, the short sequence is a regulating element such as a promoter and an RBS, and the long sequence is a replication initiation point of a gene or a carrier. The short sequence is designed to synthesize a double-stranded DNA chain, and can connect the long sequence and be used as a barcode sequence to determine the assembly sequence in an assembly process. The gene sequence in an operon is achieved by synthesizing short sequence with different cohesive ends. According to the method, multiple factors are allowed to participate in gene regulation at the same time. The method can be used to construct a combinatorial library of a biosynthesis channel, and a high-flux optimized channel is achieved.

The invention discloses a carrier inducing reversible immortalization of in vitro animal cells and an application thereof. The carrier provided by the invention contains an escherichia coli replication initiation region, an ampicillin resistance gene, an expression cassette 1 and an expression cassette 2, wherein the expression cassette 1 comprises the following components from upstream to downstream in sequence: Ptight, hTERT, IVS, IRES, Hygr and SV40polyA; the expression cassette 2 comprises the following components from upstream to downstream in sequence: PCMV, rtTA-Advanced and SV40polyA; the expression cassette 1 is opposite to the expression cassette 2. Experiments prove that reversible regulation of lives of the animal cells can be achieved and the efficiency of in vitro cell genetic operation can be improved by utilizing the annular carrier provided by the invention. The carrier is of great significance in providing safe cell materials for animal genetic modification or medical cell therapy and the like.

The invention discloses a plasmid pTsqs for synthesizing squalene through E.coli (Escherichia Coli). The plasmid pTsqs comprises a carrier pTrc99A, wherein a protein expression inhibition gene lacIq,a replication origin site pBR_322ori, an amicillin resistance gene and a squalene synthase gene ERG9 optimized according to E.coli codon preference are inserted in the carrier pTrc99A; a promoter Ptrcis located in an upstream position of the squalene synthase gene ERG9, and a downstream position of the squalene synthase gene ERG9 comprises one MCS (Multiple Cloning Site). The invention disclosesa preparation method and a using method of the plasmid pTsqs. The plasmid disclosed by the invention is capable of expressing that the E.coli is facilitated to synthesize the squalene by the squalenesynthase, the plasmid comprises the squalene synthase gene ERG9 controlled by the strong promoter Ptrc, and the squalene synthase gene ERG9 is optimized according to the E.coli codon preference. A carrier tool is provided for producing the squalene through fermentation of the E.coli.

The invention provides a saccharomyces cerevisiae DNA replication initial region identification method. The method comprises the following steps of: selecting a DNA sample from a saccharomyces cerevisiae gene sequence database; sliding on the DNA sample at equal step length by using a sliding window with the size of 3 to obtain a nucleotide triplet set; calculating the frequency of each nucleotidetriplet in the nucleotide triplet set; constructing the feature vectors of the DNA sample according to the frequency and the physicochemical properties of the nucleotide triplets; respectively inputting the feature vectors into a plurality of convolutional neural networks with different convolutional layer numbers, and selecting a target convolutional layer number according to the identificationeffect of the convolutional neural networks; performing hyper-parameter iteration on the convolutional neural networks with the target convolution layer number to obtain a target convolutional neuralnetwork with optimal hyper-parameters; and inputting a saccharomyces cerevisiae gene sequence to be detected into the target convolutional neural network to obtain a DNA replication initial region. According to the invention, the identification precision of the saccharomyces cerevisiae DNA replication initial region is improved.

The invention discloses a replication-defective recombinant lentivirus CAR-T transgenic vector targeting CD152 and a construction method of the replication-defective recombinant lentivirus CAR-T transgenic vector. The transgenic vector comprises a promoter for controlling a replication initiation site, a prokaryotic replicon for plasmid replication, a viral replicon for enhancing replication in eukaryotic cells, a resistance gene for detection, a regulatory element after viral transcription, a packaging cis element cis for lentiviral packaging and a chimeric antigen receptor CAR for recognition, transmission and initiation; the construction of the replication-defective recombinant lentiviral CAR-T transgenic vector comprises construction of a recombinant lentiviral plasmid, and packaging,concentration and purification of the recombinant lentiviral vector to obtain the replication-defective recombinant lentiviral CAR-T transgenic vector targeting CD152 with high purity. The recombinantlentivirus CAR-T transgenic vector provided by the invention can remarkably improve secretion of cytokines and specifically kill target cells CTLA-4, and provides a new treatment direction for inhibition of tumor microenvironment and attack of solid tumors.

The invention discloses a novel high-efficiency secretion and expression vector jof colibacillus, a preparation method thereof and application thereof. The expression vector is a cyclic plasmid deoxyribonucleic acid (DNA) vector, and comprises a procaryotic copy initial point, a selection marker gene and two series-wound exogenous gene expression box units. Each series-wound exogenous objective (target) gene expression box unit respectively comprises a tac strong promoter, an L-Asparaginasum signal peptide degenerate gene, an exogenous objective (target) gene and a high-transcription terminator rrnBT1T2 which are sequentially arranged with one another. The novel high-efficiency secretion and expression vector disclosed by the invention can secrete and express a polypeptide / protein objective product at a high level in the host cell of the colibacillus, and the expression product can be automatically formed into a correct intramolecular disulfide bond and secreted into cell periplasm / and culture solution, so that the invention is very good for the downstream separation, extraction and purification of the expression product, and has an important application value.

The invention provides a fish iridovirus DNA (Deoxyribonucleic Acid) vaccine which comprises an eukaryotic transcription promoter, a gene of an iridovirus major capsid protein (MCP), an ankyrinrepeats gene, an eukaryotic transcription terminator, a replication initiation site and a resistance screening marker (shown in figure 1), wherein the replication initiation site and the resistance screening marker are used for amplifying plasmids. The DNA vaccine is recommended to be injected by an intramuscular injection method and can also be taken by oral and soaking modes to enable fish to be immune.

A prokaryotic recombinant host cell comprising a heterologous replication initiation protein that activates a conditional origin of replication and an extrachromosomal DNA molecule comprising a heterologous therapeutic gene and a conditional origin of replication whose functionality in the prokaryotic recombinant host cell requires a replication initiating protein which is foreign to the host cell is described. The host cell may comprise a pir gene having at least one mutation, which may occur in the pir gene copy number control region, the pir gene leucine zipper-like motif, or the pir gene DNA binding region.

The invention relates to the technical field of genetic engineering and aims to construct a shuttle vector between escherichia coli and haemophilus parasuis. The shuttle vector is named as pSHK4 and comprises a nucleotide sequence expressed by a sequence table SEQ ID NO:1, wherein a coding area of the pSHK4 is located at the sequence corresponding to 927-1742 bit sequence of the nucleotide sequence and 271 amino acids are coded. The shuttle vector provided by the invention has the advantages that a duplicated starting locus of natural plasmids in a bacillus separating strain of the haemophilus parasuis is utilized to construct the shuttle vector suitable for the haemophilus parasuis for the first time, the shuttle vector has the characteristics of stable hereditary in pathogenic bacteria and can be used for performing genetic operation on the haemophilus parasuis, and the shuttle vector is a basic tool for researching a toxic factor of the haemophilus parasuis and has a significance in revealing a pathogenic molecular pathopoiesia mechanism.

Polypeptides and fusion proteins that bind to eukaryotic viruses, in particular, eukaryotic single-stranded DNA (ssDNA) viruses are provided. The polypeptides and fusion proteins bind to the replication initiation proteins (Rep) of ssDNA viruses and optionally inhibit viral replication and / or viral infection. The virus can be a plant pathogen or animal pathogen. Consensus sequences used to identify polypeptides that bind to eukaryotic viruses are also provided.

The invention discloses a plasmid carrier construction method applied to Shewanella, and relates to Shewanella xiamenensis BC01 with a collection number of CGMCC No.11687. The method comprises the following steps: extracting a plasmid by the Shewanella xiamenensis BC01, purifying to obtain pSXM33, submitting the pSXM33 to inspection and sequencing; comparing obtained sequence information with NCBI database information, determining a distribution condition of an open reading frame on the pSXM33 plasmid, and drawing a map of the plasmid; intercepting a fragment with a duplication original part from the pSXM33 by a genetic engineering means, and connecting the intercepted fragment with a resistance and duplication origin fragment of a pET28a plasma to construct two plasmids, namely, pETSXM1 and pETSXM2. As proved, the pETSXM1 and the pETSXM2 can pass through escherichia coli and Shewanella MR-1.

The invention discloses an Arthrobacter sp. expression plasmid, and belongs to the technical field of gene engineering. The plasmid disclosed in the invention is annular in shape, and the nucleotide sequence of the plasmid is represented by SEQ ID NO:1, comprises an Arthrobacter sp. promoter sequence, a multiple cloning site sequence, a replicating sequence in Arthrobacter sp., a kanamycin resistant gene sequence, an Escherichia coli replicating sequence and an amicillin resistant gene sequence, and can autonomously replicate in Escherichia coli and Arthrobacter sp.. The plasmid is used to express ghprt gene in the Arthrobacter sp. of CGMCC3584, and the cAMP output of the plasmid is 65.2% higher than that of original strain.

The invention relates to modifying plasmid origins of replication to create hybrid origins of replication containing nucleotide sequences from more than one plasmid. The invention also relates to a modified origin of replication cassette that is portable or exchangeable due to the creation of multiple cloning sites flanking the origin of replication. Methods and plasmids for use in exchanging origins of replication are disclosed. Such modified or hybrid plasmids provide useful cloning tools that allow for regulation of the level of expression of a desired protein.

The invention discloses a ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, a vector construction method and application and belongs to the field of biotechnology. The interference vector comprises the following sequentially connected parts: an HSV-TK expression element, A digestion locus for vector linearization, a homologous recombination arm 1 in homologous recombination with a bovine scaffold sequence upstream, a human U6 promoter sequence, a polyclonal digestion locus, a human U6 stop subsequence, a neomycin resistance gene containing a PCMV promoter, a homologous recombination arm 2 in homologous recombination with a bovine scaffold sequence downstream, a replication origin gene and an ampicillin resistance screening gene. The RNA interference vector achieves accurate target shooting on bovine derived cell bovine, and brings convenience to screening, insertion locus and insertion element do not affect the integral structure of chromosome and cause no non-research gene expression effect on genome, and RNA interference vector provides convenience for research.

The present invention relates to novel vectors, to DNA vaccines and gene therapeutics containing said vectors, to methods for the preparation of the vectors and DNA vaccines and gene therapeutics containing the vectors, and to therapeutic uses of said vectors. More specifically, the present invention relates to novel vectors comprising (a) an expression cassette of a gene of a nuclear-anchoring protein, which contains (i) a DNA binding domain capable of binding to a specific DNA sequence and (ii) a functional domain capable of binding to a nuclear component and (b) a multimerized DNA sequence forming a binding site for the anchoring protein, and optionally (c) one or more expression cassettes of a DNA sequence of interest. In particular the invention relates to vectors that lack a papilloma virus origin of replication. The nuclear-anchoring protein might be the E2 protein of Bovine Papilloma Virus type 1 or Epstein-Barr Virus Nuclear Antigen 1. The invention also relates to vectors that lack an origin of replication functional in a mammalian cell. The invention further relates to methods for expressing a DNA sequence of interest in a subject.

The invention relates to a shuttle vector beneficial to construction of long fragment DNA and a preparing method and application thereof. The shuttle vector is capable of shuttling back and forth in saccharomyces cerevisiae and escherichia coli, and has a saccharomyces cerevisiae replication origin oriV and an escherichia coli replication origin ori2, both the oriV and the ori2 are low-copy replication origins, and the shuttle vector can conduct stable replication in both the saccharomyces cerevisiae and the escherichia coli, a screening label in the saccharomyces cerevisiae is HIS3, and the screening label in the escherichia coli is chlorampenicol resistance. According to the shuttle vector beneficial to the construction of long fragment DNA and the preparing method and application thereof, the constructed saccharomyces cerevisiae-escherichia coli shuttle vector (named pSynoYAC0) is capable of being successfully assembled in a yeast system, however, plasmid extracted from the saccharomyces cerevisiae has poor quality, while the plasmid extracted from the escherichia coli has high quality, thus further experiment demands such as sequencing and enzyme digestion can be satisfied. The shuttle vector is also capable of conducting replication in the escherichia coli, and the replication belongs to a single copy; meanwhile, the shuttle vector is capable of existing stably in the replication process, thus the vectors meeting the demands can be obtained.

The invention provides a word vector and convolutional neural network based DNA replication initial region identification method. The method comprises the following steps: performing word segmentationon a DNA sequence through continuous trisection sequence word segmentation to obtain individual trinucleotides, carrying out negative sampling on the trinucleotides after word segmentation, carryingout vectorization on the trinucleotides through Word2vec iterative training to obtain word vectors, combining all word vectors to obtain a pre-training feature vector matrix which comprises a pre-training feature vector of each trinucleotide, vertically arranging the trinucleotides after word segmentation, and embedding the trinucleotides into the pre-training feature vectors of the trinucleotidesto obtain a word embedding layer, carrying out feature vectorization on the trinucleotide sequence by the word embedding layer, then carrying out convolution and pooling training to obtain a convolutional neural network, carrying out deep mining and classification identification on ORI features through the convolutional neural network added into the word embedding layer, and finally identifying ORI. The identification accuracy is greatly improved.

The object is to develop: a novel vector, preferably a novel vector which can be carried in a cell of a bacterium belonging to the genus Ralstonia, Cupriavidus or Wautersia stably in the absence of selection pressure for an antibiotic and has no conjugal transfer property; a strain capable of stably producing a polyhydroxyalkanoate using the vector; and a method for production of a polyhydroxyalkanoate using the strain. Disclosed is a novel recombinant vector having a DNA replication initiation segment which can function in a cell of a bacterium belonging to the genus Ralstonia, Cupriavidus or Wautersia, particularly a recombinant vector having both a DNA replication initiation segment which can function in a cell of a bacterium belonging to the genus Ralstonia, Cupriavidus or Wautersia and a segment which can stabilize the recombinant vector (a par segment). By using a transformant produced using the vector, the vector can be carried in a bacterium stably and a polyhydroxyalkanoate can be produced with good efficiency.

Provided is a method for intracellularly producing a large amount of a Cry protein in a Bacillus bacterium. A method for producing a Cry protein or a culture product comprising the Cry protein, comprising transforming a Bacillus bacterium with an expression plasmid incorporating a gene encoding the Cry protein operably linked to a regulatory region comprising a σA-dependent promoter or a σH-dependent promoter, and culturing the transformed cell, wherein the expression plasmid comprises a polynucleotide encoding a replication protein consisting of the amino acid sequence set forth in SEQ ID NO: 9 or a protein having an identity of 80% or more or more with the amino acid sequence of the replication protein and involved in replication initiation.

The invention discloses a shuttle plasmid based on xanthomonas and an application of the shuttle plasmid. A full-base sequence of the shuttle plasmid is as shown in a sequence 1 in a sequence table, and the shuttle plasmid has replication initiation regions in the xanthomonas and escherichia coli, resistance screening markers and multiple cloning sites. According to the shuttle plasmid, the resistance screening markers are gentamicin resistance screening markers, and genes with the gentamicin resistance screening markers are as shown in a sequence table 6. Applications of the shuttle plasmid include applications to related gene function identification of xanthomonas exopolysaccharide.

The invention discloses an artificially designed plasmid pM5500 and an application thereof in preparation of a DNA marker. The length of the plasmid is 5500 bp, the sequence of the plasmid is SEQ ID NO: 1, and the plasmid comprises a high-copy replication starting point sequence, an ampicillin resistance gene coding sequence and various artificially designed and combined DNA fragments. The plasmidcontains 12 restriction endonuclease recognition sites of the same kind within the limited length; six DNA fragments of 1500, 1000, 750, 500, 250 and 100bp suitable for medium molecular weight DNA marker preparation can be generated through single endonuclease digestion, and the mass ratio of the fragments is 3:2:3:1:1:1. The plasmid is compact and stable in structure, high in copy number and easy to prepare on a large scale, and the DNA maker developed on the basis of the plasmid can be widely applied to the fields of biological research and biotechnology.

Provided is a production method for high production of crystal protein inside bacteria cells of Bacillus bacteria. This production method is for crystal protein or a cultured product containing same and includes: transforming Bacillus bacteria using an expression plasmid wherein a gene that codes crystal protein and a control area including a sigma A-dependent promoter or a sigma H-dependent promoter are operably linked and incorporated; and said transformed cells are cultured. The expression plasmid includes a polynucleotide that codes a replication protein comprising an amino acid sequence expressed by SEQ ID NO: 9 or a protein having at least 80% homology with same in the amino acid sequence and is involved to replication initiation.