156 results about "Origin of replication" patented technology

The invention is related to a transducing particle that comprises a bacteriophage coat and a DNA core that comprises plasmid DNA comprising: a) a host-specific bacteriophage packaging site wherein the packaging site is substantially in isolation from sequences naturally occurring adjacent thereto in the bacteriophage genome, b) a reporter gene, c) a bacteria-specific promoter operably linked to said reporter gene, d) a bacteria-specific origin of replication, and optionally e) an antibiotic resistance gene. The invention includes phage transducing particles, methods of making transducing particles, and methods of using the transducing particles in bacterial detection.

Novel chimeric constructions and methods for their use are provided for expression of exogenous genes in a plant chloroplast. Particularly, expression is achieved by the use of a chloroplast or bacterial 5′ untranslated region in the expression cassette. The expression cassette may be integrated into the chloroplast genome by the use of chloroplast DNA flanking sequences, or may replicate autonomously if provided with a chloroplast origin of replication. Plants and cells containing the transformed chloroplasts are also provided. The constructs may be used with both monocotyledenous and dicotyledenous chloroplasts.

Provided herein are methods for generating human induced pluripotent stem cells free from genomic integration of exogenous transgenes by transfecting into nucleated blood cells one or more DNA expression vectors (e.g., plasmid vectors) that do not contain a mammalian origin of replication, and encode and permit expression of one or more reprogramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc). Also provided herein are the integration-free human induced pluripotent stem cells obtained by the methods described herein.

Compositions, kits, and methods are provided for use in a recombinational cloning or subcloning methods for constructing expression vectors which comprise: ligating a library of double-stranded linear donor DNAs, where each member of the library includes a donor DNA sequence, with a double-stranded linear driver DNA which includes a promoter sequence and a donor recombination site to form a single circular donor DNA, the single circular donor DNA not including an origin of replication, where the donor DNA sequence is under the transcriptional control of the promoter; and contacting the circular donor DNA and a circular acceptor acceptor vector in the presence of a recombinase to form a single fused circular vector, the circular acceptor vector comprising an origin of replication and an acceptor recombination site capable of recombining with the circular donor DNA.

We describe a regulated antigen delivery system (RADS) that has (a) a vector that includes (1) a gene encoding a desired gene product operably linked to a control sequence, (2) an origin of replication conferring vector replication using DNA polymerase III, and (3) an origin of replication conferring vector replication using DNA polymerase I, where the second origin of replication is operably linked to a control sequence that is repressible by a repressor. The RADS microorganism also has a gene encoding a repressor, operably linked to an activatible control sequence. The RADS described provide high levels of the desired gene product after repression of the high copy number origin of replication is lifted. The RADS are particularly useful as live bacterial vaccines. Also described is a delayed RADS system, in which there is a delay before the high copy number origin is expressed after the repression is lifted. The delayed RADS is also particularly useful for live bacterial vaccines. Also described are several control elements useful for these systems, as well as methods for providing immunity to a pathogen in a vertebrate immunized with the RADS microorganisms.

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.

Disclosed is a nucleic acid amplification method which is based on a new principle and enables to amplify a nucleic acid having a specific nucleotide sequence in a simple manner, within a short time and with efficiency. The nucleic acid amplification method comprises the steps of: (a) conducting a DNA polymerase chain reaction by using, as a template, DNA comprising a nucleotide sequence to be amplified and using a primer pair having a nucleotide sequence complementary to the nucleotide sequence to be amplified, thereby producing a linear DNA fragment; and (b) conducting a chain-substituting DNA polymerase chain reaction in a chaining manner by using cyclic single-stranded DNA comprising the same nucleotide sequence as that of at least one of the primer pair as a template and employing the 3′-terminus of the linear DNA fragment produced in step (a) as the replication origin.

The invention provides fowlpox virus vector shuttle plasmid pTGP3 which comprises recombinant arms TKL and TKR, a bidirectional promoter PE/L, a fluorescent protein expression cassette, and a resistant marker gene and replication origin ori; the upstream and the downstream of the bidirectional promoter PE/L are respectively provided with cloning sites MCSL and MCSR; and both ends of the fluorescent protein expression cassette are provided with loxp sequences. The plasmid of the invention has two different screening markers, and the recombinant fowlpox virus prepared with the plasmid can express 1 to 3 types of gene with different meshes in the whole processes of the early and the later periods; the strong composite promoter with expression activity in the early and the later periods is applied so as to realize the all-process high-efficiency expression of a target gene; and the loxp sequences are introduced into both ends of the fluorescent protein expression cassette, so as to knock out the exogenous recombinant fowlpox virus screening markers. The invention lays foundation for the series and the scale application of the recombinant fowlpox virus in vaccine and biological drug research and development fields.

An Actinobacillus succinogenes plasmid vector which provides a means to overexpress proteins in A. succinogenes. The plasmid can be transformed efficiently by electroporation, and replicates in a stable manner in A. succinogenes. The plasmid comprises at least one marker gene, operably linked to a first promoter functional in Actinobacillus succinogenes, an origin of replication functional in Actinobacillus succinogenes, a second promoter isolated from Actinobacillus succinogenes, and a cloning site downstream from the second promoter. Plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are disclosed. The pckA gene polypeptide sequence and nucleic acid sequence of Actinobacillus succinogenes, including the promoter and ribosome binding site, is disclosed. Furthermore, a method for producing a recombinant Actinobacillus succinogenes is described, including a method of transformation. Additionally, a recombinant Actinobacillus succinogenes is disclosed and a method for producing succinate utilizing this recombinant Actinobacillus succinogenes is described.

The present invention provides an isolated nucleic acid comprising a single retroviral LTR, a polypurine tract, a packaging signal, a primer binding site and a rev responsive element. Further provided is an isolated nucleic acid comprising a heterologous nucleotide sequence, a single retroviral long terminal repeat (LTR), a packaging signal, a rev responsive element, a polypurine tract, a eukaryotic promoter, a primer binding site, a bacterial origin of replication and a bacterial selection marker. In addition, the present invention provides an isolated nucleic acid comprising a 5′ retroviral LTR and a 3′ retroviral LTR, a heterologous nucleotide sequence, a packaging signal, a rev responsive element, a polypurine tract, a eukaryotic promoter, a primer binding site, a bacterial origin of replication and a bacterial selection marker cassette, wherein the bacterial origin of replication and bacterial selection marker are located between the two LTRs.

Disclosed herein are improved plasmid shuttle vectors, vaccines based on them, and methods related to their construction and use. Particular arrangements of functional elements of such plasmids, namely origins of replication and eukaryotic transcription/translation control elements, which give rise to generally undesirable side-products upon propagation of the plasmids in bacterial culture are disclosed. These side-products apparently arise as terminated replication intermediates. Strategies both to eliminate accumulation of these side-products, and to make them useful as a vaccine adjuvant, are described.

The invention relates to a plasmid vector and a method for building a plant population by using the plasmid vector. The plasmid vector contains, at a replication origin, a gene expression box for expressing Cas9 protein, a first promoter, nucleotide sequences containing suicide gene sequences, a gRNA scaffold element and a termination signal, and at least one first enzyme cutting site located on the nucleotide sequence 5' end containing the suicide gene sequence and at least one second enzyme cutting site located at the nucleotide sequence 3' end containing the suicide gene sequence; furthermore, no first enzyme cutting site and no second enzyme cutting site exist in other positions of the plasmid vector. The first promoter is located at the upstream of the termination signal; the nucleotide sequences containing suicide gene sequences and the gRNA scaffold element are all located between the first promoter and the termination signal.

A process for assembling a series of DNA fragments generated by PCR into an ordered circular arrangement for replication and genetic work in cells. The PCR fragments are made with a modified nucleotide in the primers that can be removed with a DNA excision repair enzyme to generate a 3′ overhang. The 3′ overhangs are designed to allow directional annealing and thus sequential PCR fragments can be assembled by annealing the overhangs and subsequent ligation. Sequential addition of PCR fragments is facilitated by growing the chain on a solid support, and the assembled chain can be removed with a site specific recombinase if the first and last primers contain the recombinase site. The circularized assembled fragment can be directly used for cell transformation if the appropriate sequences are included, such as an origin of replication and a selectable marker.

The invention relates to an adeno-associated virus (AAV) producer cell comprising nucleic acid sequences encoding: rep/cap gene; helper virus genes; and the DNA genome of the AAV vector particle, wherein the nucleic acid sequences are all integrated together at a single locus within the AAV producer cell genome. The invention also relates to nucleic acid vectors comprising a non-mammalian origin of replication and the ability to hold at least 25 kilobases (kb) of DNA, characterized in that the nucleic acid vectors comprise nucleic acid sequences encoding: rep/cap gene, and helper virus genes. The invention also relates to uses and methods using the nucleic acid vectors in order to produce stable AAV packaging and producer cell lines.

The invention provides expression vectors that support high levels of polypeptide expression in mammalian cells. The vectors contain at least one expression cassette for a target polypeptide; an expression cassette for a eukaryotic selectable marker protein; an expression cassette for a bacterial selectable marker protein, and a bacterial plasmid origin of replication.

The present invention provides a method for transferring and expressing heterologous DNA in a non-plant host cell. The vector used in this method includes a backbone having a first origin of replication capable of maintaining heterologous DNA as a single copy in an Escherichia coli host cell. The vector further includes a unique restriction endonuclease cleavage site for insertion of heterologous DNA, and left and right Agrobacterium T-DNA border sequences flanking the unique restriction endonuclease cleavage site. In certain host cells, the T-DNA border sequences allow introduction of heterologous DNA located between the left and right T-DNA border sequences into a host cell. In preferred embodiments, the vector includes a second origin of replication capable of maintaining heterologous DNA as a single copy in a host cell such as Agrobacterium species or other prokaryotic cells.