Compositions and methods for microbial production of antibiotic-free closed-ended linear DNA

Abstract

Disclosed arc vectors including i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence. Vectors may also include a bacterial replication origin or a nucleic acid sequence encoding a RNA selectable marker. Vectors may be double-tranded, linear, close-ended nucleic acid vector. Also disclosed are methods for producing double-stranded, linear, close-ended nucleic acid vectors.

Description

ALD - P2024-0647-US02 (PCT)COMPOSITIONS AND METHODS FOR MICROBIAL PRODUCTION OF ANTIBIOTIC-FREE CLOSED-ENDED LINEAR DNACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 702,993 filed October 3, 2024, the contents of which are incorporated herein in their entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure relates to close-ended linear recombinant DNA molecules. Such recombinant DNA molecules are useful in biotechnology, ex vivo gene therapy, transgenic organisms, gene therapy, therapeutic vaccination, agriculture, and DNA vaccines.BACKGROUND

[0003] Bacterial plasmids have long been an important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (e.g., gene therapies and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines. Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to a patient, wherein the desired gene product is expressed from the genome after being transposed from the plasmid into the genome (non-viral transposon vectors), or, wherein theALD - P2024-0647-US02 (PCT) desired gene product is packaged in a transducing virus particle in a production cell line and is then expressed from the virus in a target cell after viral transduction (viral vectors).

[0004] However, designing circular plasmid vectors for use in these types of applications can be challenging from a manufacturing efficiency, stability, and safety standpoint.

[0005] Close-ended linear vectors (also referred to as linear covalently closed (lee) DNA vectors) is one alternative to circular plasmids for production of safer DNA vector alternatives. See, for example, Nafissi & Slavcev. 2012. Microb Cell Fact 11, 154. However, close-ended linear vectors have traditionally been constructed by various in vitro strategies including the capping of PCR products and making “minimalistic immunogenic defined gene expression” (MIDGE) vectors. See, for example, Rodriguez et al. 2004. J Mol Med. 2004, 82: 500-509. In vitro methods for making close-ended linear vectors at scale uses multistep processes that requires considerable time and arc costly. Manufacturing costs might be kept low by using bacterial in vivo production methods; however, use of bacterial systems carry safety concerns that are absent in the in vitro methods of production. Thus, there is a clear need for safe, stable, high-yield vectors with reasonable manufacturing costs.SUMMARY OF THE INVENTION

[0006] Disclosed herein are close-ended linear (ce-linear) vectors. Ce-linear vectors are a type of double-stranded DNA molecule that contains covalently closed terminal hairpins, which increase vector stability by protecting the ce-linear DNA from exonuclease activity. Also, ce-linear DNA molecules, like ce-linear DNA vectors, are torsion-free. Being torsion-free means that the ce-linear DNA vector does not experience negative supercoiling that can occur in other DNA structures, which helps maintain the integrity of the DNA and reduces the likelihood of structural damage or mutations during replication and transcription processes. Additionally, the absence of torsionalALD - P2024-0647-US02 (PCT) stress can enhance the efficiency of transgene expression, as the DNA is more readily accessible for transcription. Therefore, the disclosed ce-linear DNA vectors are stable and high-yield vectors capable of transgene expression.

[0007] Disclosed herein are vectors comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence.

[0008] Disclosed herein are vectors comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin. In some embodiments, the spacer region further comprises a nucleic acid sequence encoding a RNA selectable marker.

[0009] Also disclosed herein are vectors comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a nucleic acid sequence encoding a RNA selectable marker. In some embodiments, the spacer region further comprises a bacterial replication origin.

[0010] Also disclosed herein are vectors comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin and a nucleic acid sequence encoding a RNA selectable marker. In some embodiments, the spacer region is less than 500 bp.

[0011] In some embodiments of any of the vectors disclosed here, the vector does not comprise a nucleic acid sequence encoding a protelomerase polypeptide.ALD - P2024-0647-US02 (PCT)

[0012] In some embodiments of any of the vectors disclosed here, the vector does not comprise a nucleic acid sequence encoding an endonuclease polypeptide.

[0013] In some embodiments of any of the vectors disclosed here, the vector comprises only one telomerase occupancy site. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5.

[0014] In some embodiments of any of the vectors disclosed here, the first region of nucleic acid sequence consists of a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends.

[0015] In some embodiments of any of the vectors disclosed here, the bacterial replication origin is a R6K bacterial origin of replication. In some embodiments, the R6K bacterial replication origin comprises a nucleic acid sequence having at least 95% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14.

[0016] In some embodiments of any of the vectors disclosed here, the RNA selectable marker is selected from the group of an RNA-OUT selectable marker that encodes and RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 25; an RNAI selectable marker that encodes an RNAII regulating RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 26; and a synthetic RNA selectable marker encoding an RNA selectable marker complement regulating RNA with at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the RNA selectable marker is a functional RNA-OUT.

[0017] In some embodiments of any of the vectors disclosed here, the bacterial replication origin is a R6K bacterial origin, and the R6K bacterial origin and the RNA-OUT RNA selectable marker have at least 95% sequence identity to SEQ ID NO: 28.ALD - P2024-0647-US02 (PCT)

[0018] In some embodiments of any of the vectors disclosed here, the first region of nucleic acid sequence and the spacer region of nucleic acid sequence form a nucleic acid structure that is double-stranded, linear, and comprises closed-ends, and wherein the closed-ends comprise a nucleic acid sequence of telR or telL.

[0019] Also disclosed here are bacterial cells comprising: a) a functional protelomerase polypeptide; and b) a vector, the vector comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence and comprises a bacterial replication origin. In some embodiments, the spacer region further comprises a nucleic acid sequence encoding a RNA selectable marker.

[0020] Also disclosed are bacterial cells comprising: a) a functional protelomerase polypeptide; and b) a vector, the vector comprising i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a nucleic acid sequence encoding a RNA selectable marker. In some embodiments, the spacer region further comprises a bacterial replication origin.

[0021] Also disclosed herein are bacterial cells comprising: a) a functional protelomerase polypeptide; and b) a vector, the vector comprising i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin and a nucleic acid sequence encoding a RNA selectable marker. In some embodiments, the spacer region is less than 500 bp.ALD - P2024-0647-US02 (PCT)

[0022] In some embodiments of any of the bacterial cells disclosed herein, the protelomerase polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.

[0023] In some embodiments of any of the bacterial cells disclosed herein, the vector does not comprise a nucleic acid encoding a protelomerase polypeptide.

[0024] In some embodiments of any of the bacterial cells disclosed herein, the vector does not comprise a nucleic acid encoding an endonuclease polypeptide.

[0025] In some embodiments of any of the bacterial cells disclosed herein, the vector comprises only one telomerase occupancy site. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5.

[0026] In some embodiments of any of the bacterial cells disclosed herein, the first region of nucleic acid sequence consists of a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends.

[0027] In some embodiments of any of the bacterial cells disclosed herein, the bacterial replication origin is a R6K bacterial replication origin, and further comprising a nucleic acid encoding a Rep protein operably linked to a PL promoter, wherein the PL promoter comprises a lambda repressor binding site (OL1). In some embodiments, the R6K bacterial replication origin comprises a nucleic acid sequence having at least 95% sequence identity to a sequence selected from the group of SEQ ID Nos: 9-14.

[0028] In some embodiments of any of the bacterial cells disclosed herein, the RNA selectable marker is selected from the group of an RNA-OUT selectable marker that encodes and RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 25; an RNAI selectable marker that encodes an RNAII regulating RNAI selectable marker encoding an RNAIIALD - P2024-0647-US02 (PCT) regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 26; and a synthetic RNA selectable marker encoding an RNA selectable marker complement regulating RNA with at least 95% sequence identity to SEQ ID NO: 27. In some embodiments, the RNA selectable marker is a functional RNA-OUT. In some embodiments, the R6K bacterial replication origin and the RNA-OUT RNA selectable marker have at least 95% sequence identity to SEQ ID NO: 28.[00291 In some embodiments of any of the bacterial cells disclosed herein, the first region of nucleic acid sequence and the spacer region of nucleic acid sequence form a nucleic acid structure that is double- stranded, linear, and comprises closed-ends, and wherein the closed-ends comprise the telomerase occupancy site.

[0030] In some embodiments of any of the bacterial cells disclosed herein, the Rep protein comprises an amino acid sequence selected from SEQ ID NOs: 38-45. In some embodiments, the Rep protein comprises a mutation selected from P42L-P113S and P42L-P106L-F107S.

[0031] In some embodiments of any of the bacterial cells disclosed herein, the Pi. promoter comprises a nucleic acid sequence selected from the group of SEQ ID NOs: 48-51.

[0032] In some embodiments of any of the bacterial cells disclosed herein, the OL1 comprises a nucleic acid sequence having a mutation compared to SEQ ID NO: 48, wherein the mutation is selected from a single base substitution or a single base deletion. In some embodiments, the mutation decreases or prevents a lambda repressor from binding to the OL1 as compared to the same lambda repressor binding a non-mutated OL1.

[0033] In some embodiments of any of the bacterial cells disclosed herein, the vector comprises a vector backbone with at least 95% sequence identity to a sequence selected from SEQ ID NOs: 29-35.ALD - P2024-0647-US02 (PCT)

[0034] In some embodiments of any of the bacterial cells disclosed herein, a nucleic acid encoding the functional protelomerase polypeptide is operably linked to a constitutive promoter. In some embodiments of any of the bacterial cells disclosed herein, a nucleic acid encoding the functional protelomerase polypeptide is operably linked to an inducible promoter.

[0035] In some embodiments of any of the bacterial cells disclosed herein, a nucleic acid encoding the functional protelomerase polypeptide is integrated into the bacterial cell’s genome.

[0036] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell is an engineered bacterial cell.

[0037] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell comprises a gene knockout of at least one gene selected from the group of sbcC, sbcD, endA, recA and pgi. In some embodiments, the bacterial cell comprises a gene knockout of at least one gene selected from the group of sbcC and sbcD, and at least one gene selected from the group of endA, recA and pgi. In some embodiments, the bacterial cell comprises a gene knockout of the group of sbcC, sbcD, endA, and recA or the group of sbcC, sbcD, endA, recA, and pgi.

[0038] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell further comprises a sbcB gene, a recB gene, a recD gene, and a red gene. In some embodiments, the sbcB gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 61, wherein the recB gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 63, wherein the recD gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 65, and wherein the red gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 67. In some embodiments, the sbcB gene encodes a functional SbcB polypeptide, the recB gene encodes a functional RecB polypeptide, the recD gene encodes a functional RecD polypeptide, and the red gene encodes a functional Red polypeptide.ALD - P2024-0647-US02 (PCT)

[0039] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell further comprises at least one region selected from the group of a uvrC gene, a mcrA gene, and a mcrBC- hsd-mrr region. In some embodiments, the uvrC gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 69, wherein the mcrA gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 70, and wherein the mcrBC-hsd-mrr region comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 71-76. In some embodiments, the uvrC gene encodes a functional UvrC polypeptide and the mcrA gene encodes a functional McrA polypeptide.

[0040] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell further comprises a gene selected from the group of fhuA and glnV. In some embodiments, the fhuA gene encodes a polypeptide having at least 90% sequence identity to SEQ ID NO: 77, and the glnV gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 78. In some embodiments, the fhuA gene encodes a functional FhuA polypeptide and the glnV gene encodes a functional GlnV tRNA. In some embodiments, the bacterial cell further comprising an fhuA gene and a glnV gene.

[0041] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell further comprising a gene knockout of a dem gene. In some embodiments, the dem gene comprises a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79. In some embodiments, the dem gene encodes a functional Dem polypeptide.

[0042] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell does not contain a supE44 gene.

[0043] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell is derived from an Escherichia coli (E. coli) cell line. In some embodiments, the E. coli cell line isALD - P2024-0647-US02 (PCT) selected from the group of DH5a, DH1 , JM107, JM108, JM109, XLIBlue, and MG1655. In some embodiments, the E. coli cell line is MG1655. In some embodiments, the bacterial cell is derived from an E. coli cell line, wherein the E. coli cell line is selected from the group of GalG20, MG1655, and MG1655 AendA ArecA, and wherein the bacterial cell comprises a gene knockout of at least one gene selected from the group of SbcC and SbcD. In some embodiments, the bacterial cell comprises the gene knockout of a SbcC gene and a SbcD gene.

[0044] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell further comprises a genomic nucleic acid sequence encoding a temperature-sensitive lambda repressor. In some embodiments, the temperature- sensitive lambda repressor is cITs857. In some embodiments, the temperature- sensitive lambda repressor comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 37. In some embodiments, the temperature-sensitive lambda repressor is a phage cp 80 attachment site chromosomally integrated copy of an arabinose inducible CITs857 gene.

[0045] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell provides improved yield of the vector as compared to a reference bacterial cell. In some embodiments, the bacterial cell provides improved yield of the vector as compared to a reference bacterial cell that does not include a gene knockout of at least one of endA, recA and pgi.

[0046] In some embodiments of any of the bacterial cells disclosed herein, the vector has increased stability and integrity in the bacterial cell as compared to in a reference bacterial cell. In some embodiments, the bacterial cell has an equal or greater growth rate as compared to a reference bacterial cell when the bacterial cell and the reference bacterial cell are grown in the same conditions.ALD - P2024-0647-US02 (PCT)

[0047] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell has an equal or greater vector yield as compared to a reference bacterial cell when the bacterial cell and the reference bacterial cell are grown in the same conditions, the vector extraction procedure is the same, and same vector yield assay is used.

[0048] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell is an E. coli bacterial cell and is isogenic to the bacterial strain from which it is derived, the strain from which it is derived being selected from the group of DH5a, DH1, JM107, JM108, JM109, MG 1655 and XL 1 Blue.

[0049] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell comprises a 16S rRNA gene having at least 97% sequence identity to SEQ ID NO: 80.

[0050] In some embodiments of any of the bacterial cells disclosed herein, the bacterial cell further comprises a nucleic acid encoding an RNA-IN sequence.

[0051] Also disclosed herein are method for producing a double-stranded, linear, close-ended nucleic acid vector. In some embodiments, the method comprises: inserting a nonlinear vector into a bacterial cell, wherein the bacterial cell can express a functional protelomerase, and wherein the nonlinear vector comprises: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin and / or a RNA selectable marker; and exposing the bacterial cell to a condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector, thereby converting the nonlinear vector to the double- stranded, linear, close- ended nucleic acid vector. In some embodiments, the step of inserting comprises transforming or transducing the nonlinear vector.ALD - P2024-0647-US02 (PCT)

[0052] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises exposing the bacterial cell to a condition sufficient to replicate the nonlinear vector prior to the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector. In some embodiments, the functional protelomerase is encoded by a nucleic acid sequence integrated into the bacterial cell genome. In some embodiments, the nonlinear vector further comprises a nucleic acid sequence encoding the functional protelomerase.

[0053] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises the step of inserting a second vector into the bacterial cell, wherein the second vector comprises a nucleic acid sequence encoding the functional pro telomerase.

[0054] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises exposing the bacterial cell to a condition sufficient to induce expression of the functional protelomerase, and wherein the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter.

[0055] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises the step of exposing the bacterial cell to a condition sufficient to replicate the double- stranded, linear, close-ended nucleic acid vector.

[0056] In some embodiments, the nonlinear vector is a plasmid vector. In some embodiments, the nucleic acid encoding the functional protelomerase is operably linked to a constitutive promoter.

[0057] In some embodiments, the condition sufficient to replicate the vector is performed by a fed-batch fermentation. In some embodiments, the fed-batch fermentation comprises culturing the bacterial cell at a first temperature of about 25°C to about 32°C during a first portion of the fed-ALD - P2024-0647-US02 (PCT) batch phase, followed by culturing the bacterial cell at a second temperature of about 37°C to about 45°C during a second portion of the fed-batch phase.

[0058] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector.

[0059] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises extracting the double-stranded, linear, close-ended nucleic acid vector from the lysate to produce an extracted vector solution.

[0060] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises purifying the double- stranded, linear, close-ended nucleic acid vector from the extracted vector solution. In some embodiments, the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

[0061] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to a condition sufficient to replicate the double-stranded, linear, close- ended nucleic acid vector.

[0062] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises extracting the double-stranded, linear, close-ended nucleic acid vector from the lysate to produce an extracted vector solution.

[0063] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises purifying the double- stranded, linear, close-ended nucleicALD - P2024-0647-US02 (PCT) acid vector from the extracted vector solution. In some embodiments, the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

[0064] In some embodiments of any of the methods described herein, the double- stranded, linear, close-ended nucleic acid vector is any one of the vectors described herein.

[0065] In some embodiments of any of the methods described herein, the bacterial cell is any one of the bacterial cells described herein.

[0066] Also disclosed herein are bacterial cells comprising: a) a nucleic acid sequence encoding a functional protelomerase polypeptide; and b) an engineered circular plasmid vector comprising a nucleic acid sequence comprising a gene of interest and a telomerase occupancy site.

[0067] Also disclosed herein are methods for producing a double- stranded, linear, close-ended nucleic acid vector, the method comprising: inserting a nonlinear vector into a bacterial cell, wherein the bacterial cell can express a functional protelomerase, and

[0068] wherein the nonlinear vector comprises a nucleic acid sequence comprising a gene of interest and a telomerase occupancy site; and exposing the bacterial cell to a condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector, thereby converting the nonlinear vector to the double- stranded, linear, close-ended nucleic acid vector. In some embodiments, the step of inserting comprises transforming or transducing the nonlinear vector.

[0069] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises exposing the bacterial cell to a condition sufficient to replicate the nonlinear vector prior to the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on theALD - P2024-0647-US02 (PCT) nonlinear vector. Tn some embodiments, the functional protelomerase is encoded by a nucleic acid sequence integrated into the bacterial cell genome.

[0070] In some embodiments, the nonlinear vector further comprises a nucleic acid sequence encoding the functional protelomerase.

[0071] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises the step of inserting a second vector into the bacterial cell, wherein the second vector comprises a nucleic acid sequence encoding the functional pro telomerase.

[0072] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises exposing the bacterial cell to a condition sufficient to induce expression of the functional protelomerase, and wherein the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter.

[0073] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises the step of exposing the bacterial cell to a condition sufficient to replicate the double- stranded, linear, close-ended nucleic acid vector.

[0074] In some embodiments, the nonlinear vector is a plasmid vector.

[0075] In some embodiments, the nucleic acid encoding the functional protelomerase is operably linked to a constitutive promoter.

[0076] In some embodiments of a method for producing a double-stranded, linear, close-ended nucleic acid vector, the condition sufficient to replicate the vector is performed by a fed-batch fermentation. In some embodiments, the fed-batch fermentation comprises culturing the bacterial cell at a first temperature of about 25°C to about 32°C during a first portion of the fed-batch phase,ALD - P2024-0647-US02 (PCT) followed by culturing the bacterial cell at a second temperature of about 37°C to about 45°C during a second portion of the fed-batch phase.

[0077] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector.

[0078] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises extracting the double-stranded, linear, close-ended nucleic acid vector from the lysate to produce an extracted vector solution.

[0079] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises purifying the double- stranded, linear, close-ended nucleic acid vector from the extracted vector solution. In some embodiments, the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

[0080] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to a condition sufficient to replicate the double-stranded, linear, close- ended nucleic acid vector.

[0081] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises extracting the double-stranded, linear, close-ended nucleic acid vector from the lysate to produce an extracted vector solution.

[0082] In some embodiments, the method for producing a double-stranded, linear, close-ended nucleic acid vector further comprises purifying the double- stranded, linear, close-ended nucleicALD - P2024-0647-US02 (PCT) acid vector from the extracted vector solution. In some embodiments, the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

[0083] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0084] The use of the term “or” in the claims and the present disclosure is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0085] Use of the term “about”, when used with a numerical value, is intended to include + / - 10%. By way of example but not limitation, if a number of amino acids is identified as about 200, this would include 200+ / - 10% (i.e., 180 to 220).

[0086] As used herein, the term “sequence identity” refers to the degree of identity between any given query sequence and a subject sequence. A subject sequence may, for example, have at least 90%, at least 95%, or at least 99% sequence identity to a given query sequence. To determine percent sequence identity, a query sequence (e.g. a nucleic acid sequence or an amino acid sequence) is aligned to one or more subject sequences using any suitable sequence alignment program that is well known in the art, for instance, the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid sequences to be carried out across their entire length (global alignment). Chema et al., 2003 Nucleic Acids Res., 31:3497-500. In a preferred method, the sequence alignment program (e.g. ClustalW) calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities, and differences can be determined. Gaps of one or more nucleotides can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pair-wise alignments of nucleic acid sequences, suitable default parameters can be selected that are appropriate for the particular alignment program. The output is a sequence alignment that reflectsALD - P2024-0647-US02 (PCT) the relationship between sequences. To further determine percent identity of a subject nucleic acid sequence to a query sequence, the sequences are aligned using the alignment program, the number of identical matches in the alignment is divided by the length of the query sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0087] The phrase “codon degenerate variant” when used with reference to a nucleic acid sequence means a nucleic acid sequence that differs from the referenced sequence, but that encodes a polypeptide having the same amino acid sequence as that encoded by the referenced sequence.

[0088] As used herein “gene of interest” refers to a gene to be expressed in the target organism.

[0089] As used herein “spacer region” refers to the region linking the 5’ and 3’ ends of the first region.

[0090] As used herein “Pol” refers to polymerase.

[0091] As used herein “Pol I” refers to E. coli DNA Polymerase I.

[0092] As used herein “Pol III” refers to E. coli DNA Polymerase III.

[0093] As used herein “Pol III dependent origin of replication” refers to an origin of replication that does uses Polymerase III, for example the rep protein dependent R6K gamma origin of replication.

[0094] As used herein “PL promoter” refers to the lambda promoter left. The PL promoter is a relatively strong promoter compared to global promoter expression that is repressed by the cl repressor binding to OL1, OL2 and OL3 repressor binding sites in the nucleic acid of the PL promoter. The temperature sensitive cI857 repressor allows control of gene expression by heatALD - P2024-0647-US02 (PCT) induction since at 30°C the cI857 repressor is functional and it represses gene expression, but at 37-42 °C the repressor is inactivated allowing the gene to be expressed.BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Fig. 1 A is a schematic of an exemplary circular vector containing a protelomerase gene coding sequence (1) operably linked to an arabinose-inducible promoter (2) and a telomerase occupancy site (TelN) (3), referred to as TelN-NP-Ara-protelomerase.

[0096] Fig. IB is a schematic of an exemplary ce-linear vector generated from the circular vector of Fig. 1A.

[0097] Figs. 2A-2C are representations of vector digestion analyses of E. co / z-R6K transformed with the TelN-NP-Ara-protelomerase vector. Fig. 2A is a representation of uncut DNA from 5 transformation clones (A-E) that showed substantially similar profiles with a predominant band running larger than 4 kb, indicating linear vector. Fig. 2B is a representation of vectors after digestion with T5 exonuclease (exonuclease) and Bglll restriction enzyme digestion. The predominant band was resistant to T5 exonuclease treatment (middle lane), indicating closed ends, and digested into 2 fragments by the Bglll restriction enzyme, further validating that the predominant product is linear. Fig. 2C is a representation of a simulated vector digest showing banding profiles of digesting circular or linear DNA with a unique restriction enzyme. The banding pattern matches that simulated for the linear vector.

[0098] Fig. 3 is an exemplary schematic of generating deactivated protelomerase. Circular vector was digested with a unique enzyme in the protelomerase coding sequence, the digest sites were filled in, and the ends were joined to make a circular- vector with a mutated protelomerase sequence. Both active and deactivated forms were used to transform E. co / z-R6K.ALD - P2024-0647-US02 (PCT)

[0099] Figs. 4A-4C show that active protelomerase generated linear vector while deactivated protelomerase generated circular vector. Fig. 4A is a vector gel analysis of each step used to produce the vectors of Fig. 3. Fig. 4B is a representation of a simulated vector digest showing banding profiles of digesting circular or linear DNA with a unique restriction enzyme. Fig. 4C is a graph of the amount of vector recovered (ng / pl) from a vector extraction.

[0100] Figs. 5A-5C are schematics of three different vectors: a vector with protelomerase operably linked to an arabinose-inducible promoter (AraBAD) named E. coZz-R6K (pUC-Tel) (Fig. 5A), a vector with no tos site that should remain circular named 260-170-2 (Fig. 5B), and a vector containing a tos site that can be linearized named 260-170-3 (Fig. 5C).

[0101] Fig. 6 is a representation of various vector digests performed in triplicate. All bacterial cells from which vectors were extracted contained pUC-TelN and also either contained 260-170-2 vector or 260-170-3 vector.

[0102] Fig. 7 is a representation of various vector digests using either an arabinose induction and / or heat shift on E. coli strains containing vectors.

[0103] Figs. 8A-8C are exemplary schematics for generating a bacterial cell with ce-linear vectors expressing a transgene.DETAILED DESCRIPTION

[0104] The present disclosure is directed to close ended-linear vectors, engineered bacteria containing the same, methods of making the same, and methods of use. Ce-linear DNA vector is double-stranded DNA molecule that contains covalently closed terminal hairpins. The covalently closed terminal hairpins increase vector stability by protecting the ce-linear DNA from exonuclease activity and DNA supercoiling, making the ce-linear DNA vectors torsion-free, which may enhance transgene expression.ALD - P2024-0647-US02 (PCT)

[0105] Typically, covalently closed terminal hairpins are formed by a protein called protelomerase, also known as telomere resolvase. Exemplary protelomerase proteins include, but are not limited to, TelN (TelN amino acid sequence - SEQ ID NO: 1, telN nucleic acid sequence - SEQ ID NO: 2), Tel (Tel amino acid sequence - SEQ ID NO: 3, tel nucleic acid sequence - SEQ ID NO: 4), TelK (UniProt Primary Accession No: Q6UAV6), and TelA (UniProt Primary Accession No: G0LXV2).

[0106] Protelomerase recognizes and cleaves a specific sequence called a telomerase occupancy site (also called a tos site) on double-stranded DNA during replication. Structurally, the telomerase occupancy site includes a 56-bp central palindromic sequence known as telRL (SEQ ID NO: 5), which is formed by a 22-base pair palindrome (telO - SEQ ID NO: 6) flanked by two 14-base pair inverted repeats (telR - SEQ ID NO: 7 and telL - SEQ ID NO: 8) and separated by 3 bps. See, for example, Daneke et al. PNAS. 2000, 97, 14:7721-7726. Other telomerase occupancy sites have been described, including a pal site (42-bp) from the Yersinia enterocolitica phage PY54. See, for example, Sefan H et al., Mol. Microbiol 2003, 48:989-1003. After cleavage, each strand is refolded into a hairpin formation and ligated to form a covalently closed hairpin. See, for example, E. Knott, Sophie, Sarah A. Milsom, and Paul J. Rothwell. 2020. ‘The Unusual Linear Plasmid Generating Systems of Prokaryotes’. Bacteriophages - Perspectives and Future. IntechOpen. doi: 10.5772 / intechopen.86882.

[0107] Vectors disclosed herein, such as ce-linear vectors or circular vectors that could be linearized, include a first region of nucleic acid sequence and a spacer region. The first region of nucleic acid sequence may include genetic components necessary for obtaining a ce-linear vector, such as one or more telomerase occupancy sites. The spacer region may include genetic components necessary for replication of the vector, either for replication of the ce-linear vector orALD - P2024-0647-US02 (PCT) a circular version of the vector before it is linearized, and can include a bacterial origin of replication, a RNA selectable marker, or both.

[0108] In one embodiment, a circular plasmid vector, such as a Nanoplasmid™ vector, can be modified to include a telomerase occupancy site as shown in Fig. 8A. This circular vector can then be introduced into a bacterial cell expressing a protelomerase and under appropriate conditions, the circular plasmid can then be replicated in the bacterial cell and then linearized and capped by the protelomerase activity in the bacterial cell as shown in Fig. 8C. Alternatively, the conditions used may first result in linearization and capping of the circular plasmid followed by replication of the linearized, capped vector (not shown).

[0109] In some embodiments, a vector comprises a first region comprising a telomerase occupancy site. Exemplary telomerase occupancy sites include, but are not limited to, telRL (SEQ ID NO: 5) and a pal site. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 80% sequence identity (e.g., at least about 85%. 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 5. For example, in some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 90% sequence identity to SEQ ID NO: 5. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO: 5. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 96% sequence identity to SEQ ID NO: 5. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 97% sequence identity to SEQ ID NO: 5. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 98% sequence identity to SEQ ID NO: 5. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least about 99%ALD - P2024-0647-US02 (PCT) sequence identity to SEQ ID NO: 5. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 10 mutations (e.g., up to 10 nucleotide substitutions, up to 10 nucleotide additions, up to 10 nucleotide deletions). In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 10 nucleotide substitutions (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 9 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 8 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 7 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 6 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 5 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 4 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 3 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by up to 2 nucleotide substitutions. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence that differs from SEQ ID NO: 5 by 1 nucleotide substitution. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence of SEQ ID NO: 5, or a codon degenerate variant thereof.ALD - P2024-0647-US02 (PCT)

[0110] In some embodiments, a vector comprises more than one telomerase occupancy site. In other embodiments, a vector comprises only one telomerase occupancy site.

[0111] Using a telomerase occupancy site to linearize a vector requires a functional protelomerase polypeptide, i.e., a protelomerase polypeptide that can generate a hairpin and covalently close the hairpin at the end of a linearized vector. In some cases, a vector comprises a nucleic acid encoding a protelomerase polypeptide. In other cases, a vector lacks a nucleic acid encoding a protelomerase polypeptide.

[0112] In some embodiments, a vector comprises genetic components necessary for expression of a gene of interest (e.g., a transgene for gene therapy) in a target organism. For example, in some embodiments, a vector comprises a first region of nucleic acid sequence comprising a gene of interest. In some embodiments, a vector comprises a first region of nucleic acid sequence comprising a gene of interest and a telomerase occupancy site. Exemplary genes of interest include, but are not limited to, gene therapies, RNA therapies, and protein therapeutics. Gene therapies may include non-mutated copies of genes for which the presence of a mutated copy of the gene is associated with a disease and vaccine constructs. RNA therapies may include short hairpin RNA (shRNA), small interfering RNA (siRNA), and microRNAs. Protein therapies may include chimeric antigen receptors, and protein antigens.

[0113] In some embodiments, the first region of nucleic acid sequence comprises 5’ and 3’ ends (e.g., a 5’ end and a 3’ end). In some embodiments, the first region of nucleic acid sequence includes a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends. In some embodiments, the first region of nucleic acid sequence consists of a gene of interest, a telomerase occupancy site, a 5’ end, and a 3’ end.ALD - P2024-0647-US02 (PCT)

[0114] The 5’ and 3’ ends of the first region are typically separated by a spacer region. That is, a spacer region typically links the 5’ and 3’ ends of the first region of the nucleic acid. The spacer region may include a bacterial origin of replication, a selectable marker, or both. In some embodiments, the spacer region is less than 1500 bp (e.g., less than 1200 bp, less than 1000 bp, less than 800 bp, or less than 500 bp). In some embodiments, the spacer region is less than 1000 bp. In some embodiments, the spacer region is less than 500 bp. In some embodiments, the spacer region is between 200 bp and 1200 bp in length (e.g., between 300 bp and 1100 bp or between 400 bp and 1000 bp). In some embodiments, the spacer region is larger than 100 bp.

[0115] In any of the foregoing embodiments, the vector may include any appropriate origin of replication. Exemplary bacterial origins of replication include, but are not limited to a R6K origin of replication, a pUC origin of replication, and a ColE2 origin of replication. In some embodiments, the bacterial origin of replication may be a R6K origin of replication (e.g., an R6K gamma origin of replication or a CpG free R6K origin of replication) comprising a nucleic acid sequence having at least about 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14. In some embodiments, the bacterial origin of replication comprises a R6K origin of replication comprising a nucleic acid sequence having at least about 90% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14. In some embodiments, the bacterial origin of replication comprises a R6K origin of replication comprising a nucleic acid sequence having at least about 95% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14. In some embodiments, the bacterial origin of replication comprises a R6K origin of replication comprising a nucleic acid sequence having at least about 98% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14. In some embodiments, the bacterial origin of replication comprises a R6KALD - P2024-0647-US02 (PCT) origin of replication comprising a nucleic acid sequence having at least about 99% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14. In some embodiments, the bacterial origin of replication comprises a R6K origin of replication comprising a nucleic acid sequence selected from the group of SEQ ID NOs: 9-14.

[0116] In some embodiments, a bacterial origin of replication comprises a ColE2 origin of replication comprising a nucleic acid sequence having at least about 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group of SEQ ID NO: 15 (ColE2 bacterial origin of replication (+7), 45 bp), SEQ ID NO: 16 (ColE2 Origin (Min), 38 bp), SEQ ID NO: 17 (ColE2 Origin (Core), 32 bp), SEQ ID NO: 18 (ColE2 Origin (+7, CpG free), 45 bp), and SEQ ID NO: 19 (ColE2 Origin (+16), 60 bp). In some embodiments, a bacterial origin of replication comprises a pUC origin of replication comprising a nucleic acid sequence having at least about 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group of, such as SEQ ID NO: 20 (minimal pUC bacterial origin of replication, 784 bp).

[0117] In some embodiments, the spacer region comprises a bacterial origin of replication, such as a Pol III dependent origin of replication (e.g., a Pol Ill-dependent R6K origin of replication). In vectors comprising a spacer region comprising Pol III dependent origin of replication, preferably, the spacer region is less than 1500 bp (e.g., less than 1200 bp, less than 1000 bp, less than 800 bp, or less than 500 bp). In some embodiments, in vectors comprising a spacer region comprising Pol III dependent origin of replication, the spacer region is less than 1200 bp. In some embodiments, in vectors comprising a spacer region comprising Pol III dependent origin of replication, the spacer region is less than 1000 bp. In some embodiments, in vectors comprising a spacer region comprising Pol III dependent origin of replication, the spacer region is less than 1000 bp. In someALD - P2024-0647-US02 (PCT) embodiments, in vectors comprising a spacer region comprising Pol ITT dependent origin of replication, the spacer region is less than 800 bp. In some embodiments, in vectors comprising a spacer region comprising Pol III dependent origin of replication, the spacer region is less than 500 bp. In some embodiments, in vectors comprising a spacer region comprising Pol III dependent origin of replication, the spacer region is between 200 bp and 1200 bp in length (e.g., between 300 bp and 1100 bp or between 400 bp and 1000 bp). In some embodiments, in vectors comprising a spacer region comprising Pol III dependent origin of replication, the spacer region is larger than 100 bp.

[0118] In any of the foregoing embodiments, the vector may include a nucleic acid sequence encoding any appropriate selectable marker. As used herein “selectable marker” refers to a marker used to select a desired host cell. For example, a selectable marker may be an antibiotic resistance gene or a RNA selectable marker. In some embodiments, a vector comprises a spacer region comprising a nucleic acid sequence encoding a selectable marker. In some embodiments, the selectable marker is not an antibiotic resistance gene. In some embodiments, the vector lacks an antibiotic resistance gene.

[0119] In some embodiments, the vector comprises a nucleic acid sequence encoding a RNA selectable marker. In some embodiments, a vector comprises a spacer region comprising a bacterial origin of replication and a nucleic acid encoding a RNA selectable marker. For example, in some embodiments, a vector comprises a spacer region that is less than 1500 bp and links the 5’ and 3’ ends of a first region of nucleic acid sequence, and the spacer region comprises a bacterial origin of replication and a nucleic acid encoding a RNA selectable marker.

[0120] For example, a vector may comprise a spacer region comprising a nucleic acid encoding a RNA selectable marker. As used herein “RNA selectable marker” refers to a plasmid bomeALD - P2024-0647-US02 (PCT) expressed non-translated RNA that regulates a chromosomally expressed target gene. Preferably, the chromosomally expressed target gene can be used as a selection marker, either as a positive selection marker (identifying desired products) or a negative selection marker (identifying or killing not desired products). The RNA selectable marker may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target. See, for example, US Patent 6,977,174. This may also be a plasmid borne antisense repressor RNA, a nonlimiting list included herein by reference includes RNA-OUT that represses RNA-IN regulated targets (See, for example, PCT Publication No. WO 2008 / 153733), pMBl plasmid origin encoded RNAI that represses RNAII regulated targets (See, for example, US Patent Publication No. 2006 / 0063232 or US Patent No. 7,611,883), IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulated targets (See, for example, Wilson et al. 1997. J Bacteriol 179:742-53), ParB locus Sok of plasmid R1 that represses Hok regulated targets, or Flm locus FlmB of F plasmid that represses HmA regulated targets (See, for example, US Patent No. 5,922,583). An RNA selectable marker may be another natural antisense repressor RNAs known in the art such as those described in Wagner et al. 2002. Adv Genet 46:361-98 and Franch T, and Gerdes K. 2000. Current Opin Microbiol 3:159-64. An RNA selectable marker may also be an engineered repressor RNAs such as synthetic small RNAs expressed SgrS, MicC or MicF scaffolds as described in Na et al. 2013. Nat Biotechnol 31:170-4. An RNA selectable marker may also be an engineered repressor RNA as part of a selectable marker that represses a target RNA fused to a target gene to be regulated such as SacB as described in US Patent Publication No. 2015 / 0275221. As used herein “SacB” refers to the structural gene encoding Bacillus subtilis levansucrase. Expression of SacB in gram negative bacteria is toxic in the presence of sucrose.ALD - P2024-0647-US02 (PCT)

[0121] By way of example, but not limitation, the RNA selectable marker can be a RNA-OUT selectable marker. As used herein “RNA-OUT” refers to an insertion sequence 10 (IS 10) encoded RNA-OUT, an antisense RNA that hybridizes to, and reduces translation of, a gene expressed downstream of RNA-IN, for example, a sacB gene that is lethal to gram negative bacteria in the presence of sucrose. The sequence of the RNA-OUT RNA (SEQ ID NO: 21) and complementary RNA-IN-SacB construct can be modified to incorporate alternative functional RNA-IN / RNA- OUT binding pairs such as those described in Mutalik et al., 2012 Nat Chem Biol 8:447, including, but not limited to, the RNA-OUT A08 / RNA-IN S49 pair, the RNA-OUT A08 / RNA-IN S08 pair, and CpG free modifications of RNA-OUT A08 that modify the CG in the RNA-OUT 5’ TTCGC sequence to a non-CpG sequence. A multitude of alternative substitutions to remove the two CpG motifs (mutating each CpG to either CpA, CpC, CpT, ApG, GpG, or TpG) may be utilized to make a CpG free RNA-OUT. In some embodiments, the RNA selectable marker is a functional RNA- OUT. For example, a functional RNA-OUT RNA selectable marker hybridizes to, and reduces translation of, a gene expressed downstream of RNA-IN.

[0122] By way of further example, but not limitation, the RNA-OUT can have at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group of SEQ ID NO: 22 and SEQ ID NO: 23, respectively. In some embodiments, the vector may include a corresponding RNA-IN sequence that regulates a downstream marker by the RNA-OUT.

[0123] In any of the foregoing embodiments, the RNA-OUT may comprise a nucleic acid sequence encoding a RNA-OUT antisense repressor RNA. By way of example, but not limitation, the RNA-OUT antisense repressor RNA can have a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21.ALD - P2024-0647-US02 (PCT)

[0124] As used herein “RNA-OUT selectable marker” refers to an RNA-OUT selectable marker DNA fragment including a promoter and terminator sequences flanking an RNA-OUT RNA. An RNA-OUT selectable marker operably linked to a promoter and a terminator sequence and flanked by Dralll and Kpnl restriction enzyme sites, and genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are described in WO 2008 / 153733, which is included herein by reference. The RNA-OUT promoter and terminator sequences that flank RNA-OUT may be replaced with heterologous promoter and terminator sequences. For example, the RNA-OUT promoter may be substituted with a CpG free promoter known in the art, for example the I-EC2K promoter or the P5 / 65 / 6 or P5 / 6 6 / 6 promoters described in WO 2008 / 153733 and included herein by reference. A 2 CpG RNA-OUT selectable marker in which the two CpG motifs in the RNA- OUT promoter are removed was given as SEQ ID NO: 23. Vectors incorporating CpG free RNA- OUT selectable marker may be selected for sucrose resistance using the RNA-IN-SacB cell lines for RNA-OUT plasmid propagation described in WO 2008 / 153733 or any cell line with RNA-IN- SacB as described in WO 2008 / 153733. Alternatively, the RNA-IN sequence in these cell lines can be modified to incorporate the 1 bp change needed to perfectly match the CpG free RNA-OUT region complementary to RNA-IN.

[0125] As used herein “RNA-IN” refers to an insertion sequence 10 (IS 10) encoded RNA-IN, an RNA complementary and antisense to a portion of RNA RNA-OUT. When RNA-IN is cloned in the untranslated leader of a mRNA, annealing of RNA-IN to RNA-OUT reduces translation of the gene encoded downstream of RNA-IN.

[0126] As used herein “RNA-IN regulated selectable marker” refers to a selectable marker that is genomically expressed and regulated by RNA-IN. For example, a nucleic acid encoding an RNA- IN regulated selectable marker may comprise the nucleic acid sequence of SEQ ID NO: 25. In theALD - P2024-0647-US02 (PCT) presence of RNA-OUT antisense repressor RNA (SEQ ID NO: 21), downstream expression of a nucleic acid encoding an RNA-IN regulated selectable marker is repressed. An RNA-IN regulated selectable marker is configured such that RNA-IN regulates the expression of mRNA encoding 1) a protein that is lethal or toxic to a cell (e.g., a bacterial cell) per se or that generates a toxic substance (e.g., SacB), or 2) a repressor protein that is lethal or toxic to a cell (e.g., a bacterial cell) by repressing the transcription of a gene that is essential for growth of the cell (e.g. repressing the murA essential gene with a RNA-IN tetR repressor gene). For example, genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid selection / propagation are described in WO 2008 / 153733. Alternative RNA-IN regulated selection markers described in the art may be substituted for SacB.

[0127] In some embodiments, the RNA selectable marker is selected from the group of i) an RNA- OUT selectable marker that encodes an RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to a sequence selected from the group of SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23; ii) an RNAI selectable marker that encodes a RNAII regulating RNAI selectable marker; and iii) an engineered repressor RNA. An engineered repressor RNA may include a synthetic RNA selectable marker encoding an RNA selectable marker complement regulating RNA. In some embodiments, a nucleic acid encoding an RNA selectable marker complement regulating RNA has at least about 90% (e.g., 95%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 27.

[0128] In some embodiments, the spacer region comprises a bacterial origin of replication and a nucleic acid sequence encoding a RNA selectable marker. For example, the spacer region may comprise a R6K bacterial origin of replication and a nucleic acid sequence encoding a RNA-OUT RNA selectable marker, wherein the nucleic acid sequence encoding the RNA-OUT RNAALD - P2024-0647-US02 (PCT) selectable marker has at least 95% sequence identity to a sequence selected from the group of SEQ ID NO: 21-23. In some embodiments, the spacer region comprises a R6K bacterial origin of replication having at least about 95% sequence identity to a sequence selected from the group of SEQ ID NO: 9-14, and a nucleic acid sequence encoding a RNA-OUT RNA selectable marker selected from the group of i) an RNA-OUT selectable marker that encodes and RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to a sequence selected from the group of SEQ ID NO: 21-23; ii) an RNAI selectable marker that encodes a RNAII regulating RNAI selectable marker; and iii) an engineered repressor RNA. In some embodiments, the spacer region comprises a R6K bacterial origin and a RNA-OUT RNA selectable marker, where the nucleic acid encoding the R6K bacterial origin and the RNA-OUT RNA selectable marker has at least 95% sequence identity to SEQ ID NO: 28.

[0129] In some embodiments, the vector comprises a nucleic acid encoding an endonuclease polypeptide. In other embodiments, the vector lacks a nucleic acid encoding an endonuclease polypeptide. For example, in some embodiments, the vector does not comprise a nucleic acid encoding an endonuclease polypeptide.

[0130] Any of the vectors described herein may have any appropriate nucleic acid structure. For example, any of the vectors described herein may be a circular vector or a ce-linear DNA vector. In some embodiments, the vector is a ce-linear DNA vector. In some embodiments, the first region of nucleic acid sequence and the spacer region form a nucleic acid structure comprising doublestranded DNA. In some embodiments, the first region of nucleic acid sequence and the spacer region form a nucleic acid structure comprising double-stranded, linear DNA having 2 closed- ends. In some embodiments, the first region of nucleic acid sequence and the spacer region form a nucleic acid structure comprising double-stranded, linear DNA having 2 closed-ends comprisingALD - P2024-0647-US02 (PCT) covalently closed hairpins. For example, a closed-end may comprise the remnants of a telomerase occupancy site after the telomerase occupancy site used to linearize the vector. In some embodiments, the first closed-end comprises telR (e.g., SEQ ID NO: 7) and the second closed-end comprises telL (e.g., SEQ ID NO: 8). In some embodiments, the first closed-end comprises the nucleic acid sequence of SEQ ID NO: 7 and the second closed-end comprises the nucleic acid sequence of SEQ ID NO: 8.

[0131] As used herein “vector backbone” refers to the nucleic acid sequence that makes up the eukaryotic and bacterial regions of a vector, excluding the transgene. The vector backbone can be derived from any appropriate vector. Exemplary vector backbones include, but are not limited to, NTC9385C, NTC9685C, NTC9385R, NTC9685R, NTC9385Ra-01, NTC9385Ra-O2, and NTC9385R-BE vector backbones. See, for example, U.S. Patent No. RE49423. A vector backbone may comprise at least about 80% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity) to the vector backbone of NTC9385C, NTC9685C, NTC9385R, NTC9685R, NTC9385Ra-01, NTC9385Ra-O2, or NTC9385R-BE. In some embodiments, a vector comprises a vector backbone having at least about 80% sequence identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity) to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 85% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 90% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 95% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 96% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In someALD - P2024-0647-US02 (PCT) embodiments, a vector comprises a vector backbone having at least about 97% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 98% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 99% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone having at least about 99.5% sequence identity to a sequence selected from SEQ ID NOs: 29-35. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 25 nucleotide mutations (e.g., 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mutation(s)). In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29- 35 by no more than 25 nucleotide substitutions (e.g., 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 20 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 15 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 10 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 9 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 8 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 7 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by noALD - P2024-0647-US02 (PCT) more than 6 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 5 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 4 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 3 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 2 nucleotide substitutions. In some embodiments, a vector comprises a vector backbone differing from a sequence selected from SEQ ID NOs: 29-35 by no more than 1 nucleotide substitution. In some embodiments, a vector comprises a vector backbone having a sequence selected from SEQ ID NOs: 29-35, or a codon degenerate variant thereof.

[0132] Also disclosed herein are bacterial cells containing any of the vectors described herein. For example, a bacterial cell comprises any of the ce-linear vectors described here. In some embodiments, a bacterial cell comprises a vector, the vector comprising: i) any of the first regions of nucleic acid sequence disclosed herein, and ii) any of the spacer regions disclosed herein. In some embodiments, a bacterial cell comprises a vector, the vector comprising: i) a first region of nucleic acid sequence comprising any of the telomerase occupancy sites disclosed herein, and ii) a spacer region that is less than 1500 bp and links the 5’ and 3’ ends of the first region of nucleic acid sequence. In some embodiments, a bacterial cell comprises a vector, the vector comprising i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends; and ii) a spacer region that is less than 1500 bp (e.g., less than 1000 bp or less than 500 bp) and links the 5’ and 3’ ends of the first region of nucleic acid sequence, and comprisesALD - P2024-0647-US02 (PCT) a bacterial origin of replication, an RNA selectable marker, or both a bacterial origin of replication and an RNA selectable marker.

[0133] A bacterial cell may also comprise a functional pro telomerase polypeptide. A functional protelomerase polypeptide may recognize a telomerase occupancy site and linearize the nucleic acid sequence comprising the telomerase occupancy site to generate closed-ends (e.g., covalently closed hairpins). Any assay that distinguishes circular vectors from linear vectors, such as those described in the Examples section herein, can be used to identify a functional protelomerase polypeptide. Exemplary protelomerase polypeptides include, but are not limited to, TelN (e.g., TelN from Escherichia coli phage N15). Additional protelomerase polypeptides include, but are not limited to, protelomerase polypeptides derived from bacterial with linear chromosomes, such as a protelomerase polypeptide derived from Boreilia burdgorferi, and protelomerase polypeptides derived from phage that exist as linear DNA, including, but not limited to, protelomerase polypeptides derived from Klebsiella oxytoca phage (pKO2, Yersinia enterocolitica phage PY54, and Vibrio parahaemolyticus phage VP882. In some embodiments, the protelomerase polypeptide is a TelN protelomerase polypeptide, or a homolog thereof. In some embodiments, the protelomerase polypeptide comprises a sequence having at least 80% sequence identity (e.g., at least about 85%. 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the protelomerase polypeptide comprises a sequence having at least 85% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the protelomerase polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the protelomerase polypeptide comprises a sequence having at least 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the protelomerase polypeptide comprises a sequence having at least 98% sequenceALD - P2024-0647-US02 (PCT) identity to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the protelomerase polypeptide comprises a sequence having at least 99% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the protelomerase polypeptide comprises a sequence differing from SEQ ID NO: 1 or SEQ ID NO: 3 by no more than 20 amino acid mutations (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation(s)). For example, the protelomerase polypeptide comprises a sequence differing from SEQ ID NO: 1 or SEQ ID NO: 3 by no more than 20 amino acid substitutions (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s)). In some embodiments, the protelomerase polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0134] The functional protelomerase polypeptide may be located on a plasmid or integrated into the genome of a bacterial cell, such as shown in Fig. 8B. In some embodiments, the protelomerase polypeptide is located on the vector in the bacterial cell (e.g., the vector comprises a nucleic acid encoding a protelomerase polypeptide). In other embodiments, the protelomerase polypeptide is not located on the vector in the bacterial cell (e.g., the vector lacks a nucleic acid encoding a protelomerase polypeptide or the vector does not comprise a nucleic acid encoding a protelomerase polypeptide). In some embodiments, the protelomerase polypeptide is integrated into the genome of the bacterial cell. In other embodiments, the protelomerase polypeptide is not integrated into the genome of the bacterial cell.

[0135] A bacterial cell may also include any genetic elements necessary for expression of a nucleic acid encoding any of the protelomerase polypeptides described herein. For example, a nucleic acid encoding a protelomerase polypeptide may be operably linked to a promoter, a terminator, or an enhancer. Promoters can include constitutive promoters and inducible promoters. In someALD - P2024-0647-US02 (PCT) embodiments, a protelomerase polypeptide is operably linked to a constitutive promoter. In some embodiments, a protelomerase is operably linked to an inducible promoter.

[0136] A bacterial cell may also comprise a nucleic acid encoding a replication (Rep) protein. Rep proteins assist in replicating certain plasmids (e.g., a Rep protein dependent plasmid). For example, a Rep protein may assist in replicating a plasmid by binding to specific sequences within a plasmid’s origin of replication, helping to unwind the plasmid DNA, or helping to assemble the replication machinery. An exemplary Rep protein amino acid is set forth in Uniprot Accession No. P03067. By way of example, but not limitation, the bacterial cell may include a nucleic acid sequence encoding a Rep protein and having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group of SEQ ID NO: 38 (nucleic acid encoding Rep protein with mutations: P42L-P106I-F107S-P113S, 918 bp), SEQ ID NO: 39 (P42L- A106-107-P113S, 912 bp), SEQ ID NO: 40 (P42L-P106L-F107S, 918 bp), and SEQ ID NO: 41 (P42L-P113S, 918 bp). In some embodiments, the bacterial cell comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 38. In some embodiments, the bacterial cell comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, the bacterial cell comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 38. In some embodiments, the bacterial cell comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 38. In some embodiments, the bacterial cell comprises a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 38. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 10 mutations (e.g., up to 10 nucleotide substitutions, up to 10 nucleotide additions, up to 10 nucleotide deletions). In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQALD - P2024-0647-US02 (PCT)ID NO: 38 by up to 10 nucleotide substitutions (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 9 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 8 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 7 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 6 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 5 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 4 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 3 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by up to 2 nucleotide substitutions. In some embodiments, the nucleic acid encoding a Rep protein comprises a nucleic acid sequence that differs from SEQ ID NO: 38 by 1 nucleotide substitution. In some embodiments, the bacterial cell comprises the nucleic acid sequence of SEQ ID NO: 38, or a codon degenerate variant thereof.

[0137] By way of further example, but not limitation, the bacterial cell can include an nucleic acid sequence encoding a Rep protein, wherein the Rep protein has at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to an amino acid sequence selected from the group of SEQ ID NO: 42 (Rep protein with mutations P42L-P106I-F107S-P113S), SEQ ID NO: 43 (P42L-A106-ALD - P2024-0647-US02 (PCT)107-P113S), SEQ ID NO: 42 (P42L-P106L-F107S), SEQ ID NO: 44 (P42L-P1 13S), SEQ ID NO: 45 (ColE2 Rep protein wild-type), SEQ ID NO: 46 (ColE2 Rep protein mutant G194D). In some embodiments, the Rep protein comprises a mutation selected from P42L-P113S, P42L-P106L- F107S, and P42L-P106I-F107S-P113S. In some embodiments, the Rep protein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, the Rep protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, the Rep protein comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 42. In some embodiments, the Rep protein comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 42. In some embodiments, the Rep protein comprises an amino acid sequence differing from SEQ ID NO: 42 by up to 10 amino acid substitutions (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s)). In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 9 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 8 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 7 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 6 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 5 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 4 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42ALD - P2024-0647-US02 (PCT) by up to 3 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by up to 2 amino acid substitutions. In some embodiments, the amino acid encoding a Rep protein comprises an amino acid sequence that differs from SEQ ID NO: 42 by 1 nucleotide substitution. In some embodiments, the Rep protein comprises the amino acid sequence of SEQ ID NO: 42.[01381 In some embodiments, the Rep protein comprises an amino acid selected from SEQ ID Nos: 38-45.

[0139] It should be understood that the nucleic acid sequences encoding the Rep protein in any of the foregoing embodiments can be under the control of (i.e., operably linked to) any appropriate promoter, include a temperature- sensitive promoter. For example, the Rep protein may be operably linked to a PL promoter. For example, the PL promoter may be temperature-sensitive to enable temperature-sensitive expression of the Rep protein. In some embodiments, the PL promoter is temperature- sensitive if there is a lambda repressor (e.g., called a cl repressor) present in the genome of the bacterial cell, such as the cITs857 lambda repressor, cl repressors binds to OL1, OL2, or OL3 repressor binding sites, if present, in the PL promoter. An exemplary PL promoter is put forth in SEQ ID NO: 36. The PL promoter may include OL1, OL2 and OL3 repressor binding sites, and -10 and -35 promoter elements for PLl and PL2 promoters. The OL1 mutations may include OL1-G and OL1-G to T alterations.

[0140] By way of example, but not limitation, the PL promoter can have a sequence having at least 95%, at least 98%, at least 99% or 100% sequence identity PL promoter OG1 (-35 to - 10) (SEQ ID NO: 48), PL promoter OL1-G (-35 to -10) (SEQ ID NO: 49), or PL promoter OL1-G to T (-35 to -10) (SEQ ID NO: 50). In some embodiments, the PL promoter comprises a nucleic acid sequence having a mutation compared to SEQ ID NO: 48. It should be further understood thatALD - P2024-0647-US02 (PCT) where the Rep protein is a R6K Rep protein (e.g., SEQ ID NOs: 39-42), a vector that is transfected into the bacterial cell may contain a R6K origin of replication and, alternatively, where the Rep protein is a ColE2 Rep protein, a vector that is transfected into the bacterial cell can contain a ColE2 origin of replication.

[0141] The PL promoter may also include an OL1 repressor site, an OL2 repressor site, an OL3 repressor site, or any combination thereof. Any of the OL repressor sites (e.g., OL1, OL2, or OL3 repressor sites) is a functional OL repressor site, meaning that the cl repressor binds to the OL repressor site. In some embodiments, the PL promoter may include an OL1 repressor site. In some embodiments, the OL1 repressor site comprises a nucleic acid sequence having at least about 80% sequence identity (e.g., 85%, 90%, 95%, 98%, 99%, or 100% sequence identity) to SEQ ID NO: 48. In some cases, the OL1 repressor site comprises a nucleic acid sequence having at least about 95% sequence identity to SEQ ID NO: 48. In some cases, the OL1 repressor site comprises a nucleic acid sequence having at least about 98% sequence identity to SEQ ID NO: 48. In some cases, the OL1 repressor site comprises a nucleic acid sequence having at least about 99% sequence identity to SEQ ID NO: 48. In some embodiments, the OL1 repressor site comprises a nucleic acid sequence differing from SEQ ID NO: 48 by no more than 10 nucleotide mutations (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide mutation(s)). In some embodiments, the OL1 repressor site comprises a nucleic acid sequence differing from SEQ ID NO: 48 by no more than 10 nucleotide substitutions (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide substitution(s)). In some embodiments, the OL1 repressor site comprises a nucleic acid sequence having a mutation compared to SEQ ID NO: 48, where the mutation is selected from a single base substitution or a single base deletion. For example, lambda repressor binding to OL1 may be altered by mutations in OL1, such as OL1-G (this is a single base deletion that also reduces the distance between the PL promoter -35 and -10 boxes from optimal 17ALD - P2024-0647-US02 (PCT) bp to 16 bp) and 0L1-G to T (this is a G to T substitution that maintains the distance between the PL promoter -35 and -10 boxes at the optimal 17 bp and corresponds to the V2 mutation described by Bailone A and Galibert F, 1980. Nucleic Acids Research 8:2147).

[0142] In some embodiments, any of the mutations to any of the OL repressor sites decreases or prevents a lambda repressor from binding to the OL as compared to the same lambda repressor binding a non-mutated OL repressor site. For example, the OL1 repressor site comprises a nucleic acid sequence having a mutation compared to SEQ ID NO: 48, where the mutation is selected from a single base substitution or a single base deletion, and the mutation decreases or prevents a lambda repressor from binding to the OL1 as compared to the same lambda repressor binding a nonmutated OL1 repressor site. In some embodiments, the OL1 repressor site is a function OL1 repressor site, meaning that the cl repressor binds to the OL1 repressor site.

[0143] When a bacterial cell comprises a vector, the vector may include a first region of nucleic acid and a spacer region. In some embodiments, the spacer region is less than 1000 bp. In some embodiments, the spacer region is less than 500 bp. In some embodiments, the vector does not comprise a nucleic acid encoding a protelomerase polypeptide. In some embodiments, the vector does not comprise a nucleic acid encoding an endonuclease polypeptide. In some embodiments, the vector comprises only one telomerase occupancy site. In some embodiments, the telomerase occupancy site comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the first region of nucleic acid sequence consists of a gene of interest, a telomerase occupancy site, a 5’ end, and a 3’ end. In some embodiments, the bacteria origin of replication is a R6K bacterial origin of replication. For example, the R6K bacterial origin of replication comprises a nucleic acid sequence having at least 95% sequence identity to a sequence selected from the group of: SEQ ID NO: 9-14.ALD - P2024-0647-US02 (PCT)

[0144] A bacterial cell may be an engineered bacterial cell. As used herein, “engineered bacterial cell” means a cell or cell line of bacterial origin that has been modified (i.e., genetically engineered) to induce a genetic change, including, but not limited to (i) deletion (e.g., knock-out) of a naturally occurring gene or portion of gene, (ii) insertion of genetic material into the genome that is not naturally present in the cell or cell line, or (iii) otherwise exposing the cell to an agent (including, but not limited to genetic material, peptides, proteins or chemical entities) that reduces or promotes the expression of a naturally-occurring gene. For example, a bacterial is an engineered bacterial cell if genetic modifications have been performed in addition to those required for having a functional protelomerase polypeptide, a vector, and a nucleic acid encoding a Rep protein operably linked to a PL promoter. The additional genetic modification may increase vector production, transgene expression, bacterial cell growth rate, or genomic or vector stability.

[0145] As used herein, “gene knockout” means (i) deletion of all or a portion of a target gene sufficient to result in silencing of expression of such target gene or a reduction of expression to a level that is insufficient to maintain the normal function of the gene product, (ii) deletion of all or a portion of a genetic element controlling expression of a target gene that results in the target gene either no longer being expressed or being expressed at a level that is insufficient to maintain its normal function, or (iii) reduction or silencing of gene expression by exposing the target gene to an agent that either acts directly on the gene or on an upstream signaling pathway. Non-limiting examples of methods to reduce or silence gene expression or to permanently delete genes or portions thereof include small-interfering RNA, microRNA, transcription activator-like effector nucleases (TALEN), the CRISPR / Cas9 system, transposons / transposases, and RNA-induced epigenetic silencing.ALD - P2024-0647-US02 (PCT)

[0146] A bacterial cell described herein (e.g., an engineered bacterial cell) may comprise one or more genes selected from the group of sbcC, sbcD, endA, recA, and pgi. In other embodiments, an engineered bacterial cell may lack one or more genes selected from the group of sbcC, sbcD, endA, rec A, and pgi. For example, a bacterial cell may comprise one or more gene knockouts (e.g., one, two, three, four, or five gene knockouts) of at least one gene selected from the group of sbcC, sbcD, endA, recA, and pgi. In some embodiments, a bacterial cell comprises a gene knockout of at least one gene from the group of sbcC and sbcD, and a second gene knockout of at least one gene selected from the group of endA, recA, and pgi. In some embodiments, a bacterial cell comprises gene knockouts of sbcC, sbcD, endA, and recA or gene knockouts sbcC, sbcD, endA, recA, and pgi. In some embodiments, a bacterial cell comprises gene knockouts of sbcC, sbcD, endA, and recA. In some embodiments, a bacterial cell comprises gene knockouts of sbcC, sbcD, endA, recA, and pgi.

[0147] As used herein, “sbcC” and “sbcD” are genes present in E. coli that each encode a polypeptide that is a subunit of the SbcCD nuclease which is involved in palindrome inviability and genetic recombination. By way of example, but not limitation, the sbcC gene can include a nucleic acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 51. For reference, a wild-type amino acid sequence of SbcC from NCBI (Reference Sequence: WP_206061808.1) for E. coli MG1655 is given by SEQ ID NO: 52. By way of example, but not limitation, the sbcD gene can include a nucleic acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 53, while a wild-type amino acid sequence of SbcD from GenBank (AAB 18122.1) for E. coli MG 1655 is given by SEQ ID NO: 54. It should be understood that these amino acid sequences areALD - P2024-0647-US02 (PCT) exemplary and that one of skill in the art can identify sbcC and sbcD genes and polypeptides, including complexes, in other bacterial strains and cell lines based on sequence homology.

[0148] As used herein, “endA” is a gene present in E. coli that encodes a DNA-specific endonuclease. The endA gene encodes an EndA polypeptide having a sequence having 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 56. For example, the endA gene can have a nucleic acid sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 55.

[0149] As used herein, “recA” is a gene present in E. coli that encodes a DNA strand exchange and recombination protein. The recA gene encodes a RecA polypeptide having the amino acid sequence of SEQ ID NO: 58. For example, the recA gene can have a nucleic acid sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 57.

[0150] As used herein, “pgi” is a gene present in E. coli that encodes a phosphoglucose isomerase, such as a glucose-6-phosphate isomerase. The pgi gene encodes a Pgi polypeptide having the sequence of SEQ ID NO: 60. For example, the pgi gene can have a nucleic acid sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 59.

[0151] A bacterial cell described herein (e.g., an engineered bacterial cell) can also comprise one or more genes (e.g., one, two, three, or four genes) selected from sbcB, recB, recD, and red. In some embodiments, a bacterial comprises a sbcB gene, a recB gene, a recD gene, and a red gene. In some embodiments, a bacterial comprises any combination of genes selected from sbcB gene, a recB gene, a recD gene, and a red gene.

[0152] As used herein, “sbcB” is a gene present in E. coli that encodes a SbcB polypeptide having 3’-5’ exonuclease activity. A functional SbcB polypeptide has 3’-5’ exonuclease activity and can be assayed using any 3’ to 5’ exonuclease activity assay. By way of example, but not limitation,ALD - P2024-0647-US02 (PCT) the sbcB gene can include a nucleic acid sequence encoding a SbcB polypeptide having the sequence of SEQ ID NO: 62. For example the sbcB gene can include a nucleic acid sequence encoding a SbcB polypeptide having at least 90%, at least 95%, at least 98%, and at least 99% or 100% sequence identity to SEQ ID NO: 62. In some embodiments, the SbcB polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 62. In some embodiments, the SbcB polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 62. In some embodiments, the SbcB polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 62. In some embodiments, the SbcB polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 62. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 20 amino acid mutations (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation(s)). In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 20 amino acid substitutions (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions(s)). In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 15 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 10 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 9 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 8 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 7 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequenceALD - P2024-0647-US02 (PCT) differing from SEQ ID NO: 62 by no more than 6 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 5 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 4 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 3 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 2 amino acid substitutions. In some embodiments, the SbcB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 62 by no more than 1 amino acid substitution. In some embodiments, the SbcB polypeptide comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the sbcB gene encodes a functional SbcB polypeptide. An exemplary nucleic acid sequence of the sbcB gene is put forth in SEQ ID NO: 61.

[0153] As used herein, “recB” is a gene present in E. coli that encodes a subunit of the RecBCD enzyme complex (Exonuclease V) which functions as a helicase-nuclease. A functional RecB polypeptide has helicase and nuclease activities, which can be measured using any helicase assay and any nuclease assay. By way of example, but not limitation, the recB gene can include a nucleic acid sequence encoding a RecB polypeptide having the sequence of SEQ ID NO: 64. For example, the recB gene can include a nucleic acid sequence encoding a RecB polypeptide having at least 90%, at least 95%, at least 98%, and at least 99% or 100% sequence identity to SEQ ID NO: 64. In some embodiments, the RecB polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 64. In some embodiments, the RecB polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 64. In some embodiments, the RecB polypeptide comprises an amino acid sequence having at least 98%ALD - P2024-0647-US02 (PCT) sequence identity to SEQ ID NO: 64. In some embodiments, the RecB polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 64. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 20 amino acid mutations (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation(s)). In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 20 amino acid substitutions (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions(s)). In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 15 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 10 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 9 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 8 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 7 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 6 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 5 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 4 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 3 amino acid substitutions. In some embodiments, the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 2 amino acid substitutions. In some embodiments,ALD - P2024-0647-US02 (PCT) the RecB polypeptide comprises an amino acid sequence differing from SEQ ID NO: 64 by no more than 1 amino acid substitution. In some embodiments, the RecB polypeptide comprises the sequence of SEQ ID NO: 64. In some embodiments, a recB gene encodes a functional RecB polypeptide. An exemplary nucleic acid sequence of the recB gene is put forth in SEQ ID NO: 63.

[0154] As used herein, “recD” is a gene present in E. coli that encodes a subunit of the RecBCD enzyme complex (Exonuclease V) which functions as a helicase-nuclease. A functional RecB polypeptide has helicase and nuclease activities, which can be measured using any helicase assay and any nuclease assay. By way of example, but not limitation, the recD gene can include a nucleic acid sequence encoding a RecD polypeptide having the sequence of SEQ ID NO: 66. For example the recD gene can include a nucleic acid sequence encoding a RecD polypeptide having at least 90%, at least 95%, at least 98%, and at least 99% or 100% sequence identity to SEQ ID NO: 66. In some embodiments, the RecD polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 66. In some embodiments, the RecD polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 66. In some embodiments, the RecD polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 66. In some embodiments, the RecD polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 66. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 20 amino acid mutations (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation(s)). In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 20 amino acid substitutions (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions(s)). In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 15 amino acidALD - P2024-0647-US02 (PCT) substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 10 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 9 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 8 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 7 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 6 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 5 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 4 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 3 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 2 amino acid substitutions. In some embodiments, the RecD polypeptide comprises an amino acid sequence differing from SEQ ID NO: 66 by no more than 1 amino acid substitution. In some embodiments, the RecD polypeptide comprises the sequence of SEQ ID NO: 66. In some embodiments, the recD gene encodes a functional RecD polypeptide. An exemplary nucleic acid sequence of the recD gene is put forth in SEQ ID NO: 65.

[0155] As used herein, “red” is a gene present in E. coli that encodes a single- stranded DNA (ssDNA) specific exonuclease. A functional Red polypeptide has exonuclease activity that is specific for ssDNA, which can be measured using any ssDNA exonuclease assay. By way of example, but not limitation, the red gene can include a nucleic acid sequence encoding a RedALD - P2024-0647-US02 (PCT) polypeptide having the sequence of SEQ ID NO: 68. For example, the red gene can include a nucleic acid sequence encoding a Red polypeptide having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 68. In some embodiments, the Red polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 68. In some embodiments, the Red polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 68. In some embodiments, the Red polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 68. In some embodiments, the Red polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 68. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 20 amino acid mutations (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation(s)). In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 20 amino acid substitutions (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions(s)). In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 15 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 10 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 9 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 8 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 7 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 6 amino acid substitutions. In some embodiments,ALD - P2024-0647-US02 (PCT) the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 5 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 4 amino acid substitutions. In some embodiments, the RecJ polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 3 amino acid substitutions. In some embodiments, the RecJ polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 2 amino acid substitutions. In some embodiments, the Red polypeptide comprises an amino acid sequence differing from SEQ ID NO: 68 by no more than 1 amino acid substitution. In some embodiments, the Red polypeptide comprises the sequence of SEQ ID NO: 68. In some embodiments, a red gene encodes a functional Red polypeptide. An exemplary nucleic acid sequence of the red gene is put forth in SEQ ID NO: 67.

[0156] In some embodiments, a bacterial cell comprises a sbcB gene encoding a SbcB polypeptide having at least 90% sequence identity to SEQ ID NO: 62, a recB gene encoding a RecB polypeptide having at least 90% sequence identity to SEQ ID NO: 64, a recD gene encoding a RecD polypeptide having at least 90% sequence identity to SEQ ID NO: 66, a red gene encoding a Red polypeptide having at least 90% sequence identity to SEQ ID NO: 68. In some embodiments, a bacterial cell comprises a sbcB gene encoding a functional SbcB polypeptide, a recB gene encoding a functional RecB polypeptide, a recD gene encoding a functional RecD polypeptide, a red gene encoding a functional Red polypeptide.

[0157] A bacterial cell described herein (e.g., an engineered bacterial cell) can also comprise one or more regions (e.g., one, two, or three regions), such as an operon, selected from a region comprising a uvrC gene, a region comprising a mcrA gene, and region comprising a mcrBC gene, a hsd gene, and a mrr gene. In some embodiments, a bacterial cell describe herein (e.g., anALD - P2024-0647-US02 (PCT) engineered bacterial cell) comprises a region comprising a uvrC gene, a region comprising a mcrA gene, and region comprising a mcrBC gene, a hsd gene, and a mrr gene

[0158] As used herein, “uvrC” is a gene present in E. coli that encodes an endonuclease and is a subunit of the UvrABC system. By way of example, but not limitation, an uvrC gene can include a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 69, or a codon degenerate variant thereof. In some embodiments, an uvrC gene encodes a functional UvrC polypeptide.

[0159] As used herein, “mcrA” is a gene present in E. coli that encodes a methylcytosine- specific nuclease. By way of example, but not limitation, the mcrA gene can include a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 70, or a codon degenerate variant thereof. In some embodiments, a mcrA gene encodes a functional McrA polypeptide.

[0160] As used herein, “a region comprising a mcrBC gene, an hsd gene, and an mrr gene” present in E. coli encodes an endonuclease complex. This region can be referred to as a mcrBC-hsd-mrr region if the mcrBC gene, the hsd gene, and the mrr gene are next to each other in the genome of the bacterial cell. By way of example, but not limitation, the region comprising a mcrBC gene, an hsd gene, and an mrr gene can include one or more sequences having at least 90%, at least 95%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 71-76, or a codon degenerate variants thereof. In some embodiments, a mcrBC gene encodes a functional McrBC polypeptide. In some embodiments, an hsd gene encodes a functional Hsd polypeptide. In some embodiments, a mrr gene encodes a functional Mrr polypeptide.

[0161] A bacterial cell described herein (e.g., an engineered bacterial cell) can also comprise one or more genes selected from the group of fhuA and glnV. In some embodiments, a bacterial cellALD - P2024-0647-US02 (PCT)(e.g., an engineered bacterial cell) comprises a fhuA gene and a glnV gene (e.g., a glnV gene comprising a glnV44 mutation).

[0162] As used herein, “fhuA” stands for ferrichrome outer membrane transporter / phage receptor and is a gene present in E. coli that encodes an outer membrane protein involved in transport and also acts as a receptor for phage. Nucleic acid and amino acid sequences for the FhuA polypeptide are known, and include, but are not limited to, the polypeptide encoded by NCBI Accession No. NP_414692.1 or UniProt Accession Nos. A0A376FS88, A0A6N8R2W7, and A0A8S7BD95, and nucleic acids encoding the same (e.g., nucleic acids associated with NCBI Gene ID: 944856). An exemplary FhuA polypeptide has the amino acid sequence of SEQ ID NO: 77. In some embodiments, a bacterial cell comprises a fhuA gene encoding an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 77. In some embodiments, a bacterial comprises a gene encoding a functional FhuA polypeptide.

[0163] As used herein, “glnV” is a gene present in E. coli that encodes a glutamine tRNA molecule, and can also be referred to as the “supE” gene. An exemplary tRNAGlncuo sequence is SEQ ID NO: 78. In some cases, glnV has a glnV44 (also called supE44) mutation which results in an amber suppressor allele of the glnV gene. GlnV44 encodes a glutamine tRNAGlncuA in which a glutamine is incorporated at a UAG codon. In some cases, glnV lacks a glnV44 (also called supE44) mutation. In some embodiments, a bacterial cell comprises a fhuA gene encoding a tRNA sequence having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 78. In some embodiments, a bacterial cell comprises tRNA sequence of SEQ ID NO: 78, or a codon degenerate variant thereof. In some embodiments, a bacterial cell comprises a gene encoding a functional FhuA tRNA.ALD - P2024-0647-US02 (PCT)

[0164] A bacterial cell described herein (e.g., an engineered bacterial cell) can also comprise a dem gene. As used herein, “dem” is a gene present in E. coli that encodes a DNA cytosine methyltransferase. Nucleic acid and amino acid sequences for a Dem polypeptide are known, and include, but are not limited to, the polypeptide encoded by NCBI Accession No. NP_416470.1 or UniProt Accession No. A0A376M657, and nucleic acids encoding the same. An exemplary dem polypeptide has the amino acid sequence of SEQ ID NO: 79. For example, a bacterial cell comprises a dem gene encoding a Dem polypeptide having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO: 79. In some embodiments, a dem gene encodes a functional Dem polypeptide. In other embodiments, a bacterial cell described herein (e.g., an engineered bacterial cell) may lack a dem gene or a homolog thereof (e.g., a bacterial cell may comprise a knockout of a dem gene or a homolog thereof). For example, a bacterial cell described herein (e.g., an engineered bacterial cell) may lack a functional dem polypeptide.

[0165] A bacterial cell described herein (e.g., an engineered bacterial cell) can also comprise a sbcC gene (e.g., SEQ ID NO: 51), a sbcD gene (e.g., SEQ ID NO: 53), or both. In other embodiments, a bacterial cell (e.g., an engineered bacterial cell) lacks a sbcC gene, a sbcD gene, or both. For example, in some embodiments the bacterial cell comprises a gene knock out of a sbcC gene, a sbcD gene, or both.

[0166] A bacterial cell described herein (e.g., an engineered bacterial cell) may also include a nucleic acid encoding an RNA-IN sequence (i.e., an RNA complementary and antisense to a portion of RNA RNA-OUT such as SEQ ID NO: 25). An RNA-IN sequence may be operably linked to an RNA-IN regulated selectable marker (e.g., a sacB gene).ALD - P2024-0647-US02 (PCT)

[0167] A bacterial cell described herein (e.g., an engineered bacterial cell) may also include a lambda repressor (referred to as cl). For example, a bacterial cell can further include a genomic nucleic acid sequence encoding a temperature-sensitive lambda repressor. By way of example, but not limitation, the temperature- sensitive lambda repressor can be cITs857. As used herein “cITs857” refers to the lambda repressor further incorporating a C to T (Ala to Thr) mutation that confers temperature sensitivity. cITs857 is a functional repressor at 28-30 °C but is mostly inactive at 37-42 °C. Also called cI857 or cI857ts.

[0168] By way of example, but not limitation, the engineered bacterial host cell can include a genomic nucleic acid sequence (which encodes the temperature-sensitive lambda repressor) that has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 81. In some embodiments, the temperature-sensitive lambda repressor comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 37.

[0169] In any of the foregoing embodiments, where the bacterial cell further includes a genomic nucleic acid sequence encoding a temperature- sensitive lambda repressor, the temperaturesensitive lambda repressor can be chromosomally integrated or expressed from a plasmid. For example, the temperature- sensitive lambda repressor can be chromosomally integrated at a phage attachment site (e.g., phage <p80 attachment site, or any other appropriate phage attachment site).

[0170] In any of the foregoing embodiments, where the bacterial cell further includes a genomic nucleic acid sequence encoding a temperature- sensitive lambda repressor, the temperaturesensitive lambda repressor can be under the control (i.e., operably linked) to a promoter. For example, a temperature- sensitive lambda repressor may be operably linked to an inducible promoter. By way of example, but not limitation, the cITs857 gene can be under the control ofALD - P2024-0647-US02 (PCT) the pBAD promoter to provide arabinose inducibility. An exemplary pBAD promoter comprises SEQ ID NO: 82.

[0171] A bacterial cell described herein (e.g., an engineered bacterial cell) may be derived from an Escherichia coli (E. coli) cell line. As used herein, “derived from” when referring to a particular bacterial cell means that the bacterial cell has descended from, or was propagated / grown from, a particular bacterial cell line or a bacterial species. Alternatively, “derived from” can refer to the base bacterial cell line, species or strain used to create the engineered bacterial cell. For example, in some embodiments a bacterial cell (e.g., an engineered bacterial cell) described herein is derived from an E. coli cell line selected from the group of DH5a, DH1, JM107, JM108, JM109, XLlBlue, and MG1655. In some embodiments, a bacterial cell (e.g., an engineered bacterial cell) described herein is derived from a MG1655 E. coli cell line.

[0172] In another embodiment, a bacterial cell (e.g., an engineered bacterial cell) comprises a vector and is otherwise isogenic to the bacterial strain from which it is derived, the bacterial strain from which it is derived being selected from the group of DH5a, DH1, IM107, IM108, JM109, XLlBlue, and MG1655. In some embodiments, a bacterial cell (e.g., an engineered bacterial cell) described herein comprises a vector and is otherwise isogenic to the bacterial strain from which it is derived, the bacterial strain from which it is derived being MG 1655.

[0173] As used herein, “DH5a” refers to an E. coli strain / cell line with the following genotype: F- cp801acZAM15 A(lacZYA-argF) U169 recAl endAl hsdR17 (rk-, mk+) gal- phoA supE44 X- thi-1 gyrA96 relAl .

[0174] As used herein, “DH1” refers to an E. coli strain / cell line with the following genotype: F " endAl rec Al relAl gyrA96 thi-1 glnV44 hsdR I7(ri< IUK )•ALD - P2024-0647-US02 (PCT)

[0175] As used herein, “JM107” refers to an E. coli strain / cell line with the following genotype: endAl glnV44 thi-1 relAl gyrA96 A(lac-proAB) [F1traD36 proAB+laclqlacZAM15] hsdR17(RK‘ mK+) '.

[0176] As used herein, “JM108” refers to an E. coli strain / cell line with the following genotype: endAl recAl gyrA96 thi-1 relAl glnV44 A(lac-proAB) hsdR17 (re mC).

[0177] As used herein, “JM109” refers to an E. coli strain / cell line with the following genotype: endAl glnV44 thi-1 relAl gyrA96 recAl mcrB+A(lac-proAB) el4- [F1traD36 proAB+laclqlacZAM15] hsdR17(rK'mK+).

[0178] As used herein, “XLlBlue” refers to an E. coli strain / cell line with the following genotype: endAl gyrA96(nalR) thi-1 recAl relAl lac glnV44 F'[ ::TnlO proAB+ laclq A(lacZ)M15] hsdR17(rK- mK+).

[0179] As used herein, “MG1655” refers to an E. coli strain / cell line with the following genotype:K-12 F’ ilvG rfb-50 rph-1. Exemplary genomic sequences of E. coli MG1655 include, but are not limited to, genomic sequences having the GenBank Accession Nos: GCA_000005845.2, GCA.000269645.2, GCA.000273425.1, GCA.000482265.1, GCA_000517165.1,GCA_000801205.1 GCA_001308065.1, GCA_001544635.1, GCA_001566335.1,GCA_002843685.1, GCA_002966145.1, GCA_003627195.1, GCA_009767645.1,GCA_011750885.1, GCA.011750905.1, GCA_011750915.1, GCA.011750925.1, GCA_011750965.1, GCA_011750975.1, GCA_011750995.1, GCA_011751015.1,GCA_013694185.1 , GCA.015291845.1 , GCA.018458765.1 , GCA.020328175.1 ,GCA.025643415.1, GCA.025643435.1, GCA.025643455.1, GCA.025643475.1, andGCA_028618655.1, or the genome of the ATCC® 700926 (also called the Migula strain, or theCastellani and Chalmers strain).ALD - P2024-0647-US02 (PCT)

[0180] In another embodiment, a bacterial cell (e.g., an engineered bacterial cell) described herein is derived from an E. coli cell line selected from the group of GalG20, MG1655, and MG1655 AendA ArecA. In some embodiments, the bacterial cell also comprises a gene knockout of at least one gene selected from the group of sbcC and sbcD.

[0181] As used herein “GALG20” refers to a MG1655 derived E. coli strain of the genotype K-12 F " ilvG r / b-50 rph-l AendA ArecA Apgi.

[0182] As used herein, “MG1655 AendA ArecA” refers to an E. coli strain / cell line with the following genotype: K-12 F ilvG rfb-50 rph-l AendA ArecA.

[0183] The lineage of a bacterial cell can also be determined using any appropriate technique, including, but not limited to, whole 16S rRNA sequencing (See, for example, Church et al. Clin Microbiol Rev 33: 10.1128 / cmr.00053-19), multilocus sequence typing, whole genome sequencing (See, for example, Schiirch et al. Clinical Microbiology and Infection, Vol. 24, Issue 4, 2018, pp. 350-354.), single nucleotide polymorphism analysis, and comparative genomics. For example, a bacterial cell described herein (e.g., an engineered bacterial cell) may comprise a 16s rRNA gene having at least 97% sequence identity (e.g., 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity) to a reference E. coli 16s rRNA gene (also referred to as the rrsB gene). In some embodiments, a bacterial cell described herein (e.g., an engineered bacterial cell) may comprise a 16s rRNA gene having at least 97% sequence identity (e.g., 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity) to a reference E. coli MG1655 16s rRNA gene. An exemplary E. coli MG1655 16s rRNA gene is set forth in NCBI Gene ID: 948466 (A Systematic Annotation Package for community analysis of genomes (ASAP) ID: ABE- 0012991v3) (SEQ ID NO: 80).ALD - P2024-0647-US02 (PCT)

[0184] A bacterial cell described herein (e.g., an engineered bacterial cell) may exhibit one or more traits (e.g., one trait, two traits, three traits, four traits, five traits or more) desirable for bacterial cell growth and vector production. For example, a bacterial cell described herein may exhibit one or more of the following: i) the bacterial cell may provide improved yield of a vector as compared to a reference bacterial cell, ii) the vector has increased stability and integrity in the bacterial cell as compared to in a reference bacterial cell, iii) the bacterial cell has an equal or greater growth rate as compared to a reference bacterial cell, when the bacterial cell and the reference bacterial cell are gown in the same conditions, iv) the bacterial cell has an equal or greater vector yield as compared to a vector in the reference bacterial cell, when the bacterial cell and the reference bacterial cell are gown in the same conditions, the vector extraction procedure is the same, and the same vector yield assay is used, and / or v) the bacterial cell has an equal or greater cell yield as compared to a reference bacterial cell, when the bacterial cell and the reference bacterial cell are gown in the same conditions. As used herein a “reference bacterial cell” may include any appropriate reference bacterial cell. For example, reference bacterial cell may be the bacterial cell without the vector or the bacterial cell without engineered mutations but with the vector.

[0185] Methods of Producing Vectors - Vectors can be produced in the bacterial cells using fermentation protocols suitable for the desired vector transfected in the appropriate engineered bacterial host cells of the present disclosure. In one embodiment, the vector can be produced by a fed-batch fermentation such as HyperGRO™ fermentation.

[0186] As used herein “HyperGRO™ fermentation” refers to fed-batch fermentation, in which plasmid-containing E. coli cells are grown at a reduced temperature during part of the fed-batch phase, during which growth rate is restricted, followed by a temperature up-shift and continuedALD - P2024-0647-US02 (PCT) growth at elevated temperature in order to accumulate plasmid; the temperature shift at restricted growth rate improved plasmid yield and purity. HyperGRO™ fermentation is described in US Patent No. 7,943,377, which is incorporated herein by reference in its entirety.

[0187] Generally, this process comprises growing the bacterial cells at a reduced temperature during a first portion of the fed-batch phase, which can be under growth-restrictive conditions, followed by a temperature up- shift to a higher temperature during a second portion of the fed- batch phase. By way of example, the reduced temperature can be about 28-30°C and the higher temperature can be about 37-42°C. By way of example, the first portion can be from about 6 hours to about 18 hours (e.g., about 12 hours) and the second portion can be from about 2 hours to about 16 hours (e.g., about 8 hours). It should be understood that where the fed-batch fermentation with a temperature upshift is used, the bacterial cell may have a lambda repressor and Rep protein that is under the control of a PL promoter that can be regulated by the lambda repressor, which can be temperature-sensitive.

[0188] For example, a method of producing a ce-linear vector may comprise inserting any of the vectors described herein into a bacterial cell. For example, the method comprises inserting a vector into a bacterial cell, where the bacterial cell can express a functional protelomerase, and where the vector comprises; i) a first region of a nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends; and ii) a spacer region that is less than 1000 bp that links the 5’ and 3’ ends of the first region of nucleic acid sequence and comprises a bacterial origin of replication and / or a RNA selectable marker; and exposing the bacterial cell to a condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the vector, thereby converting the vector to a double- stranded ce-linear nucleic acid vector.ALD - P2024-0647-US02 (PCT)

[0189] The step of inserting may comprise any appropriate method of inserting a vector into a bacterial cell. For example, the step of inserting a vector into a bacterial cell may include transforming or transducing the bacterial cell with the vector. Transfection of DNA into E. coli, commonly called transformation, is typically performed using chemical competent or electrocompetent bacterial cells (e.g., E. coli cells) using standard methodologies known in the art.

[0190] The step of exposing the bacterial cell to a condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the vector may include, for example, culturing the bacterial cell in a culture medium that allowed for expression and activity of the functional protelomerase.

[0191] Any of the methods for producing a ce-linear vector described herein may also include exposing the bacterial cell to a condition sufficient to replication the vector and optionally, replicating the vector. For example, the vector may be replicated as a circular vector, as a ce-linear vector, or both. For example, if the vector is replicating as a ce-linear vector, any of the bacterial cells described herein that comprise a vector may be exposed to a condition sufficient to activate the origin of replication (e.g., a R6K origin of replication). Such conditions may depend on whether a functional protelomerase is operably linked to a constitutive promoter or an inducible promoter.

[0192] For example, if the vector is replicated as a circular' vector, replicating the vector may occur before exposing the bacterial cell to a condition sufficient to associate the function protelomerase with the telomerase occupancy site on the vector. In addition, when the nucleic acid encoding the functional protelomerase is operably linked to a constitutive promoter, the vector may also be replicated as a ce-linear vector at substantially the same time as the vector is replicated as a circular vector. In some embodiments, the nucleic acid encoding the functional protelomerase is operablyALD - P2024-0647-US02 (PCT) linked to a constitutive promoter and the vector is replicated as a circular and ce-linear vector simultaneously.

[0193] However, the method may also include exposing the bacterial cell to a condition sufficient to induce expression of a nucleic acid encoding the functional protelomerase, if the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter. In some embodiments, the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter and the method includes exposing the bacterial cell to a condition sufficient to induce expression of a nucleic acid encoding the functional protelomerase to replicate the vector as a ce-linear vector. However, when the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter, the vector may also be replicated as a circular vector and a ce-linear vector simultaneously.

[0194] In any of the methods for producing a ce-linear vector described herein, the step of exposing the bacterial cell to a condition sufficient to replicate the vector may be performed by a fed-batch fermentation, wherein the fed-batch fermentation comprises culturing the bacterial cell at a first temperature of about 25°C to about 32°C during a first portion of the fed-batch phase, followed by culturing the bacterial cell at a second temperature of about 37°C to about 45°C during a second portion of the fed-batch phase.

[0195] Any of the methods for producing a ce-linear vector described herein may also include lysing the bacterial cell to produce a lysate. The lysate may comprise the vector as a circular vector, as a ce-linear vector, or as a combination of a circular vector and a ce-linear vector. Any appropriate method of lysing a bacterial call may be used, for example, using a lysis buffer in a DNA extraction kit, using a series of freeze / thaw steps, passing a culture of the bacterial cellsALD - P2024-0647-US02 (PCT) through a thin needle to use shearing forces, or using an intercalating molecule to destabilize the bacterial cell membranes.

[0196] Any of the methods for producing a ce-linear vector described herein may also include extracting the vector from the lysate to produce an extracted vector solution. The extracted vector solution may comprise the extracted vector and additional components of bacterial cell (e.g., cellular membranes, other proteins, other DNA, other RNA, etc). In some embodiments, the method may also include purifying or isolating the vector from the extracted vector solution. For example, any appropriate method to purify or isolate the vector from the extracted vector solution may be used. Exemplary methods include phenol-chloroform extraction and silica membrane based column purification.

[0197] In any of the foregoing embodiments, the plasmid yield after incubating the transfected cell under conditions sufficient to replicate the vector can be higher than for the bacterial cell line from which the engineered bacterial cell was derived when treated under the same conditions. In any of the foregoing embodiments, the plasmid yield after incubating the transfected host cell under conditions sufficient to replicate the vector can be higher than for SURE2, SURE, Stbl2, Stbl3, or Stbl4 cells treated under the same conditions.

[0198] It should be understood that in any of the foregoing embodiments, the engineered E. coli host cell can include a knockdown of SbcC, SbcD, or both, or a knockdown of recA, endA and pgi, rather than a knockout. The knockdown can result in reduced expression and / or reduced activity of the SbcCD complex. The reduction can be by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more.ALD - P2024-0647-US02 (PCT)

[0199] The bacterial host strains and methods of the present disclosure will now be described with reference to the following non-limiting examples.

[0200] Method of Using Vectors or Bacterial Cells Containing Vectors -

[0201] Vectors described herein and bacterial cells containing any of the vectors described herein can be used for a variety of genetic engineering and transgene production methods. For example, ce-linear vectors described herein can be used in a variety of techniques for genetic insertions and modifications of a genome (e.g., a human genome or a microbial genome). Non-limiting examples include CRISPR-HDR (Homology-Directed Repair) and non-HDR mediated knock-in of genes.

[0202] CRISPR-HDR (Homology-Directed Repair) is a precise genome editing technique that uses the CRISPR-Cas9 system to introduce double-strand breaks (DSBs) at specific genomic locations. The DSBs are then repaired using a homologous DNA template, allowing for accurate insertion or modification of target DNA sequences.

[0203] Non-HDR mediated knock-in of genes may include homology-independent targeted insertion (HITI), which uses the non-homologous end joining (NHEJ) to insert a template that has been linearized by a ribonucleoprotein (RNP), and homology mediated end joining (HMEJ), which uses micro homology (<50 bp) of the insertion / Cas9 cut site to knock-in (insert) a template that has also been linearized by a RNP.

[0204] These methods are useful for creating specific genetic changes, such as gene knock-ins or specific modifications to a target gene (e.g., removing a mutation compared to a wild-type sequence of the target gene).EXAMPLES

[0205] Example 1. Generating a ce-linear vector using a protelomerase coding sequence gene on the linearized vector.ALD - P2024-0647-US02 (PCT)

[0206] A vector, referred to as TelN-NP-Ara-protelomerase, was produced containing 1) a protelomerase gene coding sequence, for which the gene product recognized a tos site to generate close-ended hairpins, operably linked to 2) an arabinose-inducible promoter (AraBAD promoter), 3) TelN (a tos site) for the protelomerase to recognize, 4) an AraC repressor, and 5) an RNA-OUT- R6K bacterial origin of replication nucleic acid sequence (Fig. 1A). When linearized, the vector contains the same components with close-ended hairpin ends, represented by circles at the end of the linearized vector (Fig. IB).

[0207] An E. coli strain was transformed with TelN-NP-Ara-protelomerase. An analysis of uncut plasmid showed a predominant band running larger than 4 kb indicating linear DNA (Fig. 2A). The predominant band was then exposed to T5 exonuclease digestion and Bglll restriction enzyme digestion. If the vector was a linearized vector, then it should be resistant to T5 exonuclease digestion, but sensitive to Bglll digestion. The predominant band was resistant to T5 exonuclease treatment (middle lane), indicating closed ends, and digested into 2 fragments by a unique restriction enzyme, further validating that the predominant product is linear (Fig. 2B). A simulated digest showing banding profiles of digesting circular or linear DNA with Bglll restriction enzyme (Fig. 2C) were similar to the vector digestion patterns observed in the T5 exonuclease / Bglll digestion, indicating that the vector was a linear vector.

[0208] To confirm that the presence of active protelomerase was generating the linearized vectors, vectors containing active and deactive protelomerase were generated (Fig. 3). Briefly, circular vector (TelN-NP-Ara-protelomerase) was digested with a unique enzyme in the protelomerase coding sequence (Pcil restriction enzyme), the digest sites were filled in, and the ends were ligated together to make a circular vector with a mutated protelomerase coding sequence that was deactivated. In parallel, circular vector (TelN-NP-Ara-protelomerase) was digested with PcilALD - P2024-0647-US02 (PCT) restriction enzyme, and the digest sites were re-ligated to reconstitute the protelomerase, making a circular vector with an active protelomerase coding sequence. Both active and deactivated forms were used to transform E. coli-R K.

[0209] Fig. 4A shows that active protelomerase (Digest -> Ligation) generates linear vector, and deactivated protelomerase (Digest -> Blunt -> Ligation) generates circular vector (Fig. 4A). Linear vector digested differently than circular vector, as shown in Fig 4A and Fig 4B, and were resistant to T5 exonuclease due to the closed ends (Fig 4A, A & B, Digest -> Ligation). In this experiment, miniprep recoveries of the active (linear) vector were about half as much as that of the deactivated (circular) vector, although the sample sizes were small (Fig. 4C).

[0210] Taken together these data show that vectors containing a tos site are predominately converted to linear vectors when in the presence of functional protelomerase on the vector to be linearized.

[0211] Example 2. Generating a ce-linear vector using a protelomerase coding sequence gene on a different vector.

[0212] Three vectors were generated. Vector pUC-Tel containing functional protelomerase operably linked to an arabinose-inducible promoter (pAraBAD), vector 260-170-2 that lacked a tos site and should remain as a linear vector even in the presence of active protelomerase, and vector 260-170-3 that contained a tos site and should be converted to a linear vector in the presence of active pro telomerase. Vector pUC-Tel was transformed into E. co / z-R6K to generate pUC-Tel containing E. co / z-R6K. In E. c<9 / z'-R6K (pUC-Tel) protelomerase was expressed from pUC vector with no added arabinose through the leaky expression because the pUC vector is a multicopy plasmid. E. co / z-R6K (pUC-Tel) was transformed a second time with vector 260-170-2 or vector 260-170-3. Resulting transformants contained two vectors, either 1) pUC-Tel + 260-170-2ALD - P2024-0647-US02 (PCT)(referred to as 260-170-2) or 2) pUC-Tel + 260-170-3 (referred to as 260-170-3). Three clones each of 260-170-2 and 260-170-3 were digested with Eael restriction enzyme and T5 exonuclease (T5 exo), which degraded the pUC-Tel vector, but left the 260-170-2 vector and 260-170-3 vector uncut and therefore predominately circular (Fig. 6 - Eacl / T5 exo digest). Digestion with Avril and Alel restriction enzymes of DNA from the three clones of each of 260-170-2 and 260-170-3 resulted in a unique banding pattern. The pUC-TelN did not contain restriction sites for Avril or Alel restriction enzymes, and was therefore left uncut (Fig. 6, arrow). Circular vectors are shown at 3.1 kb. Linear vectors, likely close-ended linear vectors are show at 2.0 kb, 1.35 kb, and 1.13 kb (Fig. 6, Avril / Alel digest, right side).

[0213] Taken together these data show that vectors containing a tos site are predominately converted to linear vectors when in the presence of functional protelomerase on a different vector.

[0214] Example 3. Generating ce-linear vectors with protelomerase (PTelN) integrated into the genome of E. coli-R6K.

[0215] E. coli strains with protelomerase expressed from a constitutive promoter (beta-lactamase promoter, Pbia), or a heat-inducible promoters (Pciss?) with and without an element to increase nucleotide metabolism (called zwf) were generated following industry-standard protocols. The protelomerase construct was integrated into the P21 phage attachment site using the pAH121 helper plasmid.

[0216] TB + arabinose was tested as a rich medium and “sucrose select” without arabinose with and without a heat shift. Briefly, duplicate cultures for E. coli-R6K containing Pbia-PTelN vector, Pci857-PTeN vector, or Pci857-zwf-PTelN vector were incubated overnight in TB + 0.2% arabinose or “Sucrose select” no arabinose at 30°C. If heat-shifted, cultures were moved to 37°C and incubated for -7 hours before being returned to 30°C for the remaining time.ALD - P2024-0647-US02 (PCT)

[0217] Ce-linear DNA is indicated by an upwards band shift from supercoiled to linear and resistant to treatment by T5 exonuclease (Fig. 7). Also, a diagnostic digest would be expected to generate a new banding pattern for linear vectors as compared to circular vectors (indicated by the presence of -2 kb and <1.3 kb bands for circular) (Fig. 7). When protelomerase was expressed by a heat-inducible promoter the presence of a second band running higher than supercoiled in the T5 exonuclease-treated samples and 2 additional bands in the diagnostic digest in the heat-shifted samples (right lanes) were observed (Fig. 7).

[0218] Taken together these data suggest that ce-linear vector were generated in vivo with a heat shift.

[0219] Example 4. Generating an E. coli bacterial cell carrying a transgene on a ce-linear vector.

[0220] A vector is generated with an RNA-OUT-R6K construct, a tos site, and a transgene (enhanced GFP, or EGFP) (Fig. 8A). This vector is transformed into an E. coli bacterial cell previously engineered to contain a genomically-inserted nucleic acid for a function protelomerase (Fig. 8B). The vector containing the transgene is optionally amplified as a circular vector. Functional protelomerase converts circular vector into ce-linear vector, which expressed the transgene. Optionally, the ce-linear vector is amplified. Ce-linear vectors with an encoded transgene are transfected into HEK293 cells and EGFP expression is determined using flow cytometry. Ce-linear vectors achieve higher EGFP expression compared to circular plasmids.

[0221] Example 5. Generating an E. coli bacterial cell carrying a polyA tail on a ce-linear vector.

[0222] A vector is generated with an RNA-OUT-R6K construct, a tos site, and an encoded polyA tail of A100 to A500 (from about 100 to about 500 adenine in length). Natural polyA tails are 300 - 500 bp length; however, it is difficult to maintain polyA tails longer than 100 - 120 bp due toALD - P2024-0647-US02 (PCT) torsion stress when “regular” supercoiled pUC vectors are used. The polyA tails are spontaneously truncated during the plasmid replication.

[0223] PolyA tails are generated by annealing T200 and A50 oligonucleotides at equimolar and treating the annealed oligonucleotides with T4 polymerase, Klenow fragment, and T4 polynucleotide kinase (PNK) to trim flaps, fill gaps, and create blunt, phosphorylated ends, respectively. The resulting mixture is run on a 3% agarose gel and fragments in the range of 100 to 500 bp are extracted. Extracted bands are ligated with TelN-NP-T7 that was SphI digested, T4 polymerase trimmed, and T4 PNK phosphorylated. Plasmid and ce-linear vectors encoding A100 to A500 repeat are transformed into E. coli and the stability of the polyA tails are determined by sequencing. Ce-linear vectors with encoded polyA tails accommodate longer polyA tails compared to circular plasmids due to less torsional stress from supercoiling.

[0224] The encoded polyA tail is an important feature for the subsequent production of mRNA from pDNA template. The benefit is that no additional tailing is needed when mRNA drug or vaccine product is being made in an in vitro reaction and the product has increased stability within eukaryotic cells. Without being bound by theory, increased stability should increase transgene expression and duration.

[0225] An exemplary assay to determine the benefit of a longer polyA tail includes transfecting eukaryotic cells with mRNA that have varying polyA tail lengths (100 to 500 A’s) and measuring transgene expression (enhanced green fluorescent protein [EGFP]) out to seven days post transfection (arbitrary number of days).

[0226] Example 6. Generating an E. coli bacterial cell carrying AAV ITRs on a ce-linear vector.

[0227] A vector is generated with an RNA-OUT-R6K construct, a tos site, AAV ITRs, a promoter, a transgene, and a polyA signal. The resulting ce-linear AAV vector is triple transfected intoALD - P2024-0647-US02 (PCT)HEK293T cells to produce AAV virus containing the transgene. Ce-linear vectors produced higher titers of AAV compared to circular plasmids.

[0228] Example 7. Generating an E.coli bacterial cell carrying a CRISPR / Cas repair template.

[0229] A vector is generated with an RNA-0UT-R6K construct, a tos site, an encoded transgene, and / or promoter, polyA signal, splice site, homology arms, micro-homology arms, and CRISPR targeting site (CTS). The resulting ce-linear CRISPR / Cas repair template is transfected into primary human T-cells. The percent of cells containing a knock-in of the cassette was determined using a Tracking of Indels by Decomposition (TIDES) assay. For a description of a TIDES assay, see, for example, Brinkman et al. Nucleic Acids Research, Volume 42, Issue 22, 16 December 2014, Page el68. Ce-linear vectors produce higher knock-in rates in primary human T-cells compared to circular plasmids.

Claims

ALD - P2024-0647-US02 (PCT)WHAT IS CLAIMED IS:

1. A vector comprising; i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin.

2. The vector of claim 1, wherein the spacer region further comprises a nucleic acid sequence encoding a RNA selectable marker.

3. A vector comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a nucleic acid sequence encoding a RNA selectable marker.

4. The vector of claim 3, wherein the spacer region further comprises a bacterial replication origin.

5. A vector comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin and a nucleic acid sequence encoding a RNA selectable marker.

6. The vector of any one of claims 1-5, wherein the spacer region is less than 500 bp.

7. The vector of any one of claims 1-6, wherein the vector does not comprise a nucleic acid sequence encoding a protelomerase polypeptide.ALD - P2024-0647-US02 (PCT)8. The vector of any one of claims 1-7, wherein the vector docs not comprise a nucleic acid sequence encoding an endonuclease polypeptide.

9. The vector of any one of claims 1-8, wherein the vector comprises only one telomerase occupancy site.

10. The vector of any one of claims 1-9, wherein the telomerase occupancy site comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5.

11. The vector of any one of claims 1-10, wherein the first region of nucleic acid sequence consists of a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends.

12. The vector of any one of claims 1-2 or 4-11, wherein the bacterial replication origin is a R6K bacterial replication origin of replication.

13. The vector of claim 12, wherein the R6K bacterial replication origin comprises a nucleic acid sequence having at least 95% sequence identity to a sequence selected from the group of SEQ ID NOs: 9-14.

14. The vector of any one of claims 2-13, wherein the RNA selectable marker is selected from the group of an RNA-OUT selectable marker that encodes and RNA-IN regulating RNA-OUT RNA with at least 95% sequence identity to SEQ ID NO: 25; an RNAI selectable marker that encodes an RNAII regulating RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 26; and a synthetic RNA selectable marker encoding an RNA selectable marker complement regulating RNA with at least 95% sequence identity to SEQ ID NO: 27.

15. The vector of any one of claims 2-13, wherein the RNA selectable marker is a functional RNA- OUT.ALD - P2024-0647-US02 (PCT)16. The vector of claim 15, wherein the bacterial replication origin is a R6K bacterial origin, and the R6K bacterial origin and the RNA-OUT RNA selectable marker have at least 95% sequence identity to SEQ ID NO: 28.

17. The vector of any one of claims 1-16, wherein the first region of nucleic acid sequence and the spacer region of nucleic acid sequence form a nucleic acid structure that is double- stranded, linear, and comprises closed-ends, and wherein the closed-ends comprise a nucleic acid sequence of telR or telL.

18. A bacterial cell comprising: a) a functional protelomerase polypeptide; b) a vector, the vector comprising: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence and comprises a bacterial replication origin.

19. The bacterial cell of claim 18, wherein the spacer region further comprises a nucleic acid sequence encoding a RNA selectable marker.

20. A bacterial cell comprising: a) a functional protelomerase polypeptide; and b) a vector, the vector comprising i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a nucleic acid sequence encoding a RNA selectable marker.

21. The bacterial cell of claim 20, wherein the spacer region further comprises a bacterial replication origin.

22. A bacterial cell comprising: a) a functional protelomerase polypeptide; andALD - P2024-0647-US02 (PCT) b) a vector, the vector comprising i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin and a nucleic acid sequence encoding a RNA selectable marker.

23. The bacterial cell of any one of claims 18-22, wherein the spacer region is less than 500 bp.

24. The bacterial cell of any one of claims 18-23, wherein the protelomerase polypeptide comprises a sequence having at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3.

25. The bacterial cell of any one of claims 18-24, wherein the vector does not comprise a nucleic acid encoding a protelomerase polypeptide.

26. The bacterial cell of any one of claims 18-25, wherein the vector does not comprise a nucleic acid encoding an endonuclease polypeptide.

27. The bacteria cell of any one of claims 18-26, wherein the vector comprises only one telomerase occupancy site.

28. The bacteria cell of any one of claims 18-27, wherein the telomerase occupancy site comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 5.

29. The bacteria cell of any one of claims 18-28, wherein the first region of nucleic acid sequence consists of a gene of interest, a telomerase occupancy site, and 5’ and 3’ ends.

30. The bacterial cell of any one of claims 18-19, or 21-29, wherein the bacterial replication origin is a R6K bacterial replication origin, and further comprising a nucleic acid encoding a Rep protein operably linked to a PL promoter, wherein the PL promoter comprises a lambda repressor binding site (OL1).ALD - P2024-0647-US02 (PCT)31. The bacterial cell of claim 30, wherein the R6K bacterial replication origin comprises a nucleic acid sequence having at least 95% sequence identity to a sequence selected from the group of SEQ ID Nos: 9-14.

32. The bacterial cell of any one of claims 19-31, wherein the RNA selectable marker is selected from the group of an RNA-OUT selectable marker that encodes and RNA-IN regulating RNA- OUT RNA with at least 95% sequence identity to SEQ ID NO: 25; an RNAI selectable marker that encodes an RNAII regulating RNAI selectable marker encoding an RNAII regulating RNAI RNA with at least 95% sequence identity to SEQ ID NO: 26; and a synthetic RNA selectable marker encoding an RNA selectable marker complement regulating RNA with at least 95% sequence identity to SEQ ID NO: 27.

33. The bacterial cell of any one of claims 19-32, wherein the RNA selectable marker is a functional RNA-OUT.

34. The bacterial cell of claim 33, wherein the R6K bacterial replication origin and the RNA-OUT RNA selectable marker have at least 95% sequence identity to SEQ ID NO: 28.

35. The bacterial cell of any one of claims 18-34, wherein the first region of nucleic acid sequence and the spacer region of nucleic acid sequence form a nucleic acid structure that is double- stranded, linear, and comprises closed-ends, and wherein the closed-ends comprise the telomerase occupancy site.

36. The bacterial cell of any one of claims 18-35, wherein the Rep protein comprises an amino acid sequence selected from SEQ ID NOs: 38-45.

37. The bacterial cell of any one of claims 18-36, wherein the Rep protein comprises a mutation selected from P42L-P113S and P42L-P106L-F107S.

38. The bacterial cell of any one of claims 18-37, wherein the PL promoter comprises a nucleic acid sequence selected from SEQ ID NOs: 48-51.ALD - P2024-0647-US02 (PCT)39. The bacterial cell of any one of claims 30-38, wherein the OL1 comprises a nucleic acid sequence having a mutation compared to SEQ ID NO: 48, wherein the mutation is selected from a single base substitution or a single base deletion.

40. The bacterial cell of claim 39, wherein the mutation decreases or prevents a lambda repressor from binding to the OL1 as compared to the same lambda repressor binding a non-mutated OLE41. The bacterial cell of any one of claims 18-40, wherein the vector comprises a vector backbone with at least 95% sequence identity to a sequence selected from SEQ ID NOs: 29-35.

42. The bacterial cell of any one of claims 18-41, wherein a nucleic acid encoding the functional protelomerase polypeptide is operably linked to a constitutive promoter.

43. The bacterial cell of any one of claims 18-41, wherein a nucleic acid encoding the functional protelomerase polypeptide is operably linked to an inducible promoter.

44. The bacterial cell of any one of claims 18-44, wherein a nucleic acid encoding the functional protelomerase polypeptide is integrated into the bacterial cell’s genome.

45. The bacterial cell of any one of claims 18-44, wherein the bacterial cell is an engineered bacterial cell.

46. The bacterial cell of any one of claims 18-45, wherein the bacterial cell comprises a gene knockout of at least one gene selected from the group of sbcC, sbcD, endA, recA and pgi.

47. The bacterial cell of claim 46, wherein the bacterial cell comprises a gene knockout of at least one gene selected from the group of sbcC and sbcD, and at least one gene selected from the group of endA, recA and pgi.ALD - P2024-0647-US02 (PCT)48. The bacterial cell of claim 46 or claim 47, wherein the bacterial cell comprises a gene knockout selected from sbcC, sbcD, cndA, and rccA or selected from sbcC, sbcD, cndA, rccA, and pgi.

49. The bacterial cell of any one of claims 46-48, further comprising a sbcB gene, a recB gene, a recD gene, and a recJ gene.

50. The bacterial cell of claim 49, wherein the sbcB gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 61, wherein the recB gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 63, wherein the recD gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 65, and wherein the red gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 67.

51. The bacterial cell of claim 50, wherein the sbcB gene encodes a functional SbcB polypeptide, the recB gene encodes a functional RecB polypeptide, the recD gene encodes a functional RecD polypeptide, and the red gene encodes a functional Red polypeptide.

52. The bacterial cell of any one of claims 46-51, further comprising at least one region selected from the group of a uvrC gene, a mcrA gene, and a mcrBC-hsd-mrr region.

53. The bacterial cell of claim 52, wherein the uvrC gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 69, wherein the mcrA gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 70, and wherein the mcrBC-hsd-mrr region comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 71-76.

54. The bacterial cell of claim 52, wherein the uvrC gene encodes a functional UvrC polypeptide and the mcrA gene encodes a functional McrA polypeptide.

55. The bacterial cell of any one of claims 46-54, wherein the bacterial cell further comprising a gene selected from a fhuA gene and a glnV gene.ALD - P2024-0647-US02 (PCT)56. The bacterial cell of claim 55, wherein the fhuA gene encodes a polypeptide having at least 90% sequence identity to SEQ ID NO: 77, and the glnV gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 78.

57. The bacterial cell of claim 55, wherein the fhuA gene encodes a functional FhuA polypeptide and the glnV gene encodes a functional GlnV tRNA.

58. The bacterial cell of any one of claims 46-57, wherein the bacterial cell further comprising an fhuA gene and a glnV gene.

59. The bacterial cell of any one of claims 46-58, wherein the bacterial cell further comprising a gene knockout of a dem gene.

60. The bacterial cell of claim 59, wherein the dem gene comprises a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 79.

61. The bacterial cell of claim 59, wherein the dem gene encodes a functional Dem polypeptide.

62. The bacterial cell of any one of claims 46-61, wherein the bacterial cell does not contain a supE44 gene.

63. The bacterial cell of any one of claims 18-62, wherein the bacterial cell is derived from an Escherichia coli (E. coli) cell line.

64. The bacterial cell of claim 63 wherein the E. coli cell line is selected from the group of DH5a, DH1, JM107, JM108, JM109, XLlBlue, and MG1655.

65. The bacterial cell of claim 63 or claim 64, wherein the E. coli cell line is MG1655.

66. The bacterial cell of any one of claims 18-65, wherein the bacterial cell is derived from an E. coli cell line, wherein the E. coli cell line is selected from the group of GalG20, MG1655, andALD - P2024-0647-US02 (PCT)MG1655 AendA ArecA, and wherein the bacterial cell comprises a gene knockout of at least one gene selected from the group of SbcC and SbeD.

67. The bacterial cell of claim 66, wherein the bacterial cell comprises the gene knockout of a SbcC gene and a SbcD gene.

68. The bacterial cell of any one of claims 18-67, further comprising a genomic nucleic acid sequence encoding a temperature- sensitive lambda repressor.

69. The bacterial cell of claim 68, wherein the temperature- sensitive lambda repressor is cITs857.

70. The bacterial cell of claim 68 or claim 69, wherein the temperature-sensitive lambda repressor comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 37.

71. The bacterial cell of any one of claims 68-70, wherein the temperature- sensitive lambda repressor is a phage q>80 attachment site chromosomally integrated copy of an arabinose inducible CITs857 gene.

72. The bacterial cell of any one of claims 18-71, wherein the bacterial cell provides improved yield of the vector as compared to a reference bacterial cell.

73. The bacterial cell of any one of 18-72, wherein the bacterial cell provides improved yield of the vector as compared to a reference bacterial cell that does not include a gene knockout of at least one of endA, recA and pgi.

74. The bacterial cell of any one of claims 18-73, wherein the vector has increased stability and integrity in the bacterial cell as compared to in a reference bacterial cell.

75. The bacterial cell of any one of claims 18-74, wherein the bacterial cell has an equal or greater growth rate as compared to a reference bacterial cell when the bacterial cell and the reference bacterial cell are grown in the same conditions.ALD - P2024-0647-US02 (PCT)76. The bacterial cell of any one of claims 18-75, wherein the bacterial cell has an equal or greater vector yield as compared to a reference bacterial cell when the bacterial cell and the reference bacterial cell are grown in the same conditions, the vector extraction procedure is the same, and same vector yield assay is used.

77. The bacterial cell of any one of claims 18-76, wherein the bacterial cell is an E. coli bacterial cell and is isogenic to the bacterial strain from which it is derived, and wherein the strain from which it is derived is selected from the group of DH5a, DH1, JM107, JM108, JM109, MG1655 and XLlBlue.

78. The bacterial cell of any one of claims 18-77, wherein the bacterial cell comprises a 16S rRNA gene having at least 97% sequence identity to SEQ ID NO: 80.

79. The bacterial cell of any one of claims 19-78, further comprising a nucleic acid encoding an RNA-IN sequence.

80. A method for producing a double- stranded, linear, close-ended nucleic acid vector, the method comprising: inserting a nonlinear vector into a bacterial cell, wherein the bacterial cell can express a functional protelomerase, wherein the nonlinear vector comprises: i) a first region of nucleic acid sequence comprising a gene of interest, a telomerase occupancy site, and 5' and 3' ends; and ii) a spacer region that is less than 1000 bp links the 5' and 3' ends of the first region of nucleic acid sequence, and comprises a bacterial replication origin and / or a RNA selectable marker; and exposing the bacterial cell to a condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector, thereby converting the nonlinear vector to the double-stranded, linear, close-ended nucleic acid vector.ALD - P2024-0647-US02 (PCT)81. The method of claim 80, wherein the step of inserting comprises transforming or transducing the nonlinear vector.

82. The method of claim 80 or claim 81, further comprising exposing the bacterial cell to a condition sufficient to replicate the nonlinear vector prior to the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector.

83. The method of any of claims 80-82, wherein the functional protelomerase is encoded by a nucleic acid sequence integrated into the bacterial cell genome.

84. The method of any of claims 80-82, wherein the nonlinear vector further comprises a nucleic acid sequence encoding the functional protelomerase.

85. The method of any of claims 80-82, further comprising the step of inserting a second vector into the bacterial cell, wherein the second vector comprises a nucleic acid sequence encoding the functional protelomerase.

86. The method of any of claims 82-85, further comprising exposing the bacterial cell to a condition sufficient to induce expression of the functional protelomerase, and wherein the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter.

87. The method of claim 80, further comprising the step of exposing the bacterial cell to a condition sufficient to replicate the double-stranded, linear, close-ended nucleic acid vector.

88. The method of any of claims 80-87, wherein the nonlinear vector is a plasmid vector.

89. The method of any one of claims 83-85, wherein the nucleic acid encoding the functional protelomerase is operably linked to a constitutive promoter.ALD - P2024-0647-US02 (PCT)90. The method of 82 and 87, wherein the condition sufficient to replicate the vector is performed by a fcd-batch fermentation.

91. The method of claim 90, wherein the fed-batch fermentation comprises culturing the bacterial cell at a first temperature of about 25°C to about 32°C during a first portion of the fed-batch phase, followed by culturing the bacterial cell at a second temperature of about 37°C to about 45°C during a second portion of the fed-batch phase.

92. The method of claim 82, further comprising lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector.

93. The method of claim 92, further comprising extracting the double- stranded, linear, close-ended nucleic acid vector from the lysate to produce an extracted vector solution.

94. The method of claim 93, further comprising purifying the double- stranded, linear, close-ended nucleic acid vector from the extracted vector solution.

95. The method of claim 94, wherein the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

96. The method of claim 87, further comprising lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to a condition sufficient to replicate the doublestranded, linear, close-ended nucleic acid vector.

97. The method of claim 96, further comprising extracting the double- stranded, linear, close-ended nucleic acid vector from the lysate to produce an extracted vector solution.

98. The method of claim 97, further comprising purifying the double- stranded, linear, close-ended nucleic acid vector from the extracted vector solution.ALD - P2024-0647-US02 (PCT)99. The method of claim 98, wherein the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

100. The method of any one of claims 80-99, wherein the double- stranded, linear, close-ended nucleic acid vector is the vector of any one of claims 1-17.

101. The method of any one of claims 80-100, wherein the bacterial cell is the bacterial cell of any one of claims 18-79.

102. A bacterial cell comprising: a) a nucleic acid sequence encoding a functional protelomerase polypeptide; and b) an engineered circular plasmid vector comprising a nucleic acid sequence comprising a gene of interest and a telomerase occupancy site.

103. A method for producing a double- stranded, linear, close-ended nucleic acid vector, the method comprising: inserting a nonlinear vector into a bacterial cell, wherein the bacterial cell can express a functional pro telomerase, and wherein the nonlinear vector comprises a nucleic acid sequence comprising a gene of interest and a telomerase occupancy site; and exposing the bacterial cell to a condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector, thereby converting the nonlinear vector to the double-stranded, linear, close-ended nucleic acid vector.

104. The method of claim 103, wherein the step of inserting comprises transforming or transducing the nonlinear vector.

105. The method of claim 103 or claim 104, further comprising exposing the bacterial cell to a condition sufficient to replicate the nonlinear vector prior to the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector.ALD - P2024-0647-US02 (PCT)106. The method of any of claims 103-105, wherein the functional protclomcrasc is encoded by a nucleic acid sequence integrated into the bacterial cell genome.

107. The method of any of claims 103-105, wherein the nonlinear vector further comprises a nucleic acid sequence encoding the functional protelomerase.

108. The method of any of claims 103-105, further comprising the step of inserting a second vector into the bacterial cell, wherein the second vector comprises a nucleic acid sequence encoding the functional protelomerase.

109. The method of any of claims 105-108, further comprising exposing the bacterial cell to a condition sufficient to induce expression of the functional protelomerase, and wherein the nucleic acid encoding the functional protelomerase is operably linked to an inducible promoter.

110. The method of claim 103, further comprising the step of exposing the bacterial cell to a condition sufficient to replicate the double-stranded, linear, close-ended nucleic acid vector.

111. The method of any of claims 103-110, wherein the nonlinear vector is a plasmid vector.

112. The method of any one of claims 106-108, wherein the nucleic acid encoding the functional protelomerase is operably linked to a constitutive promoter.

113. The method of 105 and 110, wherein the condition sufficient to replicate the vector is performed by a fed-batch fermentation.

114. The method of claim 113, wherein the fed-batch fermentation comprises culturing the bacterial cell at a first temperature of about 25 °C to about 32°C during a first portion of the fed- batch phase, followed by culturing the bacterial cell at a second temperature of about 37°C to about 45°C during a second portion of the fed-batch phase.ALD - P2024-0647-US02 (PCT)115. The method of claim 105, further comprising lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to the condition sufficient to associate the functional protelomerase with the telomerase occupancy site on the nonlinear vector.

116. The method of claim 115, further comprising extracting the double- stranded, linear, close- ended nucleic acid vector from the lysate to produce an extracted vector solution.

117. The method of claim 116, further comprising purifying the double-stranded, linear, close- ended nucleic acid vector from the extracted vector solution.

118. The method of claim 117, wherein the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

119. The method of claim 110, further comprising lysing the bacterial cell to produce a lysate following the step of exposing the bacterial cell to a condition sufficient to replicate the doublestranded, linear, close-ended nucleic acid vector.

120. The method of claim 119, further comprising extracting the double- stranded, linear, close- ended nucleic acid vector from the lysate to produce an extracted vector solution.

121. The method of claim 120, further comprising purifying the double- stranded, linear, close- ended nucleic acid vector from the extracted vector solution.

122. The method of claim 121, wherein the purifying comprises a phenol-chloroform extraction or a silica membrane-based column purification.

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