Bacterial host strains

Abstract

Disclosed are host bacterial strains, methods of making such host bacterial strains and methods of using such host bacterial strains to improve plasmid production. In some embodiments, an engineered bacterial host cell is provided that has a knockout of SbcC, SbcD, or both, and a knockout of one or all of recA, endA, and pgi. In some embodiments, methods for replicating a vector in an engineered E. coli host cell of the present disclosure are provided.

Description

BACTERIAL HOST STRAINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 642,260, filed on May 3, 2024, the entire contents of which are incorporated herein by reference. REFERENCE TO SEQUENCE LISTING

[0002] A Sequence Listing submitted as an ST.26 XML file via the USPTO patent electronic filing system is hereby incorporated by reference. The name of the XML file for the Sequence Listing is 0802761_SequenceListing.xml, the date of the creation of the XML file is 2025-04-29, and the size of the XML file is 161 KB (165,618 bytes). BACKGROUND

[0003] Production of viral vectors and plasmids containing structured DNA sequence, such as inverted repeats and direct repeats, is challenging and often results in reduced yields and instability. Bacterial host cells used to propagate these vectors and plasmids have been engineered through genetic modifications in an attempt to solve these problems.

[0004] While certain engineered bacterial cells have incorporated mutation strategies to stabilize these types of DNA sequence structures, such as E. coli strains Stbl2, Stbl3, and Stbl4, they still suffer from reduced viability and productivity.

[0005] Therefore, there is a need for bacterial production strains for high yield manufacture of palindrome- and inverted repeat-containing vectors without ITR deletion or rearrangement which do not suffer from low stability or low viability. SUMMARY OF THE INVENTION

[0006] The present disclosure is directed to host bacterial strains, methods of making such host bacterial strains and methods of using such host bacterial strains to improve plasmid production.

[0007] In some embodiments, an engineered bacterial host cell is provided that has a knockout of SbcC, SbcD or both, and a knockout of one or all of recA, endA, and pgi.

[0008] In some embodiments, methods for replicating a vector in an engineered E. coli host cell of the present disclosure are provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1A depicts the pKD4 SbcCD targeting PCR fragment.

[0010] FIG. 1B depicts the SbcCD locus.

[0011] FIG. 1C depicts the integrated pKD4 PCR product knocking out SbcCD.

[0012] FIG. 1D depicts the scar after FRT-mediated excision of the pKD4 kanR marker. DETAILED DESCRIPTION Definitions

[0013] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0014] 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.

[0015] 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 180 to 220 (plus or minus 10%).

[0016] As used herein, “engineered bacterial host cell” means a cell or cell line of bacterial origin that has been modified by human intervention 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.

[0017] 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.

[0018] As used herein, “SbcC” and “SbcD” are genes present in E. coli that each encode a protein 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 sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 9. By way of example, but not limitation, the SbcD 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: 10. For reference, a wild-type sequence of SbcC from NCBI (Reference Sequence: WP_206061808.1) for E. coli MG1655 is given by MKILSLRLKNLNSLKGEWKIDFTREPFASNGLFAITG PTGAGKTTLLDAICLALYHETPRLSNVSQSQNDLMTRDTAECLAEVEFEVKGEAYRAF WSQNRARNQPDGNLQVPRVELARCADGKILADKVKDKLELTATLTGLDYGRFTRSMLLSQGQFAAFLNAKPKERAELLEELTGTEIYGQISAMVFEQHKSARTELEKLQAQASGVTL LTPEQVQSLTASLQVLTDEEKQLITAQQQEQQSLNWLTRQDELQQEASRRQQALQQAL AEEEKAQPQLAALSLAQPARNLRPHWERIAEHSAALAHIRQQIEEVNTRLQSTMALRASI RHHAAKQSAELQQQQQSLNTWLQEHDRFRQWNNEPAGWRAQFSQQTSDREHLRQWQ QQLTHAEQKLNALAAITLTLTADEVATALAQHAEQRPLRQHLVALHGQIVPQQKRLAQ LQVAIQNVTQEQTQRNAALNEMRQRYKEKTQQLADVKTICEQEARIKTLEAQRAQLQA GQPCPLCGSTSHPAVEAYQALEPGVNQSRLLALENEVKKLGEEGATLRGQLDAITKQLQ RDENEAQSLRQDEQALTQQWQAVTASLNITLQPLDDIQPWLDAQDEHERQLRLLSQRH ELQGQIAAHNQQIIQYQQQIEQRQQLLLTTLTGYALTLPQEDEEESWLATRQQEAQSWQ QRQNELTALQNRIQQLTPILETLPQSDELPHCEETVVLENWRQVHEQCLALHSQQQTLQ QQDVLAAQSLQKAQAQFDTALQASVFDDQQAFLAALMDEQTLTQLEQLKQNLENQRR QAQTLVTQTAETLAQHQQHRPDDGLALTVTVEQIQQELAQTHQKLRENTTSQGEIRQQL KQDADNRQQQQTLMQQIAQMTQQVEDWGYLNSLIGSKEGDKFRKFAQGLTLDNLVHL ANQQLTRLHGRYLLQRKASEALEVEVVDTWQADAVRDTRTLSGGESFLVSLALALALS DLVSHKTRIDSLFLDEGFGTLDSETLDTALDALDALNASGKTIGVISHVEAMKERIPVQIK VKKINGLGYSKLESTFAVK (SEQ ID NO: 66) and, for example, can be encoded by a gene having the sequence of SEQ ID NO: 77 (ATGAAAATTCTCAGCCTGCGCCTGAAAAA CCTGAACTCATTAAAAGGCGAATGGAAGATTGATTTCACCCGCGAGCCGTTCGCCAG CAACGGGCTGTTTGCTATTACCGGCCCAACAGGTGCGGGGAAAACCACCCTGCTGG ACGCCATTTGTCTGGCGCTGTATCACGAAACTCCGCGTCTCTCTAACGTTTCACAATC GCAAAATGATCTCATGACCCGCGATACCGCCGAATGTCTGGCGGAGGTGGAGTTTG AAGTGAAAGGTGAAGCGTACCGTGCATTCTGGAGCCAGAATCGGGCGCGTAACCAA CCCGACGGTAATTTGCAGGTGCCACGCGTAGAGCTGGCGCGCTGCGCCGACGGCAAAATTCTCGCCGACAAAGTGAAAGATAAGCTGGAACTGACAGCGACGTTAACCGGGC TGGATTACGGGCGCTTCACCCGTTCGATGCTGCTTTCGCAGGGGCAATTTGCTGCCTT CCTGAATGCCAAACCCAAAGAACGCGCGGAATTGCTCGAGGAGTTAACCGGCACTG AAATCTACGGGCAAATCTCGGCGATGGTTTTTGAGCAGCACAAATCGGCCCGCACA GAGCTGGAGAAGCTGCAAGCGCAGGCCAGCGGCGTCACGTTGCTCACGCCGGAACA AGTGCAATCGCTGACAGCGAGTTTGCAGGTACTTACTGACGAAGAAAAACAGTTAA TTACCGCGCAGCAGCAAGAACAACAATCGCTAAACTGGTTAACGCGTCAGGACGAA TTGCAGCAAGAAGCCAGCCGCCGTCAGCAGGCCTTGCAACAGGCGTTAGCCGAAGA AGAAAAAGCGCAACCTCAACTGGCGGCGCTTAGTCTGGCACAACCGGCACGAAATC TTCGTCCACACTGGGAACGCATCGCAGAACACAGCGCGGCGCTGGCGCATATTCGC CAGCAGATTGAAGAAGTAAATACTCGCTTACAGAGCACAATGGCGCTTCGCGCGAG CATTCGCCACCACGCGGCGAAGCAGTCAGCAGAATTACAGCAGCAGCAACAAAGCC TGAATACCTGGTTACAGGAACACGACCGCTTCCGTCAGTGGAACAACGAACCGGCG GGTTGGCGTGCGCAGTTCTCCCAACAAACCAGCGATCGCGAGCATCTGCGGCAATG GCAGCAACAGTTAACCCATGCTGAGCAAAAACTTAATGCGCTTGCGGCGATCACGTT GACGTTAACCGCCGATGAAGTTGCTACCGCCCTGGCGCAACATGCTGAGCAACGCC CACTGCGTCAGCACCTGGTCGCGCTGCATGGACAGATTGTTCCCCAACAAAAACGTC TGGCGCAGTTACAGGTCGCTATCCAGAATGTCACGCAAGAACAGACGCAACGTAAC GCCGCACTTAACGAAATGCGCCAGCGTTATAAAGAAAAGACGCAGCAACTTGCCGA TGTGAAAACCATTTGCGAGCAGGAAGCGCGCATCAAAACGCTGGAAGCTCAACGTG CACAGTTACAGGCGGGTCAGCCTTGCCCACTTTGTGGTTCCACCAGCCACCCGGCGG TCGAGGCGTATCAGGCGCTGGAGCCTGGCGTTAATCAGTCTCGATTACTGGCGCTGG AAAACGAAGTTAAAAAGCTCGGTGAAGAAGGTGCGACGCTACGTGGGCAACTGGACGCCATAACAAAGCAGCTTCAGCGTGATGAAAACGAAGCGCAAAGCCTCCGACAAGA TGAGCAAGCACTTACTCAACAATGGCAAGCCGTCACGGCCAGCCTCAATATCACCTT GCAGCCACTGGACGATATTCAACCGTGGCTGGATGCACAAGATGAGCACGAACGCC AGCTGCGGTTACTCAGCCAACGGCATGAATTACAAGGGCAGATTGCCGCGCATAAT CAGCAAATTATCCAGTATCAACAGCAAATTGAACAACGCCAGCAACTACTTTTAACG ACATTGACGGGTTATGCACTGACATTGCCACAGGAAGATGAAGAAGAGAGCTGGTT GGCGACACGTCAGCAAGAAGCGCAGAGCTGGCAGCAACGCCAGAACGAATTAACC GCGCTGCAAAACCGTATTCAGCAGCTGACGCCGATTCTGGAAACGTTGCCGCAAAG TGATGAACTCCCGCACTGCGAAGAAACTGTGGTATTGGAAAACTGGCGGCAGGTAC ATGAACAATGTCTCGCATTACACAGCCAGCAGCAGACGTTACAGCAACAGGATGTT CTGGCGGCGCAAAGTCTGCAAAAAGCCCAGGCGCAGTTTGACACCGCGCTACAGGC CAGCGTCTTTGACGATCAGCAGGCGTTCCTTGCGGCGCTAATGGATGAACAAACACT AACGCAGCTGGAACAGCTCAAGCAGAATCTGGAAAACCAGCGCCGTCAGGCGCAA ACTCTGGTCACTCAGACAGCAGAAACGCTGGCACAGCATCAACAACACCGACCTGA CGACGGGTTGGCTCTCACTGTGACGGTGGAGCAGATTCAGCAAGAGTTAGCGCAAA CTCACCAAAAGTTGCGTGAAAACACCACGAGTCAAGGCGAGATTCGCCAGCAGCTG AAGCAGGATGCAGATAACCGTCAGCAACAACAAACCTTAATGCAGCAAATTGCTCA AATGACGCAGCAGGTTGAGGACTGGGGATATCTGAATTCGCTAATAGGTTCCAAAG AGGGCGATAAATTCCGCAAGTTTGCCCAGGGGCTGACGCTGGATAATTTAGTCCATC TCGCTAATCAGCAACTTACCCGGCTGCACGGGCGCTATCTGTTACAGCGCAAAGCCA GCGAGGCGCTGGAAGTCGAGGTTGTTGATACCTGGCAGGCAGATGCGGTACGCGAT ACCCGTACCCTTTCCGGCGGCGAAAGTTTCCTCGTTAGTCTGGCGCTGGCGCTGGCG CTTTCGGATCTGGTCAGCCATAAAACACGTATTGACTCGCTGTTCCTTGATGAAGGTTTTGGCACGCTGGATAGCGAAACGCTGGATACCGCCCTTGATGCGCTGGATGCCCTG AACGCCAGTGGCAAAACCATCGGTGTGATTAGCCACGTAGAAGCGATGAAAGAGCG TATTCCGGTGCAGATCAAAGTGAAAAAGATCAACGGCCTGGGCTACAGCAAACTGG AAAGTACGTTTGCAGTGAAATAA), while a wild-type sequence of SbcD from GenBank (AAB18122.1) for E. coli MG1655 is given by MLFRQGTVMRILHTSDWHLGQNFYSK SREAEHQAFLDWLLETAQTHQVDAIIVAGDVFDTGSPPSYARTLYNRFVVNLQQTGCHL VVLAGNHDSVATLNESRDIMAFLNTTVVASAGHAPQILPRRDGTPGAVLCPIPFLRPRDII TSQAGLNGIEKQQHLLAAITDYYQQHYADACKLRGDQPLPIIATGHLTTVGASKSDAVR DIYIGTLDAFPAQNFPPADYIALGHIHRAQIIGGMEHVRYCGSPIPLSFDECGKSKYVHLV TFSNGKLESVENLNVPVTQPMAVLKGDLASITAQLEQWRDVSQEPPVWLDIEITTDEYL HDIQRKIQALTESLPVEVLLVRRSREQRERVLASQQRETLSELSVEEVFNRRLALEELDES QQQRLQHLFTTTLHTLAGEHEA (SEQ ID NO: 67) and, for example, can be encoded by a gene having a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 78 (ATGCGCATCCTTCACACCTCAGACTGGCATCTCGGCCAGAACTT CTACAGTAAAAGCCGCGAAGCTGAACATCAGGCTTTTCTTGACTGGCTGCTGGAGAC AGCACAAACCCATCAGGTGGATGCGATTATTGTTGCCGGTGATGTTTTCGATACCGG CTCGCCGCCCAGTTACGCCCGCACGTTATACAACCGTTTTGTTGTCAATTTACAGCA AACTGGCTGTCATCTGGTGGTACTGGCAGGAAACCATGACTCGGTCGCCACGCTGAA TGAATCGCGCGATATCATGGCGTTCCTCAATACTACCGTGGTCGCCAGCGCCGGACA TGCGCCGCAAATCTTGCCTCGTCGCGACGGGACGCCAGGCGCAGTGCTGTGCCCCAT TCCGTTTTTACGTCCGCGTGACATTATTACCAGCCAGGCGGGGCTTAACGGTATTGA AAAACAGCAGCATTTACTGGCAGCGATTACCGATTATTACCAACAACACTATGCCGA TGCCTGCAAACTGCGCGGCGATCAGCCTCTGCCCATCATCGCCACGGGACATTTAACGACCGTGGGGGCCAGTAAAAGTGACGCCGTGCGTGACATTTATATTGGCACGCTGG ACGCGTTTCCGGCACAAAACTTTCCACCAGCCGACTACATCGCGCTCGGGCATATTC ACCGCGCACAGATTATTGGCGGCATGGAACATGTTCGCTATTGCGGCTCCCCCATTC CACTGAGTTTTGATGAATGCGGTAAGAGTAAATATGTCCATCTGGTGACATTTTCAA ACGGCAAATTAGAGAGCGTGGAAAACCTGAACGTACCGGTAACGCAACCCATGGCA GTGCTGAAAGGCGATCTGGCGTCGATTACCGCACAGCTGGAACAGTGGCGCGATGT ATCGCAGGAGCCACCTGTCTGGCTGGATATCGAAATCACTACTGATGAGTATCTGCA TGATATTCAGCGCAAAATCCAGGCATTAACCGAATCATTGCCTGTCGAAGTATTGCT GGTACGTCGGAGTCGTGAACAGCGCGAGCGTGTGTTAGCCAGCCAACAGCGTGAAA CCCTCAGCGAACTCAGCGTCGAAGAGGTGTTCAATCGCCGTCTGGCACTGGAAGAA CTGGATGAATCGCAGCAGCAACGTCTGCAGCATCTTTTCACCACGACGTTGCATACC CTCGCCGGAGAACACGAAGCATGA). It should be understood that these amino acid sequences are exemplary and that one of skill in the art can identify SbcC and SbcD genes and proteins, including complexes, in other strains and cell lines based on homology.

[0019] As used herein, “a viability- or yield-reducing mutation” refers to a mutation which reduces the viability or yield, respectively, of a cell line with respect to the cell line from which the mutated cell line is derived under the same culture conditions. It should be understood that such mutations can be engineered or naturally-occurring.

[0020] As used herein, “endA” is a gene present in E. coli that encodes a DNA-specific endonuclease. The endA gene encodes a protein having a sequence having 95% or more sequence identity to the sequence of SEQ ID NO: 73 (MYRYLSIAAVVLSAAFSGPALAEGINSFSQA KAAAVKVHADAPGTFYCGCKINWQGKKGVVDLQSCGYQVRKNENRASRVEWEHVVP AWQFGHQRQCWQDGGRKNCAKDPVYRKMESDMHNLQPSVGEVNGDRGNFMYSQWNGGEGQYGQCAMKVDFKEKAAEPPARARGAIARTYFYMRDQYNLTLSRQQTQLFNA WNKMYPVTDWECERDERIAKVQGNHNPYVQRACQARKS). For example, the endA gene can have the sequence of SEQ ID NO: 74 (ATGTACCGTTATTTGTCTATTGCTGCGGTGG TACTGAGCGCAGCATTTTCCGGCCCGGCGTTGGCCGAAGGTATCAATAGTTTTTCTC AGGCGAAAGCCGCGGCGGTAAAAGTCCACGCTGACGCGCCCGGTACGTTTTATTGC GGATGTAAAATTAACTGGCAGGGCAAAAAAGGCGTTGTTGATCTGCAATCGTGCGG CTATCAGGTGCGCAAAAATGAAAACCGCGCCAGCCGCGTAGAGTGGGAACATGTCG TTCCCGCCTGGCAGTTCGGTCACCAGCGCCAGTGCTGGCAGGACGGTGGACGTAAA AACTGCGCTAAAGATCCGGTCTATCGCAAGATGGAAAGCGATATGCATAACCTGCA GCCGTCAGTCGGTGAGGTGAATGGCGATCGCGGCAACTTTATGTACAGCCAGTGGA ATGGCGGTGAAGGCCAGTACGGTCAATGCGCCATGAAGGTCGATTTCAAAGAAAAA GCTGCCGAACCACCAGCGCGTGCACGCGGTGCCATTGCGCGCACCTACTTCTATATG CGCGACCAATACAACCTGACACTCTCTCGCCAGCAAACGCAGCTGTTCAACGCATG GAACAAGATGTATCCGGTTACCGACTGGGAGTGCGAGCGCGATGAACGCATCGCGA AGGTGCAGGGCAATCATAACCCGTATGTGCAACGCGCTTGCCAGGCGCGAAAGAGC TAA).

[0021] 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 protein having the sequence of SEQ ID NO: 79 (MAIDENKQKALAAALGQIEKQFGKGSIMRLGEDRSMDVETISTGSL SLDIALGAGGLPMGRIVEIYGPESSGKTTLTLQVIAAAQREGKTCAFIDAEHALDPIYARK LGVDIDNLLCSQPDTGEQALEICDALARSGAVDVIVVDSVAALTPKAEIEGEIGDSHMGL AARMMSQAMRKLAGNLKQSNTLLIFINQIRMKIGVMFGNPETTTGGNALKFYASVRLDI RRIGAVKEGENVVGSETRVKVVKNKIAAPFKQAEFQILYGEGINFYGELVDLGVKEKLIEKAGAWYSYKGEKIGQGKANATAWLKDNPETAKEIEKKVRELLLSNPNSTPDFSVDDSE GVAETNEDF). For example, the recA gene can have the sequence of SEQ ID NO: 80 (ATGGCTATCGACGAAAACAAACAGAAAGCGTTGGCGGCAGCACTGGGCCAGATTG AGAAACAATTTGGTAAAGGCTCCATCATGCGCCTGGGTGAAGACCGTTCCATGGAT GTGGAAACCATCTCTACCGGTTCGCTTTCACTGGATATCGCGCTTGGGGCAGGTGGT CTGCCGATGGGCCGTATCGTCGAAATCTACGGACCGGAATCTTCCGGTAAAACCAC GCTGACGCTGCAGGTGATCGCCGCAGCGCAGCGTGAAGGTAAAACCTGTGCGTTTA TCGATGCTGAACACGCGCTGGACCCAATCTACGCACGTAAACTGGGCGTCGATATC GACAACCTGCTGTGCTCCCAGCCGGACACCGGCGAGCAGGCACTGGAAATCTGTGA CGCCCTGGCGCGTTCTGGCGCAGTAGACGTTATCGTCGTTGACTCCGTGGCGGCACT GACGCCGAAAGCGGAAATCGAAGGCGAAATCGGCGACTCTCACATGGGCCTTGCGG CACGTATGATGAGCCAGGCGATGCGTAAGCTGGCGGGTAACCTGAAGCAGTCCAAC ACGCTGCTGATCTTCATCAACCAGATCCGTATGAAAATTGGTGTGATGTTCGGTAAC CCGGAAACCACTACCGGTGGTAACGCGCTGAAATTCTACGCCTCTGTTCGTCTCGAC ATCCGTCGTATCGGCGCGGTGAAAGAGGGCGAAAACGTGGTGGGTAGCGAAACCCG CGTGAAAGTGGTGAAGAACAAAATCGCTGCGCCGTTTAAACAGGCTGAATTCCAGA TCCTCTACGGCGAAGGTATCAACTTCTACGGCGAACTGGTTGACCTGGGCGTAAAAG AGAAGCTGATCGAGAAAGCAGGCGCGTGGTACAGCTACAAAGGTGAGAAGATCGG TCAGGGTAAAGCGAATGCGACTGCCTGGCTGAAAGATAACCCGGAAACCGCGAAAG AGATCGAGAAGAAAGTACGTGAGTTGCTGCTGAGCAACCCGAACTCAACGCCGGAT TTCTCTGTAGATGATAGCGAAGGCGTAGCAGAAACTAACGAAGATTTTTAA).

[0022] 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 protein having the sequence ofSEQ ID NO: 75 (MKNINPTQTAAWQALQKHFDEMKDVTIADLFAKD GDRFSKFSATFDDQMLVDYSKNRITEETLAKLQDLAKECDLAGAIKSMFSGEKINRTEN RAVLHVALRNRSNTPILVDGKDVMPEVNAVLEKMKTFSEAIISGEWKGYTGKAITDVV NIGIGGSDLGPYMVTEALRPYKNHLNMHFVSNVDGTHIAEVLKKVNPETTLFLVASKTF TTQETMTNAHSARDWFLKAAGDEKHVAKHFAALSTNAKAVGEFGIDTANMFEFWDW VGGRYSLWSAIGLSIVLSIGFDNFVELLSGAHAMDKHFSTTPAEKNLPVLLALIGIWYNN FFGAETEAILPYDQYMHRFAAYFQQGNMESNGKYVDRNGNVVDYQTGPIIWGEPGTNG QHAFYQLIHQGTKMVPCDFIAPAITHNPLSDHHQKLLSNFFAQTEALAFGKSREVVEQE YRDQGKDPATLDYVVPFKVFEGNRPTNSILLREITPFSLGALIALYEHKIFTQGVILNIFTF DQWGVELGKQLANRILPELKDDKEISSHDSSTNGLINRYKAWRG). For example, the pgi gene can have a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 76 (ATGAAAAACATCAATCCAACGCAGACCGCTGCCTGGCAGG CACTACAGAAACACTTCGATGAAATGAAAGACGTTACGATCGCCGATCTTTTTGCTA AAGACGGCGATCGTTTTTCTAAGTTCTCCGCAACCTTCGACGATCAGATGCTGGTGG ATTACTCCAAAAACCGCATCACTGAAGAGACGCTGGCGAAATTACAGGATCTGGCG AAAGAGTGCGATCTGGCGGGCGCGATTAAGTCGATGTTCTCTGGCGAGAAGATCAA CCGCACTGAAAACCGCGCCGTGCTGCACGTAGCGCTGCGTAACCGTAGCAATACCC CGATTTTGGTTGATGGCAAAGACGTAATGCCGGAAGTCAACGCGGTGCTGGAGAAG ATGAAAACCTTCTCAGAAGCGATTATTTCCGGTGAGTGGAAAGGTTATACCGGCAA AGCAATCACTGACGTAGTGAACATCGGGATCGGCGGTTCTGACCTCGGCCCATACAT GGTGACCGAAGCTCTGCGTCCGTACAAAAACCACCTGAACATGCACTTTGTTTCTAA CGTCGATGGGACTCACATCGCGGAAGTGCTGAAAAAAGTAAACCCGGAAACCACGC TGTTCTTGGTAGCATCTAAAACCTTCACCACTCAGGAAACTATGACCAACGCCCATAGCGCGCGTGACTGGTTCCTGAAAGCGGCAGGTGATGAAAAACACGTTGCAAAACAC TTTGCGGCGCTTTCCACCAATGCCAAAGCCGTTGGCGAGTTTGGTATTGATACTGCC AACATGTTCGAGTTCTGGGACTGGGTTGGCGGCCGTTACTCTTTGTGGTCAGCGATT GGCCTGTCGATTGTTCTCTCCATCGGCTTTGATAACTTCGTTGAACTGCTTTCCGGCG CACACGCGATGGACAAGCATTTCTCCACCACGCCTGCCGAGAAAAACCTGCCTGTA CTGCTGGCGCTGATTGGCATCTGGTACAACAATTTCTTTGGTGCGGAAACTGAAGCG ATTCTGCCGTATGACCAGTATATGCACCGTTTCGCGGCGTACTTCCAGCAGGGCAAT ATGGAGTCCAACGGTAAGTATGTTGACCGTAACGGTAACGTTGTGGATTACCAGACT GGCCCGATTATCTGGGGTGAACCAGGCACTAACGGTCAGCACGCGTTCTACCAGCT GATCCACCAGGGAACCAAAATGGTACCGTGCGATTTCATCGCTCCGGCTATCACCCA TAACCCGCTCTCTGATCATCACCAGAAACTGCTGTCTAACTTCTTCGCCCAGACCGA AGCGCTGGCGTTTGGTAAATCCCGCGAAGTGGTTGAGCAGGAATATCGTGATCAGG GTAAAGATCCGGCAACGCTTGACTACGTGGTGCCGTTCAAAGTATTCGAAGGTAACC GCCCGACCAACTCCATCCTGCTGCGTGAAATCACTCCGTTCAGCCTGGGTGCGTTGA TTGCGCTGTATGAGCACAAAATCTTTACTCAGGGCGTGATCCTGAACATCTTCACCT TCGACCAGTGGGGCGTGGAACTGGGTAAACAGCTGGCGAACCGTATTCTGCCAGAG CTGAAAGATGATAAAGAAATCAGCAGCCACGATAGCTCGACCAATGGTCTGATTAA CCGCTATAAAGCGTGGCGCGGTTAA).

[0023] As used herein, “sbcB” is a gene present in E. coli that encodes a 3’-5’ exonuclease. By way of example, but not limitation, the sbcB gene can include a sequence encoding a protein having the sequence of SEQ ID NO: 81 (MMNDGKQQSTFLFHDYETFGTHPALDRPAQFA AIRTDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGITPQEARAKGENEAAFAARIHSLFT VPKTCILGYNNVRFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDVMRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYL FTHRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTSWVAPLAWHPENRNAVIMVDL AGDISPLLELDSDTLRERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRL GINRQHCLDNLKILRENPQVREKVVAIFAEAEPFTPSDNVDAQLYNGFFSDADRAAMKI VLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLDYAEQQRWLEHRRQVFTPEFLQG YADELQMLVQQYADDKEKVALLKALWQYAEEIV). For example the sbcB gene can include a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 11.

[0024] 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. By way of example, but not limitation, the recB gene can include a sequence encoding a protein having the sequence of SEQ ID NO: 82 (MSDVAETLDPLRLPLQGERLIEASAGTGKTFTIAALYL RLLLGLGGSAAFPRPLTVEELLVVTFTEAATAELRGRIRSNIHELRIACLRETTDNPLYER LLEEIDDKAQAAQWLLLAERQMDEAAVFTIHGFCQRMLNLNAFESGMLFEQQLIEDESL LRYQACADFWRRHCYPLPREIAQVVFETWKGPQALLRDINRYLQGEAPVIKAPPPDDET LASRHAQIVARIDTVKQQWRDAVGELDALIESSGIDRRKFNRSNQAKWIDKISAWAEEE TNSYQLPESLEKFSQRFLEDRTKAGGETPRHPLFEAIDQLLAEPLSIRDLVITRALAEIRET VAREKRRRGELGFDDMLSRLDSALRSESGEVLAAAIRTRFPVAMIDEFQDTDPQQYRIFR RIWHHQPETALLLIGDPKQAIYAFRGADIFTYMKARSEVHAHYTLDTNWRSAPGMVNS VNKLFSQTDDAFMFREIPFIPVKSAGKNQALRFVFKGETQPAMKMWLMEGESCGVGDY QSTMAQVCAAQIRDWLQAGQRGEALLMNGDDARPVRASDISVLVRSRQEAAQVRDAL TLLEIPSVYLSNRDSVFETLEAQEMLWLLQAVMTPERENTLRSALATSMMGLNALDIET LNNDEHAWDVVVEEFDGYRQIWRKRGVMPMLRALMSARNIAENLLATAGGERRLTDILHISELLQEAGTQLESEHALVRWLSQHILEPDSNASSQQMRLESDKHLVQIVTIHKSKGL EYPLVWLPFITNFRVQEQAFYHDRHSFEAVLDLNAAPESVDLAEAERLAEDLRLLYVAL TRSVWHCSLGVAPLVRRRGDKKGDTDVHQSALGRLLQKGEPQDAAGLRTCIEALCDD DIAWQTAQTGDNQPWQVNDVSTAELNAKTLQRLPGDNWRVTSYSGLQQRGHGIAQDL MPRLDVDAAGVASVVEEPTLTPHQFPRGASPGTFLHSLFEDLDFTQPVDPNWVREKLEL GGFESQWEPVLTEWITAVLQAPLNETGVSLSQLSARNKQVEMEFYLPISEPLIASQLDTLI RQFDPLSAGCPPLEFMQVRGMLKGFIDLVFRHEGRYYLLDYKSNWLGEDSSAYTQQAM AAAMQAHRYDLQYQLYTLALHRYLRHRIADYDYEHHFGGVIYLFLRGVDKEHPQQGIY TTRPNAGLIALMDEMFAGMTLEEA). For example, the recB gene can include a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 12.

[0025] 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. By way of example, but not limitation, the recD gene can include a sequence encoding a protein having the sequence of SEQ ID NO: 83 (MKLQKQLLEAVEHKQLRPLDVQFALTVAGDEHPAVT LAAALLSHDAGEGHVCLPLSRLENNEASHPLLATCVSEIGELQNWEECLLASQAVSRGD EPTPMILCGDRLYLNRMWCNERTVARFFNEVNHAIEVDEALLAQTLDKLFPVSDEINWQ KVAAAVALTRRISVISGGPGTGKTTTVAKLLAALIQMADGERCRIRLAAPTGKAAARLT ESLGKALRQLPLTDEQKKRIPEDASTLHRLLGAQPGSQRLRHHAGNPLHLDVLVVDEAS MIDLPMMSRLIDALPDHARVIFLGDRDQLASVEAGAVLGDICAYANAGFTAERARQLSR LTGTHVPAGTGTEAASLRDSLCLLQKSYRFGSDSGIGQLAAAINRGDKTAVKTVFQQDF TDIEKRLLQSGEDYIAMLEEALAGYGRYLDLLQARAEPDLIIQAFNEYQLLCALREGPFG VAGLNERIEQFMQQKRKIHRHPHSRWYEGRPVMIARNDSALGLFNGDIGIALDRGQGTR VWFAMPDGNIKSVQPSRLPEHETTWAMTVHKSQGSEFDHAALILPSQRTPVVTRELVYTAVTRARRRLSLYADERILSAAIATRTERRSGLAALFSSRE). For example the recD gene can include a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 13.

[0026] As used herein, “recJ” is a gene present in E. coli that encodes an ssDNA specific exonuclease. By way of example, but not limitation, the recJ gene can include a sequence encoding a protein having the sequence of SEQ ID NO: 84 (MKQQIQLRRREVDETADLPAELPPLLR RLYASRGVRSAQELERSVKGMLPWQQLSGVEKAVEILYNAFREGTRIIVVGDFDADGAT STALSVLAMRSLGCSNIDYLVPNRFEDGYGLSPEVVDQAHARGAQLIVTVDNGISSHAG VEHARSLGIPVIVTDHHLPGDTLPAAEAIINPNLRDCNFPSKSLAGVGVAFYLMLALRTF LRDQGWFDERNIAIPNLAELLDLVALGTVADVVPLDANNRILTWQGMSRIRAGKCRPGI KALLEVANRDAQKLAASDLGFALGPRLNAAGRLDDMSVGVALLLCDNIGEARVLANE LDALNQTRKEIEQGMQIEALTLCEKLERSRDTLPGGLAMYHPEWHQGVVGILASRIKER FHRPVIAFAPAGDGTLKGSGRSIQGLHMRDALERLDTLYPGMMLKFGGHAMAAGLSLE EDKFKLFQQRFGELVTEWLDPSLLQGEVVSDGPLSPAEMTMEVAQLLRDAGPWGQMFP EPLFDGHFRLLQQRLVGERHLKVMVEPVGGGPLLDGIAFNVDTALWPDNGVREVQLAY KLDINEFRGNRSLQIIIDNIWPI). For example, the recJ gene can include a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 65.

[0027] 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, the uvrC gene can include a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NO: 14.

[0028] 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 99% or 100% sequence identity to SEQ ID NO: 15.

[0029] As used herein, “mcrBC-hsd-mrr” is a gene present in E. coli that encodes an endonuclease complex. By way of example, but not limitation, the mcrBC-hsd-mrr gene can include a sequence having at least 90%, at least 95%, at least 99% or 100% sequence identity to SEQ ID NOS: 16-21.

[0030] As used herein, “fhuA” is a gene present in E. coli that encodes an outer membrane protein involved in transport and also acts as a receptor.

[0031] 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. 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.

[0032] As used herein, “dcm” is a gene present in E. coli that encodes a DNA cytosine methyltranferase.

[0033] As used herein, “derived from” when referring to a particular cell means that the cell has been descended from a particular cell line or a general species. Alternatively, “derived from” can refer to the base cell line, species or strain used to create the engineered bacterial host cells.

[0034] As used herein, “DH5α” refers to an E. coli strain / cell line with the following genotype: F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ- thi-1 gyrA96 relA1.

[0035] As used herein, “DH1” refers to an E. coli strain / cell line with the following genotype: F–λ–endA1 recA1 relA1 gyrA96 thi-1 glnV44 hsdR17(rK–mK–).

[0036] As used herein, “JM107” refers to an E. coli strain / cell line with the following genotype: endA1 glnV44 thi-1 relA1 gyrA96 Δ(lac-proAB) [F' traD36 proAB+lacIqlacZΔM15] hsdR17(RK- mK+) λ-.

[0037] As used herein, “JM108” refers to an E. coli strain / cell line with the following genotype: endA1 recA1 gyrA96 thi-1 relA1 glnV44 Δ(lac-proAB) hsdR17 (rK- mK+).

[0038] As used herein, “JM109” refers to an E. coli strain / cell line with the following genotype: endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+Δ(lac-proAB) e14- [F' traD36 proAB+lacIqlacZΔM15] hsdR17(rK-mK+).

[0039] As used herein, “MG1655” refers to an E. coli strain / cell line with the following genotype: K-12 F–λ–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, and GCA_028618655.1, or the genome of the ATCC® 700926 (also called the Migula strain, or the Castellani and Chalmers strain).

[0040] As used herein, “MG1655 ΔendA ΔrecA” refers to an E. coli strain / cell line with the following genotype: K-12 F–λ–ilvG–rfb-50 rph-1 ΔendA ΔrecA.

[0041] As used herein, “XL1Blue”: refers to an E. coli strain / cell line with the following genotype: endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB+lacIqΔ(lacZ)M15] hsdR17(rK- mK+).

[0042] As used herein “GALG20” refers to a MG1655 derived E. coli strain of the genotype K-12 F–λ–ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi.

[0043] As used herein, “REVIVER-pUC” refers to an E. coli strain / cell line with the following genotype: fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi- 1 hsdR17 ΔSbcDC Δdcm.

[0044] As used herein, “engineered mutation” should be understood a mutation that did not occur naturally and was instead the product of direct, human intervention.

[0045] 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%, at least 98%, at least 99% or 100% sequence identity to a given query sequence. To determine percent sequence identity, a query sequence (e.g. a nucleic 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 particularalignment program. The output is a sequence alignment that reflects 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

[0046] As used herein “HyperGROTMfermentation” 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 continued growth at elevated temperature in order to accumulate plasmid; the temperature shift at restricted growth rate improved plasmid yield and purity. HyperGROTMfermentation is described in US Patent No. 7,943,377, which is incorporated herein by reference in its entirety.

[0047] As used herein, a “Rep protein” means a protein found in E. coli cells involved in the replication process.

[0048] As used herein, the term “genomic” refers to nucleic acid sequences incorporated into the genome of a bacterial cell.

[0049] As used herein, “AAV vector” refers to an adeno-associated virus vector or episomal viral vector. By way of example, but not limitation, “AAV vector” includes self-complementary adeno- associated virus vectors (scAAV) and single-stranded adeno-associated virus vectors (ssAAV).

[0050] As used herein, “amp” refers to ampicillin.

[0051] As used herein, “ampR” refers to an ampicillin resistance gene.

[0052] As used herein “bacterial region” refers to the region of a vector, such as a plasmid, required for prorogation and selection in a bacterial host.

[0053] As used herein “CatR” refers to a chloramphenicol resistance gene.

[0054] As used herein “ccc” or “CCC” means “covalently closed circular” unless used in the context of a nucleotide or amino acid sequence.

[0055] As used herein, “cI” means lambda repressor.

[0056] 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 ºC but is mostly inactive at 37-42 ºC. Also called cI857 or cI857ts.

[0057] As used herein “cmv” or “CMV” refers to cytomegalovirus.

[0058] As used herein “copy cutter host strain” refers to R6K origin production strains containing a phage φ80 attachment site chromosomally integrated copy of an arabinose inducible CI857ts gene. Addition of arabinose to plates or media (e.g. to 0.2-0.4% final concentration) induces pARA mediated CI857ts repressor expression which reduces copy number at 30° C through CI857ts mediated downregulation of the R6K Rep protein expressing pL promoter [i.e. additional CI857ts mediates more effective downregulation of the pL (OL1-G to T) promoter at 30 °C]. Copy number induction after temperature shift to 37-42 °C is not impaired since the CI857ts repressor is inactivated at these elevated temperatures. Copy cutter host strains increase the R6K vector temperature upshift copy number induction ratio by reducing the copy number at 30 °C. This is advantageous for production of large, toxic, or dimerization prone R6K origin vectors.

[0059] As used herein “dcm methylation” refers to methylation by E. coli methyltransferase that methylates the sequences CC(A / T)GG at the C5 position of the second cytosine.

[0060] As used herein “eukaryotic region” refers to the region of a plasmid that encodes eukaryotic sequences and / or sequences required for plasmid function in the target organism. This includes the region of a plasmid vector required for expression of one or more transgenes in the target organism including RNA Pol II enhancers, promoters, transgenes and polyA sequences. This also includes the region of a plasmid vector required for expression of one or more transgenes in the target organism using RNA Pol I or RNA Pol III promoters, RNA Pol I or RNA Pol III expressed transgenes or RNAs. The eukaryotic region may optionally include other functional sequences, such as eukaryotic transcriptional terminators, supercoiling-induced DNA duplex destabilized (SIDD) structures, S / MARs, boundary elements, and the like. In a Lentiviral or Retroviral vector, the eukaryotic region contains flanking direct repeat LTRs, in a AAV vector the eukaryotic region contains flanking inverted terminal repeats, while in a Transposon vector the eukaryotic region contains flanking transposon inverted terminal repeats or IR / DR termini (e.g., Sleeping Beauty). In genome integration vectors, the eukaryotic region may encode homology arms to direct targeted integration.

[0061] As used herein “expression vector” refers to a vector for expression of a gene of interest including, but not limited to, mRNA, protein antigens, protein therapeutics, shRNA, RNA or microRNA genes in a target organism.

[0062] As used herein “gene of interest” refers to a gene to be expressed in the target organism.

[0063] As used herein “inverted repeat” refers to a single-stranded sequence of nucleotides followed downstream by its reverse complement. The intervening sequence of nucleotides between the initial sequence and the reverse complement can be any length including zero. When the intervening length is zero, the composite sequence is a palindrome. It should be understoodthat inverted repeats can occur in double-stranded DNA and that other inverted repeats can occur within the intervening sequence.

[0064] As used herein “IR / DR” refers to inverted repeats which are directly repeated twice. For example, Sleeping Beauty transposon IR / DR repeats.

[0065] As used herein “iteron” refers to directly repeated DNA sequences in an origin of replication that are required for replication initiation. R6K origin iteron repeats are 22 bp such and are described in WO 2019 / 183248 (see SEQ ID NOs 19-23 therein).

[0066] As used herein “ITR” refers to an inverted terminal repeat.

[0067] As used herein “lentiviral vector” refers to an integrative viral vector that can infect dividing and non-dividing cells. Also called a Lentiviral transfer plasmid. The plasmid encodes Lentiviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with Lentiviral envelope and packaging plasmids required to make viral particles.

[0068] As used herein “lentiviral envelope vector” refers to a plasmid encoding envelope glycoprotein.

[0069] As used herein “lentiviral packaging vector” refers to one or two plasmids that express gag, pol and Rev gene functions required to package the lentiviral transfer vector.

[0070] As used herein “minicircle” refers to covalently closed circular plasmid derivatives in which the bacterial region has been removed from the parent plasmid by in vivo or in vitro site- specific recombination or in vitro restriction digestion / ligation. Minicircle vectors are replication incompetent in bacterial cells.

[0071] As used herein “NanoplasmidTMvector” refers to a vector combining an RNA selectable marker with a R6K, ColE2 or ColE2 related replication origin as described in WO2014077866 and WO 2014 / 035457, each of which is incorporated herein by reference in its entirety.

[0072] As used herein “NTC9385R” refers to the NTC9385R NanoplasmidTMvector described in WO 2014 / 035457 and has a spacer region encoded NheI- trpA terminator-R6K origin RNA-OUT –KpnI bacterial region linked through the flanking NheI and KpnI sites to the eukaryotic region.

[0073] As used herein “PCR” refers to “polymerase chain reaction.”

[0074] As used herein “pDNA” refers to plasmid DNA.

[0075] As used herein “piggyback transposon” refers to a transposon system that integrates an ITR flanked PB transposon into the genome by a simple cut and paste mechanism mediated by PB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the PB ITRs which is excised and integrated into the genome.

[0076] As used herein “PL promoter” refers to the lambda promoter left. PL is a strong promoter that is repressed by the cI repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30ºC the cI857 repressor is functional and it represses gene expression, but at 37-42 ºC the repressor is inactivated so expression of the gene ensues.

[0077] As used herein “PL (OL1 G to T) promoter” refers to the lambda promoter left with an OL1 G to T mutation. PL is a strong promoter that is repressed by the cI repressor binding to OL1, OL2 and OL3 repressor binding sites. The temperature sensitive cI857 repressor allows control of gene expression by heat induction since at 30 ºC the cI857 repressor is functional and it represses gene expression, but at 37-42 ºC the repressor is inactivated so expression of the gene ensues. The cI repressor binding to OL1 is reduced by the OL1 G to T mutation resulting in increased promoter activity at 30 ºC and 37-42 ºC as described in WO 2014 / 035457.

[0078] As used herein “plasmid” refers to an extra chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently from the chromosomal DNA.

[0079] As used herein “plasmid copy number” refers to the number of copies of plasmid per cell. Increases in plasmid copy number indicate an increase in plasmid production yield.

[0080] As used herein “Pol” refers to polymerase.

[0081] As used herein “Pol I” refers to E. coli DNA Polymerase I.

[0082] As used herein “Pol III” refers to E. coli DNA Polymerase III.

[0083] As used herein “Pol III dependent origin of replication” refers to a replication origin that doesn’t require Pol I, for example the rep protein dependent R6K gamma replication origin. Numerous additional Pol III dependent replication origins are known in the art, many of which are summarized in del Solar et al., Supra, 1998 which is included herein by reference.

[0084] As used herein “polyA” refers to a polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. The polyadenylation signal contains the sequence motif recognized by the RNA cleavage complex. Most human polyadenylation signals contain an AAUAAA motif and conserved sequences 5’ and 3’ to it. Commonly utilized polyA signals are derived from the rabbit β globin, bovine growth hormone, SV40 early, or SV40 late polyA signals.

[0085] As used herein a “polyA repeat” refers to a consecutive sequence of adenine nucleotides as a direct repeat. Similarly, a “polyG repeat” refers to a consecutive sequence of guanine nucleotides as a direct repeat, a “polyC repeat” refers to a consecutive sequence of cytosine nucleotides as a direct repeat, and a “polyT repeat” refers to a consecutive sequence of thymine nucleotides as a direct repeat. An “mRNA vector” contains polyA repeats.

[0086] As used herein “pUC origin” refers to a pBR322-derived replication origin, with G to A transition that increases copy number at elevated temperature and deletion of the ROP negative regulator.

[0087] As used herein “pUC free” refers to a plasmid that does not contain the pUC origin.

[0088] As used herein “pUC plasmid” refers to a plasmid containing the pUC origin.

[0089] As used herein “R6K plasmid” refers to a plasmid with a R6K or R6K-derived origin of replication such as NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a- O1, NTC9385R2a-O2, NTC9385R2b-O1, NTC9385R2b-O2, NTC9385Ra-O1, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors as well as modifications and alternative vectors containing a R6K replication origin that were described in WO 2014 / 035457 and WO2019 / 183248. Alternative R6K vectors known in the art including, but not limited to, pCOR vectors (Gencell), pCpGfree vectors (Invivogen), and CpG free University of Oxford vectors including pGM169.

[0090] As used herein “R6K replication origin” refers to a region which is specifically recognized by the R6K Rep protein to initiate DNA replication, including, but not limited to, R6K gamma replication origin sequence (SEQ ID NOs: 43-44, 46 and 60, respectively). Also included are CpG free versions as described in Drocourt et al., United States Patent 7244609, which is incorporated herein by reference (SEQ ID NO: 63).

[0091] As used herein “R6K replication origin-RNA-OUT bacterial origin” contains a R6K replication origin for propagation and the RNA-OUT selectable marker (SEQ ID NOs: 50-59, respectively).

[0092] As used herein “Rep protein dependent plasmid” refers to a plasmid in which replication is dependent on a replication (Rep) protein provided in Trans. For example, R6K replication origin, ColE2-P9 replication origin and ColE2 related replication origin plasmids in which the Rep protein is expressed from the host strain genome. Numerous additional Rep protein dependent plasmids are known in the art, many of which are summarized in del Solar et al., Supra, 1998, Microbiol. Mol. Biol. Rev. 62:44-464 which is incorporated herein by reference.

[0093] As used herein “retroviral vector” refers to integrative viral vector that can infect dividing cells. Also call transfer plasmid. Plasmid encodes Retroviral LTR flanked expression unit. Transfer plasmid is transfected into production cells along with envelope and packaging plasmids required to make viral particles.

[0094] As used herein “retroviral envelope vector” refers to a plasmid encoding envelope glycoprotein.

[0095] As used herein “retroviral packaging vector” refers to a plasmid that encodes retroviral gag and pol genes required to package the retroviral transfer vector.

[0096] As used herein “RNA-IN” refers to an insertion sequence 10 (IS10) 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.

[0097] As used herein “RNA-IN regulated selectable marker” refers to a genomically expressed RNA-IN regulated selectable marker. In the presence of plasmid borne RNA-OUT antisense repressor RNA (SEQ ID NO: 48), expression of a protein encoded downstream of RNA-IN (e.g. having sequence gccaaaaatcaataatcagacaacaagatg (SEQ ID NO: 68)) is repressed. An RNA-IN regulated selectable marker is configured such that RNA-IN regulates either 1) a protein that is lethal or toxic to said cell per se or by generating a toxic substance (e.g., SacB), or 2) a repressor protein that is lethal or toxic to said bacterial cell by repressing the transcription of a gene that is essential for growth of said cell (e.g. murA essential gene regulated by 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 selection markers described in the art may be substituted for SacB.

[0098] As used herein “RNA-OUT” refers to an insertion sequence 10 (IS10) encoded RNA-OUT, an antisense RNA that hybridizes to, and reduces translation of, the transposon gene expressed downstream of RNA-IN. The sequence of the RNA-OUT RNA (SEQ ID NO: 48) and complementary RNA-IN SacB genomically expressed RNA-IN-SacB cell lines 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.

[0099] As used herein “RNA-OUT selectable marker” refers to an RNA-OUT selectable marker DNA fragment including E. coli transcription promoter and terminator sequences flanking an RNA-OUT RNA. An RNA-OUT selectable marker, utilizing the RNA-OUT promoter and terminator sequences, that is flanked by DraIII and KpnI restriction enzyme sites, and designer genomically expressed RNA-IN-SacB cell lines for RNA-OUT plasmid propagation, are described in WO 2008 / 153733 and included herein by reference. The RNA-OUT promoter and terminator sequences that flank the RNA-OUT RNA 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 / 66 / 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: 49. 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 propagationdescribed 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.

[0100] As used herein “RNA selectable marker” refers to a plasmid borne expressed non- translated RNA that regulates a chromosomally expressed target gene to afford selection. This may be a plasmid borne nonsense suppressing tRNA that regulates a nonsense suppressible selectable chromosomal target as described by Crouzet J and Soubrier F 2005 US Patent 6,977,174 included herein by reference. This may also be a plasmid borne antisense repressor RNA, a non-limiting list included herein by reference includes RNA-OUT that represses RNA-IN regulated targets (WO 2008 / 153733), pMB1 plasmid origin encoded RNAI that represses RNAII regulated targets (Grabherr R, Pfaffenzeller I. 2006 US patent publication US20060063232; Cranenburgh RM. 2009; US Patent 7,611,883), IncB plasmid pMU720 origin encoded RNAI that represses RNA II regulated targets (Wilson IW, Siemering KR, Praszkier J, Pittard AJ. 1997. J Bacteriol 179:742- 53), ParB locus Sok of plasmid R1 that represses Hok regulated targets, Flm locus FlmB of F plasmid that represses flmA regulated targets (Morsey MA, 1999 US patent US5922583). An RNA selectable marker may be another natural antisense repressor RNAs known in the art such as those described in Wagner EGH, Altuvia S, Romby P. 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 D, Yoo SM, Chung H, Park H, Park JH, Lee SY.2013. Nat Biotechnol 31:170- 4. An RNA selectable marker may also be an engineered repressor RNA as part of a selectablemarker that represses a target RNA fused to a target gene to be regulated such as SacB as described in US 2015 / 0275221.

[0101] 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.

[0102] As used herein “selectable marker” or “selection marker” refer to a selectable marker, for example, a kanamycin resistance gene or a RNA selectable marker.

[0103] As used herein “shRNA” refers to short hairpin RNA.

[0104] As used herein “Sleeping Beauty Transposon” refers to a transposon system that integrates an IR / DR flanked SB transposon into the genome by a simple cut and paste mechanism mediated by SB transposase. The transposon vector typically contains a promoter-transgene-polyA expression cassette between the IR / DRs which is excised and integrated into the genome.

[0105] As used herein “spacer region” refers to the region linking the 5’ and 3’ ends of the eukaryotic region sequences. The eukaryotic region 5’ and 3’ ends are typically separated by the bacterial replication origin and bacterial selectable marker in plasmid vectors (bacterial region) so many spacer regions consist of the bacterial region. In Pol III dependent origin of replication vectors of the invention, this spacer region preferably is less than 1000 bp.

[0106] As used herein “structured DNA sequence” refers to a DNA sequence that is capable of forming replication inhibiting secondary structures (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71:13-35). This includes but is not limited to inverted repeats, palindromes, direct repeats, IR / DRs, homopolymeric repeats or repeat containing eukaryotic promoter enhancers, or repeat containing eukaryotic origin of replications.

[0107] As used herein “SV40 origin” refers to Simian Virus 40 genomic DNA that contains the origin of replication.

[0108] As used herein “SV40 enhancer” refers to Simian Virus 40 genomic DNA that contains the 72 bp and optionally the 21 bp enhancer repeats.

[0109] As used herein “TE Buffer” refers to a solution containing approximately 10mM Tris pH 8 and 1 mM EDTA.

[0110] As used herein “TetR” refers to a tetracycline resistance gene.

[0111] As used herein “transfection” refers to a method to deliver nucleic acids into cells [e.g. poly(lactide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes, virosomes, block copolymers, Pluronic block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleofection, piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound, SQZ high speed cell deformation mediated membrane disruption, corona plasma, plasma facilitated delivery, tissue tolerable plasma, laser microporation, shock wave energy, magnetic fields, contactless magneto-permeabilization, gene gun, microneedles, microdermabrasion, hydrodynamic delivery, high pressure tail vein injection, etc] as known in the art and included herein by reference. Transfection of DNA into E. coli, commonly called transformation, is typically performed using chemical competent E. coli or electrocompetent E. coli cells using standard methodologies as known in the art and included herein by reference.

[0112] As used herein “transgene” refers to a gene of interest that is cloned into a vector for expression in a target organism.

[0113] As used herein “transposase vector” refers to a vector which encodes a transposase.

[0114] As used herein “transposon vector” refers to a vector which encodes a transposon which is a substrate for transposase-mediated gene integration.

[0115] As used herein “ts” means temperature-sensitive.

[0116] As used herein “UTR” refers to an untranslated region of mRNA (5’ or 3’ to the coding region).

[0117] As used herein “vector” refers to a gene delivery vehicle, including viral (e.g. Alphavirus, Poxvirus, Lentivirus, Retrovirus, Adenovirus, Adenovirus related virus, etc.) and non-viral (e.g. plasmid, MIDGE, transcriptionally active PCR fragment, minicircles, bacteriophage, NanoplasmidTM, etc.) vectors. These are well known in the art and are included herein by reference.

[0118] As used herein “vector backbone” refers to the eukaryotic and bacterial region of a vector, without the transgene or target antigen coding region. Engineered Bacterial Host Cells

[0119] The present disclosure is directed to engineered bacterial host strains with improved stability and replication of transfected plasmids, including, but not limited to those plasmids containing structured DNA sequences such as ITRs and AAV vectors. The engineered bacterial host strains comprise at least one gene knockout selected from the group consisting of SbcC and SbcD, and at least one gene knockout selected from the group consisting of endA, recA and pgi. The engineered bacterial host strains may also comprise a gene knockout of SbcC, SbcD, endA, recA, and pgi.

[0120] In certain embodiments, the engineered bacterial host cells do not include an engineered viability- or yield-reducing mutation in any of sbcB, recB, recD, and recJ and, optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr and combinations thereof. In certain embodiments, the engineered bacterial host cells do not include a viability- or yield-reducing mutation in any of sbcB, recB, recD, and recJ and, optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr and combinations thereof. In certain embodiments, the engineered bacterial host cells include sbcB,recB, recD, and recJ genes, and, optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr and combinations thereof, wherein the sbcB, recB, recD, and recJ genes, and, optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr and combinations thereof, if present, encodes a functional gene product.

[0121] In certain embodiments, the engineered bacterial host cells are derived from an Escherichia coli (E. coli) cell line and in some embodiments, are isogenic to the cell line / strain from which it is derived. In certain embodiments, the E. coli cell line is selected from the group consisting of DH5α, DH1, JM107, JM108, JM109, XL1Blue, and MG1655.

[0122] In some embodiments, engineered bacterial host cell includes a gene knockout of one or both of the SbcC and SbcD genes and is otherwise isogenic to the strain from which it is derived, the strain from which it is derived being selected from the group consisting of MG1655, MG1655 ΔendA ΔrecA, and MG1655 ΔendA ΔrecA Δpgi (GalG20). To the extent not inconsistent with any of the foregoing embodiments, the engineered E. coli host cell can further not include an engineered viability- or yield-reducing mutation in any or all of sbcB, recB, recD, and recJ, and at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof. Thus, in some embodiments, the engineered E. coli host cell further does not include an engineered viability- or yield-reducing mutation, engineered mutation, or any mutation in uvrC. In other embodiments, the engineered E. coli host cell further does not include an engineered viability- or yield-reducing mutation, engineered mutation, or any mutation in mcrA. In still other embodiments, the engineered E. coli host cell further does not include an engineered viability- or yield-reducing mutation, engineered mutation, or any mutation in mcrBC-hsd-mrr. In yet other embodiment, the engineered E. coli host cell further does not include an engineered viability- or yield-reducing mutation, engineered mutation, or any mutation in mcrA and mrBC-hsd-mrr.

[0123] To the extent not inconsistent with any of the foregoing embodiments, the engineered E. coli host cell can further not include a viability- or yield-reducing mutation in any or all of sbcB, recB, recD, and recJ, and at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof. Thus, in some embodiments, the engineered E. coli host cell further does not include a viability- or yield-reducing mutation, engineered mutation, or any mutation in uvrC. In other embodiments, the engineered E. coli host cell further does not include a viability- or yield-reducing mutation, engineered mutation, or any mutation in mcrA. In still other embodiments, the engineered E. coli host cell further does not include a viability- or yield-reducing mutation, engineered mutation, or any mutation in mcrBC-hsd-mrr. In yet other embodiment, the engineered E. coli host cell further does not include a viability- or yield-reducing mutation, engineered mutation, or any mutation in mcrA and mrBC-hsd-mrr.

[0124] Additionally, to the extent not inconsistent with any of the foregoing embodiments, the engineered bacterial host cell includes sbcB, recB, recD, and recJ genes, and, optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr and combinations thereof, wherein the sbcB, recB, recD, and recJ genes, and, optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr and combinations thereof, if present, encodes a functional gene product. Thus, in some embodiments, the engineered E. coli host cell includes a sbcB gene, wherein the sbcB gene encodes a functional gene product. In some embodiments, the engineered E. coli host cell includes a recB gene, wherein the recB gene encodes a functional gene product. In some embodiments, the engineered E. coli host cell includes a recD gene, wherein the recD gene encodes a functional gene product. In some embodiments, the engineered E. coli host cell includes a recJ gene, wherein the recJ gene encodes a functional gene product. Additional, in some embodiments, the engineered further includes an uvrC gene, wherein the uvrC gene encodes a functional gene product. In other embodiments, the engineered E. colihost cell includes a mcrA gene, wherein the mcrA gene encodes a functional gene product. In still other embodiments, the engineered E. coli host cell further includes a mcrBC-hsd-mrr gene operon, wherein the mcrBC-hsd-mrr gene operon encodes a functional gene product. In yet other embodiment, the engineered E. coli host cell further includes a mcrA gene and an mrBC-hsd-mrr gene operon, wherein the mcrA gene encodes a functional gene product and the mrBC-hsd-mrr gene operon encodes a functional gene product.

[0125] In any of the foregoing embodiments, the gene knockout of the engineered bacterial host cell can be a knockout of SbcC. Alternatively, in some embodiments, the gene knockout of the engineered bacterial host cell can be a knockout of SbcD. In any of the foregoing embodiments, the gene knockout of the engineered bacterial host cell can be a knockout of both SbcC and SbcD.

[0126] In any of the foregoing embodiments, the engineered bacterial host cell can further include a genomic antibiotic resistance marker. By way of example, but not limitation, the genomic antibiotic resistance marker can be kanR comprising a sequence having at least 90%, at least 95%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23 (kanR, 795 bp). By way of further example, but not limitation, the genomic antibiotic resistance marker can be kanR comprising a sequence encoding a protein having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36 (kanR). By way of still further example, the genomic antibiotic resistance marker can be a chloramphenicol resistance marker, gentamicin resistance marker, kanamycin resistance marker, spectinomycin and streptomycin resistance marker, trimethoprim resistance marker, or a tetracycline resistance marker. Alternatively, in any of the foregoing embodiments, the bacterial host cell cannot include a genomic antibiotic resistance marker.

[0127] In any of the foregoing embodiments, the engineered bacterial host cell can further include a Rep protein suitable for culturing a Rep protein dependent plasmid. By way of example, but not limitation, the engineered bacterial host cell can include a genomic nucleic acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 26 (P42L-P106I-F107S-P113S, 918 bp), SEQ ID NO: 27 (P42L-Δ106-107-P113S, 912 bp), SEQ ID NO: 28 (P42L-P106L-F107S, 918 bp), and SEQ ID NO: 29 (P42L-P113S, 918 bp). By way of further example, but not limitation, the engineered bacterial host cell can include a genomic nucleic acid sequence encoding a Rep protein having at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39 (P42L-P106I-F107S-P113S), SEQ ID NO: 40 (P42L-Δ106-107-P113S), SEQ ID NO: 42 (P42L-P106L-F107S), SEQ ID NO: 41 (P42L-P113S), SEQ ID NO: 34 (ColE2 wild-type), SEQ ID NO: 35 (ColE2 mutant G194D). 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 a PL promoter and that such PL promoter can enable temperature-sensitive expression of the Rep protein if there is a lambda repressor present in the genome, such as cITs857. 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 to ttgacataaa taccactggc ggtgatact (PL promoter (-35 to -10); SEQ ID NO: 68), ttgacataaa taccactggc gtgatact (PL promoter OL1-G (-35 to -10); SEQ ID NO: 69), or ttgacataaa taccactggc gttgatact (PL promoter OL1-G to T (-35 to -10); SEQ ID NO: 70). It should be further understood that where the Rep protein is a R6K Rep protein such as SEQ ID NOs: 39-42, a vector that is transfected into the engineered bacterial host cell can contain a R6K origin of replication and, alternatively, wherethe Rep protein is a ColE2 Rep protein, a vector that is transfected into the engineered bacterial host cell can contain a ColE2 origin of replication.

[0128] In any of the foregoing embodiments, the engineered bacterial host cell can further include a genomic nucleic acid sequence encoding a genomically expressed RNA-IN regulated selectable marker. By way of example, but not limitation, the engineered bacterial host cell can include a genomic nucleic acid sequence (which encodes the selectable marker) that has at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25 (SacB, 1422 bp). By way of further example, but not limitation, the engineered bacterial host cell can include a genomic nucleic acid sequence that encodes the selectable marker which has an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 38 (SacB). By way of still further example, but not limitation, the engineered bacterial host cell can include a RNA-IN regulated selectable marker having an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 38 (SacB). In any of the foregoing embodiments, the RNA-IN regulated selectable marker can be downstream of an RNA-IN having the sequence gccaaaaatcaataatcagacaacaagatg (SEQ ID NO: 68); in embodiments where this RNA-IN is used, the corresponding RNA-OUT in a vector can be that of SEQ ID NO: 48. Thus, for SacB, the RNA-IN SacB sequence can be gccaaaaatcaataatcagacaacaagatgaacatcaaaaagtttgcaaaacaagcaacagtattaacctttactaccgcactgctggcagg aggcgcaactcaagcgtttgcgaaagaaacgaaccaaaagccatataaggaaacatacggcatttcccatattacacgccatgatatgctg caaatccctgaacagcaaaaaaatgaaaaatatcaagttcctgaattcgattcgtccacaattaaaaatatctcttctgcaaaaggcctggacg tttgggacagctggccattacaaaacgctgacggcactgtcgcaaactatcacggctaccacatcgtctttgcattagccggagatcctaaaa atgcggatgacacatcgatttacatgttctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctggccgcgtctttaaagaca gcgacaaattcgatgcaaatgattctatcctaaaagaccaaacacaagaatggtcaggttcagccacatttacatctgacggaaaaatccgtttattctacactgatttctccggtaaacattacggcaaacaaacactgacaactgcacaagttaacgtatcagcatcagacagctctttgaacatc aacggtgtagaggattataaatcaatctttgacggtgacggaaaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctca ggcgacaaccatacgctgagagatcctcactacgtagaagataaaggccacaaatacttagtatttgaagcaaacactggaactgaagatg gctaccaaggcgaagaatctttatttaacaaagcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaacttctgcaaagcg ataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacgatgattacacactgaaaaaagtgatgaaaccgct gattgcatctaacacagtaacagatgaaattgaacgcgcgaacgtctttaaaatgaacggcaaatggtacctgttcactgactcccgcggatc aaaaatgacgattgacggcattacgtctaacgatatttacatgcttggttatgtttctaattctttaactggcccatacaagccgctgaacaaaact ggccttgtgttaaaaatggatcttgatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagcgaaaggaaacaatgtcgtgatt acaagctatatgacaaacagaggattctacgcagacaaacaatcaacgtttgcgccaagcttcctgctgaacatcaaaggcaagaaaacat ctgttgtcaaagacagcatccttgaacaaggacaattaacagttaacaaataa (SEQ ID NO: 71). It should be understood that any suitable RNA-IN regulated selected marker and RNA-IN can be used and these are known in the art.

[0129] In any of the foregoing embodiments, the engineered bacterial host 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. 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: 24 (cITs857, 714 bp). By way of further example, but not limitation, the engineered bacterial host cell can further include a genomic nucleic acid sequence encoding cITs857 having an amino acid sequence with at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37 (cITs857). By way of still further example, but not limitation, the engineered bacterial host cell can further include a temperature-sensitive lambda repressor having an amino acid sequencewith at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37 (cITs857). In any of the foregoing embodiments, where the engineered bacterial host cell further includes a genomic nucleic acid sequence encoding a temperature-sensitive lambda repressor, the temperature-sensitive lambda repressor can be a phage φ80 attachment site chromosomally integrated copy of an arabinose inducible CITs857 gene. By way of example, but not limitation, the cITs857 gene can be under the control of the pBAD promoter to provide arabinose inducibility (pBAD promoter, ctgcataatgtgcctgtcaaatggacgaagcagggattctgcaaaccctatgctactccgtcaagccgtcaattgtctgattcgttaccaattat gacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgaga aatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacg ttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgc gacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcg actcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgtt aatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaa gccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctc tcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattga gaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggca ttaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattg catcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcggga ccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgctatg ccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgtttttttggctcgactaga aataattttgtttaactttaagaaggagatataacc, SEQ ID NO: 72).

[0130] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆SbcDC::kanR.

[0131] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆SbcDC.

[0132] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆dcm ∆SbcDC::kanR

[0133] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆dcm ∆SbcDC

[0134] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; ∆SbcDC::kanR.

[0135] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; ∆SbcDC.

[0136] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC::kanR.

[0137] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC.

[0138] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC::kanR ∆dcm.

[0139] In some embodiments, an engineered bacterial host cell is provided having the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC ∆dcm.

[0140] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)dcm-; ∆SbcDC.

[0141] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)dcm-; ∆SbcDC::kanR.

[0142] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC.

[0143] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC::kanR.

[0144] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attφ80::pARA- CI857ts Pc-RNA-IN- SacB, tetR; ∆SbcDC.

[0145] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attφ80::pARA- CI857ts Pc-RNA-IN- SacB, tetR; ∆SbcDC::kanR.

[0146] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts, tetR; ∆SbcDC.

[0147] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts, tetR; ∆SbcDC::kanR.

[0148] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts Pc-RNA- IN- SacB, tetR; ∆SbcDC.

[0149] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts Pc-RNA- IN- SacB, tetR; ∆SbcDC::kanR.

[0150] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20) dcm- attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA- CI857ts Pc-RNA-IN- SacB, tetR; ∆SbcDC.

[0151] In some embodiments, an engineered bacterial host cell is provided having the following genotype: MG1655 ΔendA ΔrecA Δpgi (GalG20) dcm- attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA- CI857ts Pc-RNA-IN- SacB, tetR; ∆SbcDC::kanR.

[0152] It should be understood that any of the above embodiments, to the extent applicable, could be incorporated into DH5α, DH1, JM107, JM108, JM109, and XL1Blue strains that include gene knockouts of endA, recA, pgi, SbcD and SbcC. It should also be understood that any of the above embodiments, to the extent applicable, could be incorporated into REVIVER-pUC strains that include gene knockouts of endA, recA, and pgi,

[0153] In any of the foregoing embodiments, the engineered bacterial host cell can further include a vector. By way of example, but not limitation, the vector can be a non-viral transposon vectorsuch as a transposase vector, a Sleeping Beauty transposon vector, a Sleeping Beauty transposase vector, a PiggyBac transposon vector, a PiggyBac transposase vector, an expression vector, and the like, a non-viral gene editing vector such as Homology-Directed Repair (HDR) / CRISPR-Cas9 vectors or a viral vector such as an AAV vector, an AAV rep cap vector, an AAV helper vector, an Ad helper vector, a Lentivirus vector, a Lentiviral envelope vector, a Lentiviral packaging vector, a Retroviral vector, a Retroviral envelope vector, a Retroviral packaging vector, a mRNA vector, or the like.

[0154] In any of the foregoing embodiments, where the bacterial host cell further includes a vector, the vector can include a nucleic acid sequence having a palindrome. A palindrome can be understood as a nucleic acid sequence in a double-stranded DNA molecule wherein reading in a certain direction on one strand matches the sequence reading in the opposite direction on the complementary strand, such that there are complementary portions along the one strand, where there is no intervening sequence between the complementary portions. By of example, but not limitation, the complementary sequences of the palindrome can each include about 10 to about 200 basepairs, about 15 and to about 200 basepairs, about 20 to about 200 basepairs, about 25 to about 200 basepairs, about 30 to about 200 basepairs, about 40 to about 200 basepairs, about 50 to about 200 basepairs, about 75 to about 200 basepairs, about 100 to about 200 base pairs, about 15 to about 200 basepairs, about 10 to about 150 basepairs, about 15 to about 150 basepairs, about 20 to about 150 base pairs, about 25 to about 150 basepairs, about 30 to about 150 basepairs, about 30 to about 150 basepairs, about 40 to about 150 basepairs, about 50 to about 150 basepairs, about 100 to about 150 base pairs, about 10 to about 140 basepairs, about 15 to about 140 basepairs, about 20 to about 140 basepairs, about 25 to about 140 basepairs, about 30 to about 140 basepairs, about 30 to about 140 basepairs, about 40 to about 140 basepairs, about 50 to about 140 basepairs,about 100 to about 140 basepairs, about 10 to about 100 basepairs, about 15 to about 100 basepairs, about 20 to about 100 basepairs, about 25 to about 100 base pairs, about 30 to about 100 basepairs, about 40 to about 100 basepairs, about 50 to about 100 basepairs, or about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 basepairs.

[0155] In any of the foregoing embodiments, where the bacterial host cell further includes a vector, the vector can include a nucleic acid sequence having at least one direct repeat. By way of example, but not limitation, the at least one direct repeat can include about 40 to 150 nucleotides, about 60 to about 120 nucleotides or about 90 nucleotides. By way of further example, but not limitation, the at least one direct repeat can be a simple repeat including a short sequence of DNA consisting of multiple repetitions of a single base, such as a polyA repeat, a polyT repeat, a polyC repeat or a polyG repeat, where the simple repeat includes about 40 to about 150 consecutive repeats of the same base, about 60 to about 120 consecutive repeats of the same base, or about 90 consecutive repeats of the same base. By way of further example, but not limitation, the polyA repeat can include 40 to 150 consecutive adenine nucleotides, 60 to 120 consecutive adenine nucleotides, or about 90 adenine nucleotides.

[0156] In any of the foregoing embodiment, where the bacterial host cell further includes a vector, the vector can include an inverted repeat sequence, a direct repeat sequence, a homopolymeric repeat sequence, a eukaryotic origin of replication, and a eukaryotic promoter enhancer sequence. By way of further example, the vector can include a sequence selected from the group consisting of a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR / DR repeat, a Sleeping Beauty transposon IR / DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer. By way of example, but not limitation, an AAV vector can contain an AAV ITR. In some embodiments, where the bacterial host cell further includes a vector, thevector can include a nucleic acid sequence having at least one inverted repeat sequence, which can also be an inverted terminal repeat such as, by way of example, but not limitation, an AAV ITR. Thus, in any of the foregoing embodiments, the vector can include an AAV ITR. It should be understood that an inverted repeat sequence is a single stranded sequence of nucleotides followed downstream by its reverse complement. It should be further understood that the single stranded sequence can be part of a double-stranded vector. The intervening sequence of nucleotides between the initial sequence and the reverse complement can be any length including zero. When the intervening length is zero, the composite sequence is a palindrome. When the intervening length is greater than zero, the composite sequence is an inverted repeat. In any of the foregoing embodiments, the intervening sequence can be 1 to about 2000 basepairs. By way of example, but not limitation, the inverted repeat, which can also be an inverted terminal repeat, can be separated by an intervening sequence comprising about 1 to about 2000 basepairs, about 5 to about 2000 basepairs, about 10 to about 2000 basepairs, about 25 to about 2000 basepairs, about 50 to about 2000 basepairs, about 100 to about 2000 basepairs, about 250 to about 2000 basepairs, about 500 to about 2000 basepairs, about 750 to about 2000 basepairs, about 1000 to about 2000 basepairs, about 1250 to about 2000 basepairs, about 1500 to about 2000 basepairs, about 1750 to about 2000 basepairs, about 1 to about 100 basepairs, about 1 to about 50 basepairs, about 1 to about 25 basepairs, about 1 to about 20 basepairs, about 1 to about 10 basepairs, about 1 to about 5 basepairs, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 basepairs. By of example, but not limitation, the complementary portions of the inverted repeat can each include about 10 to about 200 basepairs, about 15 and to about 200 basepairs, about 20 to about 200 basepairs, about 25 to about 200 basepairs, about 30 to about 200 basepairs, about 40 to about200 basepairs, about 50 to about 200 basepairs, about 75 to about 200 basepairs, about 100 to about 200 base pairs, about 15 to about 200 basepairs, about 10 to about 150 basepairs, about 15 to about 150 basepairs, about 20 to about 150 base pairs, about 25 to about 150 basepairs, about 30 to about 150 basepairs, about 30 to about 150 basepairs, about 40 to about 150 basepairs, about 50 to about 150 basepairs, about 100 to about 150 base pairs, about 10 to about 140 basepairs, about 15 to about 140 basepairs, about 20 to about 140 basepairs, about 25 to about 140 basepairs, about 30 to about 140 basepairs, about 30 to about 140 basepairs, about 40 to about 140 basepairs, about 50 to about 140 basepairs, about 100 to about 140 basepairs, about 10 to about 100 basepairs, about 15 to about 100 basepairs, about 20 to about 100 basepairs, about 25 to about 100 base pairs, about 30 to about 100 basepairs, about 40 to about 100 basepairs, about 50 to about 100 basepairs, or about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 basepairs. By way of example, but not limitation, the at least one inverted repeat can include an AAV ITR repeat that comprises sequences having at least 95%, at least 95%, at least 98%, at least 99% or 100% sequence identity to ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctc agtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct (5’ AAV ITR; SEQ ID NO: 73) and aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccg ggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa (3’ AAV ITR; SEQ ID NO: 74).

[0157] Alternatively, in any of the foregoing embodiments, where the bacterial host cell further includes a vector, the vector cannot include a nucleic acid sequence having a palindrome, direct repeat, or inverted repeat.

[0158] In any of the foregoing embodiments, the vector can be an AAV vector. In some embodiments, where the vector is an AAV vector, the AAV vector comprises an AAV ITR. Inother embodiments, the vector can be a lentiviral vector, lentiviral envelope vector or lentiviral packaging vector. In still other embodiments, the vector can be a retroviral vector, retroviral envelope vector or a retroviral packaging vector. In yet other embodiments, the vector can be a transposase vector or a transposon vector. In still further embodiments, the vector can be an mRNA vector. By way of example, but not limitation, the mRNA vector can include a polyA repeat as described in the present disclosure.

[0159] In any of the foregoing embodiments, the vector can be a plasmid. In any of the foregoing embodiments, the vector can be a Rep protein dependent plasmid.

[0160] In any of the foregoing embodiments, the vector can further include a RNA selectable marker. By way of example, but not limitation, the RNA selectable marker can be a RNA-OUT. 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 consisting of SEQ ID NO: 47 (gtagaattgg taaagagagt cgtgtaaaat atcgagttcg cacatcttgt tgtctgatta ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatg attttgataa aaatcatta) and SEQ ID NO: 49 (gtagaattgg taaagagagt tgtgtaaaat attgagttcg cacatcttgt tgtctgatta ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatg attttgataa aaatcatta), respectively. In some embodiments, the engineered bacterial host cell can include a corresponding RNA-IN sequence to permit regulation of a downstream marker by the RNA-OUT and that the RNA-OUT sequence corresponds to the RNA- IN.

[0161] In any of the foregoing embodiments, the vector can further include 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: 48.

[0162] In any of the foregoing embodiments, the vector can further include a bacterial origin of replication. By way of example, but not limitation, the bacterial origin of replication can be selected from the group consisting of R6K, pUC and ColE2. By way of further example, but not limitation, the bacterial origin of replication can be a R6K gamma replication origin with at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 43 (ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacat gagagcttag tacgtactat caacaggttg aactgctgat c), SEQ ID NO: 44 (ggcttgttgt ccacaaccat taaaccttaa aagctttaaa agccttatat attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacattag ccatgagagc ttagtacatt agccatgagg gtttagttca ttaaacatga gagcttagta cattaaacat gagagcttag tacatactat caacaggttg aactgctgat c), SEQ ID NO: 45 (aaaccttaaa acctttaaaa gccttatata ttcttttttt tcttataaaa cttaaaacct tagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt acatgaaaca tgagagctta gtacattagc catgagagct tagtacatta gccatgaggg tttagttcat taaacatgag agcttagtac attaaacatg agagcttagt acatactatc aacaggttga actgctgatc), SEQ ID NO: 46 (tgtcagccgt taagtgttcc tgtgtcactg aaaattgctt tgagaggctc taagggcttc tcagtgcgtt acatccctgg cttgttgtcc acaaccgtta aaccttaaaa gctttaaaag ccttatatat tctttttttt cttataaaac ttaaaacctt agaggctatt taagttgctg atttatatta attttattgt tcaaacatga gagcttagta cgtgaaacat gagagcttag tacgttagcc atgagagctt agtacgttag ccatgagggt ttagttcgtt aaacatgaga gcttagtacg ttaaacatga gagcttagta cgtgaaacat gagagcttag tacgtactat caacaggttg aactgctgat cttcagatc) and SEQ ID NO: 60 (ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtact atcaacaggt tgaactgctg atc), SEQID NO: 30 (ColE2 Origin (+7), 45 bp), SEQ ID NO: 31 (ColE2 Origin (+7, CpG free), 45 bp), SEQ ID NO: 32 (ColE2 Origin (Min), 38 bp), SEQ ID NO: 33 (ColE2 Origin (+16), 60 bp), and SEQ ID NO: 22 (pUC, 784 bp).

[0163] In any of the foregoing embodiments, the engineered bacterial host cell can further include a eukaryotic pUC-free minicircle expression vector that can include: (i) a eukaryotic region sequence encoding a gene of interest and having 5’ and 3’ ends; and (ii) a spacer region having a length of less than 1000, preferably less than 500, basepairs that links the 5’ and 3’ ends of the eukaryotic region sequence and that comprises a R6K bacterial replication origin and a RNA selectable marker. By way of example, but not limitation, the R6K bacterial replication origin and RNA selectable marker can have sequences as described in the present disclosure and as known in the art. Alternatively, in any of the foregoing embodiments, the engineered bacterial cell can further include a covalently closed circular plasmid having a backbone including a Pol III- dependent R6K origin of replication and an RNA-OUT selectable marker, where the backbone is less than 1000 bp, preferably less than 500 bp, and an insert including a structured DNA sequence. By way of example, but not limitation, the structured DNA sequence can include a sequence selected from the group consisting of an inverted repeat sequence, a direct repeat sequence, a homopolymeric repeat sequence, an eukaryotic origin of replication, and a eukaryotic promoter enhancer sequence. By way of further example, the structured DNA sequence can include a sequence selected from the group consisting of a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR / DR repeat, a Sleeping Beauty transposon IR / DR repeat, an AAV ITR, a CMV enhancer, and a SV40 enhancer. By way of example, but not limitation, the insert can be a transposase vector, an AAV vector, or a lentiviral vector. By way of example, but not limitation the Pol III-dependent R6K origin of replication canhave a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 60. By way of example, but not limitation, the RNA-OUT selectable marker can be an RNA-IN regulating RNA-OUT functional variant with at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 47 or SEQ ID NO: 49. By way of further example, the RNA-OUT selectable marker can be 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: 48.

[0164] As disclosed herein, methods for the knockout or knockdown of a gene are well-known in the art, including, by way of example not limitation, the method disclosed in the Examples herein (recombineering), as well as P1 phage transduction, genome mass transfer, and CRISPR / Cas9. It should be understood that a gene knockout can result in either abolished expression of a protein or expression of a non-functional protein. Thus, the SbcCD complex may or may not be present in the bacterial host strains of the present disclosure, however, if present it is non-functional in the case of a knockout or has reduced activity as a nuclease in the case of a knockdown. It should be understood that embodiments of the disclosure can include a knockout or knockdown of SbcC, SbcD or both.

[0165] It is expected, without being bound to theory, that a knockout of SbcC or SbcD alone is sufficient to achieve the desired effect of the present invention because both proteins are essential subunits of the SbcCD nuclease (Connelly JC and Leach DR, Genes Cells 1:285, 1996). The sbcC and sbcD genes of bacterial encode a nuclease involved in palindrome inviability and genetic recombination. (Connelly JC and Leach DR, Genes Cells 1:285, 1996).Methods of Vector Production in Engineered Bacterial Host Cells

[0166] Vectors can be produced in the engineered bacterial host 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 HyperGROTMfermentation. Generally, this process comprises growing the engineered bacterial host 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 28-30oC and the higher temperature can be about 37-42oC. By way of example, the first portion can be about 12 hours and the second portion can be about 8 hours. It should be understood that where the fed-batch fermentation with a temperature upshift is used, the engineered bacterial host cell can have a lambda repressor and Rep protein that is under the control of a PLpromoter that can be regulated by the lambda repressor, which can be temperature-sensitive.

[0167] 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 the cell line from which the engineered bacterial host cell was derived 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.

[0168] 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.

[0169] The bacterial host strains and methods of the present disclosure will now be described with reference to the following non-limiting examples. EXAMPLES

[0170] In the following examples, for shake flask production proprietary Plasmid+®shake culture medium was used. The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 μg / mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates were grown at 30-32 °C; cells were resuspended in media and used to provide approximately 2.5 OD600 inoculums for the 500 mL Plasmid+®shake flasks that contained 50 μg / mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose to select for RNA-OUT plasmids. Flask were grown with shaking to saturation at the growth temperatures as indicated.

[0171] In the following examples, HyperGROTMfermentations were performed using proprietary fed-batch media (NTC3019, HyperGROTMmedia) in New Brunswick BioFlo 110 bioreactors as described in U.S. Patent No. 7,943,377. The seed cultures were started from glycerol stocks or colonies and streaked onto LB medium agar plates containing 50 μg / mL antibiotic (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates were grown at 30-32 °C; cells were resuspended in media and used to provide approximately 0.1% inoculums for the fermentations that contained 50 μg / mL antibiotic for ampR or kanR selection plasmids or 0.5% sucrose for RNA-OUT plasmids. HyperGROTMtemperature shifts were as indicated.

[0172] In the following examples, culture samples were taken at key points and regular intervals during all fermentations. Samples were analyzed immediately for biomass (OD600) and for plasmid yield. Where plasmid yield was determined, the analysis was performed by quantification of plasmid obtained from Qiagen Spin Miniprep Kit preparations as described in U.S. Patent No. 7,943,377. Briefly, cells were alkaline lysed, clarified, plasmid was column purified, and eluted prior to quantification. Plasmid quality was determined by agarose gel electrophoresis analysis (AGE) and was performed on 0.8-1% Tris / acetate / EDTA (TAE) gels as described in U.S. Patent No. 7,943,377. EXAMPLE 1: Preparation of SbcCD Knockout Strains

[0173] SbcCD knockout strains were produced using Red Gam recombination cloning as described in Datsenko and Wanner, PNAS USA 97:6640-6645 (2000). The pKD4 plasmid (Datsenko and Wanner, 2000) was PCR amplified with the following primers to introduce SbcC and SbcD targeting homology arms. SEQ ID NO 1 (SbccR-pKD4): CCCTCTGTATTCATTATCCTGCTGAATAGTTATTTCACTGCAAACGTACTCATA TGAATATCCTCCTTAG SEQ ID NO 2 (SbcdF-pKD4): TCTGTTTGGGTATAATCGCGCCCATGCTTTTTCGCCAGGGAACCGTTATGTGT AGGCTGGAGCTGCTTCG

[0174] The 1.6 kb PCR product (SEQ ID NO: 5)(FIG. 1A) was purified and DpnI digested (to eliminate template plasmid). The host strain in which the SbcCD genes were to be knocked out was transformed with pKD46-RecA recombineering plasmid (WO 2008 / 153731, which is incorporated by reference herein in its entirety) and transformants selected for ampicillinresistance. Electrocompetent cells of the transformed cell line were made by growth in LB medium including 50 μg / mL ampicillin, at approximately 0.05 OD600, arabinose was added to 0.2% to induce recombineering gene expression, the cells were grown to mid-log phase and electrocompetent cells made by centrifugation and resuspension in 10% glycerol at 1 / 200 original volume. 5 μL of DpnI-digested, purified PCR product was electroporated into 25 μL electrocompetent cells after which 1 mL of SOC medium was added. The cells were outgrown for 2 hours at 30 °C, plated on LB agar plates containing 20 μg kanamycin and grown at 37 °C overnight. Individual kanR colonies were screened for ΔSbcDC::kanR by using SbcDF and SbcCR primers as described below. SEQ ID NO 3 (SbcDF primer): cgtctcgccatgatttgccctg SEQ ID NO 4 (SbcCR primer): cgttatgcgccagctccgtgag Host: Product of SbcDF and SbcCR primers = 4.8 kb (FIG. 1B) (SEQ ID NO: 6) Host ΔSbcDC::kanR: Product of SbcDF and SbcCR primers = 1.9 kb (FIG. 1C) (SEQ ID NO: 7)

[0175] The temperature-sensitive pKD46-recApa plasmid was cured from the cell lines by growing at 37-42 °C. Ampicillin sensitivity of the individual kanR colonies was also verified.

[0176] For host strains for antibiotic resistance plasmids (e.g. pUC replication origin; antibiotic selection; R6K replication origin; antibiotic selection) the kanR chromosomal marker was removed from ∆SbcDC::kanR using FRT recombination as described (Datsenko and Wanner, Supra, 2000). Briefly the ∆SbcDC::kanR cell line was transformed with pCP20 FRT plasmid (Datsenko and Wanner, Supra, 2000) and transformants grown at 30 °C and selected for ampicillin resistance. Individual colonies were streaked for single colonies on LB medium plates (without ampicillin) and grown at 43 °C to cure the temperature sensitive pCP20 plasmid. Single colonieson the 43 °C LB plate were streaked on LB amp and LB kan plates to verify loss of ampR pCP20 plasmid and kanR excision respectively. Individual amp and kan sensitive colonies were screened for ∆SbcDC by PCR using SbcDF and SbcCR primers (FIG. 1D). For the PCR product of the SbcDF primer and SbcCR primer, the size was 0.53 kb as shown in FIG. 1D (SEQ ID NO: 8).

[0177] Alternative genes can be deleted using Red Gam recombination with pKD4 gene specific PCR products, and pKD4 kanR chromosomal marker removed using pCP20 mediated FRT excision as described above. Example genes deleted in this manner include dcm and fhuA. pUC Replication Origin For MG1655 ΔendA ΔrecA Δpgi (GalG20)

[0178] The starting strain had the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi, and is referred to as GalG20. Following knockout of SbcCD and kanR excision, the knockout strain (GalG20 ∆SbcDC) has the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi∆SbcDC. This strain can be used for the production of plasmids having a pUC bacterial origin of replication. pUC Replication Origin

[0179] An additional strain was produced from GalG20 ∆SbcDC. Following knockout of dcm and kanR excision, the knockout strain (GalG20 ∆SbcDC ∆dcm) has the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆SbcDC ∆dcm (GALG20-REVIVER-pUC). This strain can be used for the production of plasmids having a pUC bacterial origin of replication.

[0180] Additional ∆SbcDC strains for production of pUC replication origin constructed in this manner include DH5α ∆SbcDC disclosed in WO 2021 / 183827 (starting strain DH5α; ∆SbcDC strain genotype F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ- thi-1 gyrA96 relA1 ∆SbcDC) and E. cloni 5-alpha ∆SbcDC Δdcm (starting strainE. cloni 5-alpha; ∆SbcDC strain genotype F- fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 ∆SbcDC Δdcm). R6K Replication Origin

[0181] An additional strain was produced from GalG20 ∆SbcDC ∆dcm by integrating a heat- inducible R6K rep protein cassette (attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3- ), SpecR StrepR) into the host genome as described in WO 2014 / 035457 to yield a new strain, GalG20 R6K Rep ∆SbcDC ∆dcm, which will have the genotype: GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; ∆SbcDC ∆dcm. This strain can be used for the production of plasmids having a R6K bacterial origin of replication. R6K Replication Origin with RNA-OUT Selection

[0182] An additional strain was produced from GalG20 attHK022::pL (OL1-G to T) P42L-P106I- F107S P113S (P3-), SpecR StrepR; ∆SbcDC ∆dcm by integrating a constitutively expressed RNA- IN sacB cassette (Pc-RNA-IN-SacB, catR;) into the host genome as described in WO 2014 / 035457 to yield a new strain which has the genotype GalG20 attHK022::pL (OL1-G to T) P42L-P106I- F107S P113S (P3-), SpecR StrepR; attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC ∆dcm. An additional strain was produced from GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC ∆dcm by integrating an arabinose inducible CI857ts lambda repressor that includes a second copy of the RNA-IN-SacB expression cassette (::pARA-CI857ts, tetR) into the host genome as described in WO 2014 / 035457 to yield a new strain which has the genotype GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attλ:: Pc-RNA-IN-SacB, catR; attφ80::pARA-CI857ts, tetR; ∆SbcDC ∆dcm (GALG20-REVIVER-R6K). This strain can be used for the production of NanoplasmidTMvectors having an R6K bacterial origin of replication and RNA-OUT antibiotic free selection marker.Additional ∆SbcDC strains for production of R6K replication origin / RNA-OUT vectors constructed in this manner include DH5α ∆SbcDC (REVIVER-NP) disclosed in WO 2021 / 183827 (starting strain DH5α ∆SbcDC; final REVIVER-NP strain genotype F- φ80lacZΔM15 Δ(lacZYA- argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ- thi-1 gyrA96 relA1 ∆SbcDC; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; 2) attλ:: Pc-RNA- IN-SacB, catR; and 3) attφ80::pARA-CI857ts, tetR) and E. cloni 5-alpha ∆SbcDC Δdcm (REVIVER-R6K) (starting strain E. cloni 5-alpha; final REVIVER-R6K strain genotype F- ΔfhuA Δ(argF-lacZ)U169 phoA ΔPhoE glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; 2) attλ:: Pc- RNA-IN-SacB, catR; and 3) attφ80::pARA-CI857ts, tetR). pUC Replication Origin with RNA-OUT Selection

[0183] An additional strain may be produced from GalG20 ∆SbcDC ∆dcm by integrating a constitutively expressed RNA-IN sacB cassette (Pc-RNA-IN-SacB, catR;) into the host genome as described in WO 2014 / 035457 to yield a new strain with the genotype GalG20 attλ:: Pc-RNA- IN-SacB, catR; ∆SbcDC ∆dcm. An additional strain may be produced from GalG20 ∆SbcDC by integrating a constitutively expressed RNA-IN sacB cassette (Pc-RNA-IN-SacB, catR;) into the host genome as described in WO 2014 / 035457 to yield a new strain with the genotype GalG20 attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC. These strains can be used for the production of vectors having a pUC bacterial origin of replication and RNA-OUT antibiotic free selection marker. EXAMPLE 2: SbcCD Knockout Strain Performance with AAV ITR Vectors: ITR stability

[0184] The application of GALG20 ∆SbcDC host strains to improve AAV ITR containing vector stability was evaluated with: the 5.5 kb AAV2 R6K origin-RNA-OUT marker NanoplasmidTMvector in GALG20 ∆SbcDC ∆dcm R6K Replication Origin with RNA-OUT Selection host; and a 12 kb pUC origin-kanR AAV vector in GALG20 ∆SbcDC ∆dcm.

[0185] To evaluate the GALG20 ∆SbcDC host strains ability to stabilize AAV ITRs, the AAV ITR vectors were transformed into the GALG20 ∆SbcDC host strains. Individual colonies were screened for intact ITRs by SmaI and AhdI digestion, then correct clones were submitted to Mass General Hospital (MGH) CCIB DNA Core (Cambridge MA) for Complete Plasmid Sequencing by Next Generation Sequencing. For the 5.5 kb AAV2 R6K origin-RNA-OUT marker NanoplasmidTMvector in GALG20 ∆SbcDC ∆dcm R6K Replication Origin with RNA-OUT Selection host, 10 / 10 screened colonies had intact ITRs by restriction digestion, and 9 / 9 screened colonies had intact ITRs by Next Generation Sequencing. For the 12 kb pUC origin-kanR AAV vector in GALG20 ∆SbcDC ∆dcm pUC origin vector host, 6 / 6 screened colonies had intact ITRs by restriction digestion, and 6 / 6 screened colonies had intact ITRs by Next Generation Sequencing. This demonstrates ITR stability during transformation in the pUC and R6K origin GALG20 ∆SbcDC host strains. EXAMPLE 3: SbcCD Knockout Strain Performance with AAV ITR Vectors: HyperGROTMFermentation

[0186] The application of GALG20 ∆SbcDC host strains to improve AAV ITR containing vector stability and production was evaluated in HyperGROTMfermentation with: the 5.5 kb AAV2 R6K origin-RNA-OUT marker NanoplasmidTMvector in GALG20 ∆SbcDC ∆dcm R6K Replication Origin with RNA-OUT Selection host (GALG20-REVIVER-R6K) compared to DH5α ∆SbcDC and E. cloni 5-alpha ∆SbcDC ∆dcm R6K Replication Origin with RNA-OUT Selection host hosts; and a 12 kb pUC origin-kanR AAV vector in GALG20 ∆SbcDC ∆dcm (GALG20-REVIVER- pUC) compared to DH5α, DH5α ∆SbcDC, E. cloni 5-alpha ∆SbcDC ∆dcm and Stbl3. The results are summarized in Tables 1 and 2.Table 1: pAAV NanoplasmidTMvector (5.5 kb) (R6K origin, RNA-OUT selection) HyperGROTMfermentation evaluation Cell linebHyperGROTMHarvest Plasmid Plasmid Plasmid Plasmid ITR Ferm OD600 yield yield specific quality integrity inb All cell lines are R6K Replication Origin with RNA-OUT Selection derivates with genomically integrated copies of 1) attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; 2) attλ:: Pc-RNA-IN-SacB, catR; and 3) attφ80::pARA-CI857ts, tetR c The DH5α ∆SbcDC strain was disclosed in WO 2021 / 183827 ‘Bacterial Host Strains’ d E. cloni 5-alpha ∆SbcDC Δdcm genotype: F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal-phoA supE44 λ- thi-1 gyrA96 relA1 fhuA2 glnV44 ∆SbcDC Δdcm Table 2: pAAV vector (12 kb pUC origin-kanR) HyperGROTMfermentation evaluation Cell HyperGROTMHarvest Plasmid Plasmid Plasmid Plasmid ITR tyStbl3a20 171 4.3 8.7 CCC √ b 27 214 3.6 7.9 monomerb 30-->37 °C ramp 24-36h c 30 °C, Shift to 37 °C at 55 OD600 until OD drops or lysis, 25 °C Hold d 30 °C, Shift to 37 °C at 30 h until OD drops or lysis, 25 °C Hold e E. cloni 5-alpha ∆SbcDC Δdcm genotype: F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ- thi-1 gyrA96 relA1 fhuA2 glnV44 ∆SbcDC Δdcm f The DH5α ∆SbcDC strain was disclosed in WO 2021 / 183827

[0187] The GALG20 ∆SbcDC ∆dcm host strains GALG20-REVIVER-R6K and GALG20- REVIVER-pUC showed improved plasmid production and / or plasmid quality compared to the Stbl3 , DH5α, DH5α ∆SbcDC or E. cloni 5-alpha ∆SbcDC ∆dcm hosts with AAV ITR vectors. The GALG20 ∆SbcDC ∆dcm host additionally stabilized AAV ITRs during cell line transformation. The improved yield with GALG20 ∆SbcDC ∆dcm is unexpected since plasmid productivity in fed batch HyperGROTMfermentation has been reported to be similar with GALG20 and DH5α (Goncalves, et al., (2014) Journal of Biotechnology 186:119-127).

[0188] The foregoing examples may be repeated using MG1655 derivates with additional gene knockouts, for example relA, ackA, topA, pta, poxB, fruR, pykA, pykF, purR, stress inducible DNA polymerases, transposon or transposase gene deletions, rac prophage deletion, etc, or additional gene overexpression through promoter up mutations or ectopic gene copies of polA, ligA, gyrAB, trxA grx1, zwf, rpiA, prsA, priA, priB, dnaT, and priC.

[0189] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0190] The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0191] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS What is claimed is:

1. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises a gene knockout of at least one gene selected from SbcC and SbcD, and at least one gene selected from the group consisting of endA, recA and pgi.

2. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises a gene knockout of SbcC and SbcD, and at least one gene selected from endA, recA and pgi.

3. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises a gene knockout of SbcC, SbcD, endA, and recA.

4. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises a gene knockout of SbcC, SbcD, endA, recA, and pgi.

5. The engineered bacterial host cell of any of claims 1-4, further comprising a sbcB gene, a recB gene, a recD gene, and a recJ gene, and wherein there are no engineered viability- or yield-reducing mutations in any of the sbcB, recB, recD, and recJ genes.

6. The engineered bacterial host cell of any of claims 1-5, further comprising at least one gene selected from the group consisting of uvrC, mcrA, and mcrBC-hsd-mrr, wherein the engineered bacterial host cell does not include any engineered viability- or yield-reducing mutations in at least one gene selected from uvrC, mcrA, and mcrBC-hsd-mrr.

7. The engineered bacterial host cell of any of claims 1-6, further comprising a gene selected from fhuA and glnV.

8. The engineered bacterial host cell of any of claims 1-6, further comprising an fhuA gene and a glnV gene.

9. The engineered bacterial host cell of any of claims 1-8, further comprising a gene knockout of a dcm gene.

10. The engineered bacterial host cell of any of claims 1-9, wherein the engineered bacterial host cell does not contain a supE44 gene.

11. The engineered bacterial host cell of any of claims 1-10, wherein the engineered bacterial host cell is derived from an Escherichia coli (E. coli) cell line.

12. The engineered bacterial host cell of claim 11, wherein the E. coli cell line is selected from the group consisting of DH5α, DH1, JM107, JM108, JM109, XL1Blue, and MG1655.

13. The engineered bacterial host cell of claim 11, wherein the E. coli cell line is MG1655.

14. An engineered bacterial host cell, wherein the engineered bacterial host cell is derived from an E. coli cell line, wherein the E. coli cell line is selected from the group consisting of GalG20, MG1655, and MG1655 ΔendA ΔrecA, and wherein the engineered bacterial host cell comprises a gene knockout of at least one gene selected from the group consisting of SbcC and SbcD.

15. An engineered bacterial host cell, wherein the engineered bacterial host cell is derived from an E. coli cell line, wherein the E. coli cell line is selected from the group consisting of GalG20, MG1655, and MG1655 ΔendA ΔrecA, and wherein the engineered bacterial host cell comprises a gene knockout of a SbcC gene and a SbcD gene.

16. The engineered host cell of any of claims 14-15, wherein the engineered bacterial host cell comprises a sbcB gene, a recB gene, a recD gene, and a recJ gene, and wherein there are no engineered viability- or yield-reducing mutations in any of the sbcB, recB, recD, and recJ genes.

17. The engineered bacterial host cell of any of claims 1-16, wherein the engineered bacterial host cell further comprises a genomic nucleic acid sequence encoding a Rep protein, wherein the Rep protein comprises an amino acid sequence of at least 90% sequence identity to a sequence selected from SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:

28. SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:

41. SEQ ID NO: 42, SEQ ID NO: 34, and SEQ ID NO:

35.

18. The engineered bacterial host cell of any of claims 1-17, further comprising a genomic nucleic acid sequence encoding a temperature-sensitive lambda repressor.

19. The engineered bacterial host cell of claim 18, wherein the temperature-sensitive lambda repressor is cITs857.

20. The engineered bacterial host cell of claim 18, wherein the temperature-sensitive lambda repressor comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:

37.

21. The engineered bacterial host cell of claim 18, wherein the temperature-sensitive lambda repressor is a phage φ80 attachment site chromosomally integrated copy of an arabinose inducible CITs857 gene.

22. The engineered bacterial host cell of any of claims 5 and 16, wherein sbcB gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 11, wherein the recB gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 12, wherein the recD gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 13, and wherein the recJ gene comprises a sequence having at least 90% sequence identity to SEQ ID NO: 65.

23. The engineered bacterial host cell of any of claims 1-22, further comprising a vector, wherein the vector comprises a nucleic acid sequence having an inverted repeat, a direct repeat, or a palindrome.

24. The engineered bacterial host cell of any of claims 1-22, further comprising a vector, wherein the vector is an AAV vector, a lentiviral vector, a retroviral vector, or a mRNA vector.

25. The engineered bacterial host cell of any of claims 1-24, further comprising a plasmid vector.

26. The engineered bacterial host cell of claim 25, wherein the plasmid vector is a eukaryotic pUC-free minicircle expression vector that comprises: (i) a eukaryotic region sequence encoding a gene of interest and having 5’ and 3’ ends; and (ii) a spacer region having a length of less than 1000 basepairs that links the 5’ and 3’ ends of the eukaryotic region sequence and that comprises a R6K bacterial replication origin and a RNA selectable marker.

27. The engineered bacterial host cell of claim 26, wherein the gene of interest comprises a structured DNA sequence selected from an inverted repeat sequence, a direct repeat sequence, a homopolymeric repeat sequence, an eukaryotic origin of replication, a polyA repeat, a SV40 origin of replication, a viral LTR, a Lentiviral LTR, a Retroviral LTR, a transposon IR / DR repeat, a Sleeping Beauty transposon IR / DR repeat, and an AAV ITR.

28. A method for improved vector production, comprising: providing an engineered bacterial host cell of any of claims 23-27; and incubating the engineered bacterial host cell under conditions sufficient to replicate the vector.

29. The method of claim 28, wherein the step of incubating the engineered bacterial host cell under conditions sufficient to replicate the vector is performed by a fed-batch fermentation, wherein the fed-batch fermentation comprises growing the engineered bacterial host cell at a first temperature of about 25oC to about 32oC during a first portion of the fed-batch phase, followed by a temperature up-shift to a second temperature of about 37oC to about 42oC during a second portion of the fed-batch phase.

30. The engineered bacterial host cell of claim 25, wherein the engineered bacterial host cell provides improved yield of the plasmid vector as compared to a reference engineered bacterial host cell that does not include a gene knockout of at least one of endA, recA and pgi, without a loss of stability and integrity of the plasmid vector as compared to the reference engineered bacterial host cell.

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