BACTERIAL STRAINS FOR DNA PRODUCTION

1 . An engineered nucleic acid vector comprising a stationary-phase-induced promoter and a primosome assembly site (PAS).

2 . The engineered nucleic acid vector of claim 1 , further comprising point-mutations causing the formation of a critical stem-loop on RNAII, SL4.

3 . The engineered nucleic acid vector of claim 1 or 2 , wherein a native promoter for RNAII has been disrupted.

4 . The engineered nucleic acid vector of claim 1 or 2 , wherein a native promoter for RNAII has been deleted.

5 . The engineered nucleic acid vector of claim 1 or any one of claims 2 - 4 , wherein the stationary-phase-induced promoter is P(osmY).

6 . The engineered nucleic acid vector of claim 5 , wherein the P(osmY) has a sequence of SEQ ID NO: 27.

7 . The engineered nucleic acid vector of any one of claims 1 - 6 , wherein the PAS has a sequence of SEQ ID NO: 28.

8 . The engineered nucleic acid vector of claim 2 or any one of claims 3 - 7 , wherein the SL4 has a sequence of SEQ ID NO: 29.

9 . The engineered nucleic acid vector of claim 8 , wherein the vector is Plasmid 1 (+PAS+P(osmY)).

10 . The engineered nucleic acid vector of claim 8 or claim 9 , wherein the vector is Plasmid 2 (+PAS+P(osmY)+SL4).

11 . The engineered nucleic acid vector of claim 1 , wherein the vector has a sequence of at least 70% sequence identity to SEQ ID NO: 19.

12 . The engineered nucleic acid vector of claim 1 , wherein the vector has a sequence of at least 70% sequence identity to SEQ ID NO: 20.

13 . The engineered nucleic acid vector of any one of claims 1 - 12 , comprising in the following 5′ to 3′ configuration:

(a) an origin of replication;

(b) the promoter; and

(c) an antibiotic resistance gene.

14 . The engineered nucleic acid vector of any one of claims 1 - 13 , further comprising an open reading frame (ORF) encoding an mRNA of interest.

15 . A recombinant plasmid comprising the geneotype:|<repA|ori_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)>|gamma>|beta>|exo>|a>|.

16 . A recombinant plasmid comprising a nucleic acid sequence with at least 70% identity to SEQ ID NO: 19.

17 . A recombinant plasmid comprising a nucleic acid sequence with at least 70% identity to SEQ ID NO: 20.

18 . A method of performing an in vitro transcription reaction using the engineered nucleic acid vector of any one of claims 1 - 17 .

19 . A nucleic acid comprising a prsA variant.

20 . The nucleic acid of claim 19 , wherein the nucleic acid has 70%-99% sequence identity to prsA* (SEQ ID NO: 23).

21 . The nucleic acid of claim 19 , wherein the nucleic acid has at least 70% sequence identity to prsA* (SEQ ID NO: 23)

22 . The nucleic acid of claim 19 , wherein the nucleic acid has at least 80%, 90%, or 95% sequence identity to prsA* (SEQ ID NO: 23).

23 . The nucleic acid of claim 19 , wherein the nucleic acid encodes a protein having at least 95% sequence identity to prsA* (SEQ ID NO: 24).

24 . The nucleic acid of claim 19 , wherein the nucleic acid has 100% sequence identity to SEQ ID NO: 23 or encodes a protein having 100% sequence identity to SEQ ID NO: 24.

25 . A genetically modified microorganism comprising a prsA variant, wherein the microorganism has a genome in which a repressor gene purR has been disrupted.

26 . The genetically modified microorganism of claim 25 , wherein the prsA variant has 70%-99% sequence identity to prsA.

27 . The genetically modified microorganism of claim 25 , wherein the prsA variant has least 90% sequence identity to prsA* (SEQ ID NO: 23).

28 . The genetically modified microorganism of claim 25 , wherein the prsA variant comprises a sequence of SEQ ID NO: 23.

29 . The genetically modified microorganism of any one of claims 25 - 28 , wherein the purR has been deleted.

30 . The genetically modified microorganism of claim 29 , wherein the purR comprises a sequence of SEQ ID NO: 25.

31 . The genetically modified microorganism of any one of claims 25 - 30 , wherein an EcoKI restriction system has been deleted from the genome.

32 . The genetically modified microorganism of any one of claims 25 - 31 , wherein endA has been deleted from the genome.

33 . The genetically modified microorganism of any one of claims 25 - 32 , wherein recA has been deleted from the genome.

34 . The genetically modified microorganism of any one of claims 25 - 33 , wherein the genetically modified microorganism is a recombinant strain of Escherichia coli ( E. coli ).

35 . A recombinant strain of Escherichia coli ( E. coli ), comprising: an E. coli genome with at least the following gene deletions: endA (ΔendA) and recA (ΔrecA).

36 . The recombinant strain of claim 35 , wherein the E. coli is derived from MG1655.

37 . The recombinant strain of claim 35 or claim 36 , wherein the E. coli genome comprises a nucleic acid sequence of MG1655 genome including at least the following gene deletions: endA (ΔendA) and recA (ΔrecA) with respect to the MG1655 genome.

38 . The recombinant strain of claim 35 or any one of claims 36 - 37 , wherein the E. coli genome comprises a nucleic acid sequence of at least 95% sequence identity with MG1655 genome.

39 . The recombinant strain of any one of claim 35 - 38 , wherein an EcoKI restriction system has been deleted from the genome of the E. coli.

40 . The recombinant strain of claim 39 , wherein the E. coli genome comprises a nucleic acid sequence with at least 80% identity to MG1655 genome.

41 . The recombinant strain of claim 39 or claim 40 , wherein the E. coli genome comprises a nucleic acid sequence of wherein the E. coli genome comprises a nucleic acid sequence of MG1655 genome including the EcoKI restriction system deletion with respect to the MG1655 genome.

42 . The recombinant strain of any one of claim 35 - 41 , wherein the E. coli comprises a prsA variant.

43 . The recombinant strain of claim 42 , wherein the E. coli genome comprises a nucleic acid sequence with at least 80% identity to MG1655 genome.

44 . The recombinant strain of claim 43 , wherein the E. coli genome comprises a nucleic acid sequence of SEQ ID NO: 23.

45 . The recombinant strain of any one of claim 35 - 44 , wherein a purR sequence has been deleted from the genome of the E. coli.

46 . The recombinant strain of claim 45 , wherein the E. coli genome comprises a nucleic acid sequence with at least 80% identity to MG1655 genome.

47 . The recombinant strain of claim 46 , wherein the E. coli genome has a nucleic acid sequence of SEQ ID NO: 25 deleted with respect to the MG1655 genome.

48 . The recombinant strain of any one of claim 35 - 47 , wherein the E. coli genome further comprises: at least one of gene deletions selected from the group comprising: mrr; hsdR; hsdM; hsdS; symE; and mcrBC.

49 . The recombinant strain of any one of claim 35 - 48 , the E. coli genome is derived from the strain MG or KS.

50 . A genetically modified microorganism comprising Strain 3.

51 . A genetically modified microorganism comprising Strain 4.

52 . An engineered nucleic acid vector comprising a nucleic acid having at least 70% sequence identity to SEQ ID NO: 21.

53 . An engineered nucleic acid vector comprising a nucleic acid having at least 80% sequence identity to SEQ ID NO: 21.

54 . An engineered nucleic acid vector comprising a nucleic acid having at least 90% sequence identity to SEQ ID NO: 21.

55 . An engineered nucleic acid vector comprising a nucleic acid having at least 95% sequence identity to SEQ ID NO: 21.

56 . An engineered nucleic acid vector comprising a nucleic acid having SEQ ID NO: 21.

57 . An engineered nucleic acid vector comprising a nucleic acid having at least 70% sequence identity to SEQ ID NO: 22.

58 . An engineered nucleic acid vector comprising a nucleic acid having at least 80% sequence identity to SEQ ID NO: 22.

59 . An engineered nucleic acid vector comprising a nucleic acid having at least 90% sequence identity to SEQ ID NO: 22.

60 . An engineered nucleic acid vector comprising a nucleic acid having at least 95% sequence identity to SEQ ID NO: 22.

61 . An engineered nucleic acid vector comprising a nucleic acid having SEQ ID NO: 22.

62 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NO: 1-15.

63 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 1-15.

64 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 1-15.

65 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 1-15.

66 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 99% sequence identity to any one of SEQ ID NO: 1-15.

67 . An engineered nucleic acid vector comprising a nucleic acid sequence of any one of SEQ ID NO: 1-15.

68 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 10.

69 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 11.

70 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 10.

71 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 11.

72 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 10.

73 . An engineered nucleic acid vector comprising a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 11.

74 . An engineered nucleic acid vector comprising a nucleic acid sequence of SEQ ID NO: 10.

75 . An engineered nucleic acid vector comprising a nucleic acid sequence of SEQ ID NO: 11.